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破坏神经发育过程的突变体，并随后鉴定致病分子，对于理解神经系统发育的遗传基础至关重要。然而，由于通过传统的基因定位技术来识别被破坏的基因需要花费大量的时间和人力，因此，通过前向基因筛选来识别神经生物学过程所需的基因总补体的全部前景很少实现。近年来，利用全基因组测序（WGS）来鉴定化学诱变秀丽隐杆线虫或果蝇菌株的致病点突变的原理证明研究已经出现。在这两种情况下，该战略都是快速且具有成本效益的。我们建议采用WGS技术来识别我们在初步研究中产生的大量果蝇神经肌肉连接（NMJ）突触突变体中的分子病变。利用传统的遗传作图技术，我们已经在这些突变体的子集中确定了被破坏的基因，并随后表征了新的突触调节途径以及人类疾病相关蛋白的果蝇同源物中的突变。我们将在另外40个选定的突触结构突变体中确定致病遗传缺陷，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位点进行深入分析提供指导。此外，全基因组测序的重复、常规应用将为该方法的可重复性和可靠性提供有价值的信息，并将该技术建立为果蝇和其他神经遗传模型系统中神经系统突变体的最先进克隆技术。

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.