Deciphering the Genetics of Synapse Development by Whole Genome Sequencing

描述(由申请人提供)：在果蝇等模型系统中筛选扰乱神经发育过程的突变并随后鉴定致病分子一直是了解神经系统发育的遗传基础的核心。然而，正向基因筛查识别神经生物学过程所需的全部基因组合的全部承诺很少实现，部分原因是通过传统的基因图谱技术识别中断的基因需要时间和劳动力。近年来，在使用全基因组测序(WGS)来识别化学诱变的线虫或果蝇品系中的致病点突变方面进行了原则证明研究。在这两种情况下，这一战略都是快速和具有成本效益的。我们建议利用WGS技术来鉴定我们在初步研究中产生的大量果蝇神经肌肉接头(NMJ)突触突变的分子损伤。使用传统的遗传作图技术，我们先前已经在这些突变体的子集中确定了中断的基因，随后描述了新的突触调节途径以及人类疾病相关蛋白的果蝇同源基因的突变。我们将确定另外40个突触结构突变的致病遗传缺陷，目的是增加我们对突触发育的分子调控的理解，并为未来对未发现的基因座的深入分析提供指导。此外，重复、常规的全基因组测序将为该方法的重复性和可靠性提供有价值的信息，并将该技术确立为果蝇和其他神经遗传学模型系统中神经系统突变的最先进的克隆技术。 公共卫生相关性：我们将采用最新的高通量全基因组测序和克隆技术，并将其应用于神经发育突变。建立这项技术作为一种最先进的方法来识别调节神经发育的基因，将迅速扩大与正常和异常神经系统发育相关的分子概要，其中许多分子可能在人类神经疾病中发挥重要作用。

DESCRIPTION (provided by applicant): Screening for mutants that disrupt neurodevelopmental processes in model systems such as Drosophila and the subsequent identification of the causative molecules have been central to understanding of the genetic basis of nervous system development. However, the full promise of forward genetic screening to discern the total complement of genes required for a neurobiological process is rarely realized, in part due to the time and labor required to identify the disrupted genes through conventional genetic mapping techniques. Recent years have seen proof-of-principle studies on the use of Whole Genome Sequencing (WGS) to identify causative point mutations in chemically mutagenized C.elegans or Drosophila strains. In both cases, the strategy was fast and cost-effective. We propose to recruit WGS technology to identify the molecular lesions in a large collection of Drosophila neuromuscular junction (NMJ) synapse mutants we have generated in preliminary studies. Using conventional genetic mapping techniques, we have previously identified the disrupted genes in subset of these mutants and subsequently characterized both novel synaptic regulatory pathways as well mutations in the Drosophila orthologs of human disease relevant proteins. We will determine the causative genetic defect in an additional forty selected synaptic structure mutants with the goal to both increase our understanding of the molecular regulation of synapse development and provide a guide for future, in-depth analysis of the uncovered loci. Furthermore, the repeated, routine application of Whole Genome Sequencing will supply valuable information on the reproducibility and reliability of this approach and establish the technology as a state-of-the-art cloning technique for nervous system mutants in Drosophila and other neurogenetic model systems. PUBLIC HEALTH RELEVANCE: We will conscript the most current high throughput whole genome sequencing and cloning technology and apply it to neurodevelopment mutants. Establishment of this technology as a state-of-the-art method to identify genes that regulate neurodevelopment will rapidly expand the compendium of molecules associated with normal and aberrant nervous system development, many of which are likely to play important roles in human neurological disease.