Human genetics and molecular mechanisms of congenital hydrocephalus

项目摘要 先天性脑积水（CH），即充满脑脊液（CSF）的脑室的原发性扩大， 影响1/1000的新生儿，是发病率和死亡率的主要原因。尽管所有CH病例中约60%为 预测具有遗传病因，已知基因占CH病例的<5%。我们的重大差距 对CH分子发病机制的理解阻碍了预防、诊断和治疗的发展， 治疗措施。使用传统遗传学方法发现CH基因的根本障碍 包括基因座异质性、表型复杂性和大多数CH病例的散发性。 全外显子组测序（WES）有可能克服这些障碍，并导致前所未有的 自闭症和结构性脑疾病的基因发现的机会。我们最近使用WES识别了四个 新的CH基因，占研究的CH病例的~10%（Furey等，Neuron，2018）。这四个基因都是 神经干细胞（NSC）是神经管发育所必需的，并调节神经干细胞（NSC）命运。这些结果表明 在一个重要的CH亚组的发病机制中，神经发生受损，而不是CSF过度蓄积 患者，具有潜在的范式改变诊断和治疗意义。许多因果CH 基因仍然未被发现，我们的目标是利用功能基因组学方法来发现， 验证，并获得对新发现的CH引起的突变的机理洞察。我们的假设是WES 将鉴定出多种新的CH基因，其中许多将集中在调节NSC的途径上 发展根据我们成功识别几种CH和结构脑的经验， 在过去的几年里，我们已经发现了一些基因，现在我们建议确定额外的散发性CH病例-父母 trios和土耳其血缘家族CH形式，并在我们的大型，良好的表型CH上进行WES 队列发现新的从头和传播CH基因突变。随后将进行分析， 确定哺乳动物大脑发育过程中新发现的优先基因的表达模式。 然后，我们将快速和廉价的功能筛选优先CH候选基因突变， 使用我们的新的、经验证的利用活爪蟾胚胎的平台重现脑积水的能力， CRISPR/Cas9基因编辑、定量光学相干断层扫描和实时CSF颗粒跟踪 (Date例如，Sci Rep，2019，已接受）。对于选择验证的基因，我们将建立爪蟾系，以阐明 人类CH突变对纤毛调节的CSF动力学和NSC的生物学后果 生长/分化和图案化。这种功能基因组学方法将阐明遗传结构 的CH，并设置了更详细的未来生物学研究的范围以外的小鼠模型的阶段， 提议最终，这些知识有可能改善临床管理，诊断， 监测和遗传咨询;刺激对新的非手术疗法的研究;提高 我们对CH患者及其家属的支持。

PROJECT SUMMARY Congenital hydrocephalus (CH), the primary enlargement of the cerebrospinal (CSF)-filled brain ventricles, affects 1/1000 births and is a major cause of morbidity and mortality. Although ~60% of all CH cases are predicted to have a genetic etiology, known genes account for <5% of CH cases. Significant gaps in our understanding of the molecular pathogenesis of CH impede the development of preventive, diagnostic, and therapeutic measures. Fundamental obstacles to CH gene discovery using traditional genetic approaches include locus heterogeneity, phenotypic complexity, and the sporadic nature of the majority of CH cases. Whole exome sequencing (WES) has the potential to overcome these obstacles and has led to unprecedented opportunities for gene discovery in autism and structural brain disorders. We recently used WES to identify four novel CH genes, accounting for ~10% of studied CH cases (Furey et al., Neuron, 2018). All four genes are required for neural tube development and regulate neural stem cell (NSC) fate. These results implicate impaired neurogenesis, rather than CSF over-accumulation, in the pathogenesis of a significant subset of CH patients, with potentially paradigm-changing diagnostic and therapeutic implications. As many causal CH genes remain undiscovered, our objective here is to utilize a functional genomics approach to discover, validate, and gain mechanistic insight into newly identified CH-causing mutations. Our hypothesis is that WES will identify multiple novel CH genes, many of which will converge on pathways that regulate the NSC development. Based on our experience that has been successful in identifying several CH and structural brain disorder genes over the past several years, we now propose to ascertain additional sporadic CH case-parent trios and Turkish consanguineous familial CH forms and perform WES on our large, well-phenotyped CH cohort to discover novel de novo and transmitted CH gene mutations. This will be followed by analyses to determine the expression patterns of newly identified prioritized genes during mammalian brain development. We will then rapidly and inexpensively functionally screen prioritized CH candidate gene mutations for their ability to recapitulate hydrocephalus using our novel, validated platform that utilizes live Xenopus embryos, CRISPR/Cas9 gene editing, quantitative optical coherence tomography, and real-time CSF particle tracking (Date et al., Sci Rep, 2019, Accepted). For select validated genes, we will establish Xenopus lines to elucidate the biological consequences of human CH mutations on cilia-regulated CSF dynamics and NSC growth/differentiation and patterning. This functional genomics approach will elucidate the genetic architecture of CH, and set the stage for more detailed future biological studies in mouse models beyond the scope of this proposal. Ultimately, such knowledge has the potential to improve clinical management, prognostication, surveillance, and genetic advice; stimulate research into new non-surgical therapies; and improve the quality of our support for CH patients and their families.