Human genetics and molecular mechanisms of congenital hydrocephalus

项目总结 先天性脑积水(CH)，即充满脑脊液的脑室的原发性增大， 影响1/1000的新生儿，是发病率和死亡率的主要原因。尽管~60%的CH病例是 据预测有遗传病因，已知的基因占CH病例的5%。我们在以下方面存在重大差距 对CH分子发病机制的了解阻碍了预防、诊断和治疗的发展 治疗措施。用传统遗传学方法发现CH基因的根本障碍 包括基因座的异质性、表型的复杂性和大多数CH病例的散发性。 全外显子组测序(WES)具有克服这些障碍的潜力，并导致了前所未有的 自闭症和结构性脑部疾病的基因发现机会。我们最近用WES识别出了四个 新的CH基因，约占所研究CH病例的10%(Furey等人，Neuron，2018年)。这四个基因都是 它是神经管发育和调节神经干细胞(NSC)命运所必需的。这些结果表明 在一个重要的CH亚型的发病机制中，神经发生受损，而不是脑脊液过度蓄积 患者，具有潜在的范式改变的诊断和治疗意义。许多因果关系的问题 基因仍未被发现，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导致CH的突变的机械性洞察。我们的假设是韦斯 将确定多个新的CH基因，其中许多将会聚在调节NSC的途径上 发展。根据我们的经验，已经成功地识别了几个CH和结构脑 疾病基因在过去几年中，我们现在建议确定额外的散发性CH病例-父母 三人组和土耳其血亲家族性CH形成并在我们的大型、表型良好的CH上执行WES 队列以发现新的从头开始和传递的CH基因突变。在此之后将分析 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，我们将快速和廉价地筛选优先的CH候选基因突变 能够使用我们的新型验证平台重现脑积水，该平台利用活的非洲爪哇胚胎， CRISPR/Cas9基因编辑、定量光学相干断层扫描和实时脑脊液颗粒跟踪 (Date等人，科学代表，2019年，接受)。对于选择有效的基因，我们将建立非洲爪哇品系来阐明 人类CH突变对纤毛调节的脑脊液动力学和神经干细胞的生物学影响 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 ，并为未来在小鼠模型中进行更详细的生物学研究奠定了基础 求婚。归根结底，这种知识有可能改善临床管理、预测、 监测和遗传建议；促进对新的非手术疗法的研究；并提高 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.