Disease Gene Discovery in Structural Brain Disorders

描述(申请人提供)：下一代DNA测序平台的引入为结构性脑疾病(SBDS)的基因发现带来了前所未有的机会，从而识别了几个对人类大脑和小脑发育至关重要的新基因。尽管取得了成功，但这些研究也揭示了与这些疾病的疾病基因发现相关的重大挑战，再次证实了它们的基因位点和表型异质性。根据我们在过去几年中成功识别出几个SBD基因的经验，我们现在建议确定更多的病例和生物样本，然后对以前与SBD相关的基因进行突变筛查。我们将对SBD‘突变阴性’队列进行外显子组测序，以发现新的基因，然后进行全面的研究，以研究这些新发现的基因，使用生物信息学分析来确定它们在胚胎大脑发育期间的时空表达模式，通过实验验证原位杂交和免疫染色，并通过使用现有的患者衍生材料(如皮肤成纤维细胞培养)以及过度表达和抑制实验，在蛋白质水平上研究变异的影响。对于数量有限的基因，我们的目标是建立诱导多能干细胞系，并使用果蝇RNAi系和小鼠宫内电穿孔来进一步检测这些突变在神经发育过程中的生物学后果。这些研究将继续揭开导致人脑形成的复杂事件背后的分子途径，为未来更详细的生物学研究奠定基础。

DESCRIPTION (provided by applicant): The introduction of next-generation DNA sequencing platforms led to unprecedented opportunities for gene discovery in structural brain disorders (SBDs), resulting in the identification of several novel genes fundamental in human cerebral and cerebellar development. Despite their success, these studies also revealed significant challenges associated with disease gene discovery in these disorders confirming, once again, their locus and phenotypic heterogeneity. Based on our experience that has been successful in identifying several SBD genes over the past several years, we now propose to ascertain additional cases and biological samples, followed by mutational screening for genes previously associated with SBDs. We will perform exome sequencing of the SBD 'mutation negative' cohort to discover novel genes followed by comprehensive studies to investigate these newly identified genes, using bioinformatics analyses to determine their temporal and spatial expression patterns during embryonic brain development, experimentally validating with in situ hybridization and immunostaining and studying the effects of the variants at the protein level, both by using available patient derived material, such as skin fibroblast cultures, as well as over-expression and inhibition experiments. For a limited number of genes, we aim to establish induced pluripotent stem cell lines and use Drosophila melanogaster RNAi lines and in utero electroporation in mouse to further examine the biological consequences of the mutations during neural development. These studies will continue to unravel the molecular pathways that underlie the complex events that result in formation of the human brain, setting the stage for more detailed future biological studies.