Genetic and Functional Studies of Human Ciliary Syndromes

纤毛病是一组>100种遗传性疾病，通过重叠的结构和/或功能 纤毛和基体的缺陷。对这些疾病的研究强调了 发展和稳态机制，同时提供发展计算和 功能工具，以发现新的致病基因;扩大已知基因的表型谱;和 设计合理的治疗模式。在过去二十年取得显著进展的背景下， 挑战和机遇依然存在。具体来说，尽管一些纤毛病变的诊断率 接近90%时，主隐性基因座基因型的预测能力仍然不足以 告知临床表现。这部分是因为我们对多效性病理机制的理解 和可变表现力仍然知之甚少。此外，迄今为止，几乎所有的纤毛病发现工作都有： (a)集中于编码突变和基因组重排;和（B）假设 等位基因在所有细胞环境中是恒定的。在这次更新中，我们渴望弥补这些知识差距。 首先，我们将利用基因组范围的siRNA筛选，对BBS 4切除的细胞进行筛选，这些细胞报告异常的 Wnt信号。这个实验收集了81个基因，当这些基因被抑制时， 表型，因此代表事实上的候选人窝藏因果或上位突变， 睫状体疾病患者。我们将通过对我们的表型多样性患者进行测序来验证这一假设。 使用最先进的体内工具进行群组和功能性测试所得候选基因和等位基因。 重要的是，这81个基因中的大多数不是睫状体的组成部分，这提供了机会， 在纤毛功能障碍和其他细胞过程之间建立新的生物学联系。与此同时，我们将继续 研究将探索等位基因致病性的新现象，其中真正的致病性错义 变体在某些剪接异构体中表现为对蛋白质功能有害，但在其它剪接异构体中表现为良性。使用 结合体内互补试验和体外生化研究，我们将询问 这些现象是否是亚细胞定位或稳定性可能代表信息生化 这些现象的驱动因素。最后，通过全基因组筛选调节纤毛发生的miRNAs，我们 发现let-7 b作为纤毛长度的调节剂。随后的转录组学分析确定了42个纤毛基因 差异表达与let-7 b剂量相关，并且在其3' UTR中含有预测的let-7 b位点。 我们将利用这一丰富的数据集来研究已知纤毛病变基因的3'UTR中的这种miRNA结合位点是否 有助于因果关系和修改这组疾病。总之，我们的研究将告知遗传学 通过发现可能调节疾病表达性的位点来构建纤毛病变疾病实体; 通过探索致病变异的背景依赖效应;并通过询问 一种很大程度上未被探索的影响调节机制的变异类型的疾病。

The ciliopathies are a group of >100 genetic disorders unified by overlapping structural and/or functional defects of the cilium and the basal body. The study of these disorders has highlighted fundamental developmental and homeostatic mechanisms, while offering the opportunity to develop computational and functional tools to discover new causal genes; to expand the phenotypic spectrum of known genes; and to design rational therapeutic paradigms. In the backdrop of striking progress during the past two decades, major challenges and opportunities remain. Specifically, despite a diagnostic rate that for some ciliopathies approaches 90%, the predictive power of the genotype at the primary recessive locus remains insufficient to inform clinical manifestation. This is in part because our understanding of the pathomechanisms of pleiotropy and variable expressivity remain poorly understood. Moreover, almost all ciliopathy discovery work to date has: (a) focused on coding mutations and genomic rearrangements; and (b) assumed that the pathogenicity of alleles is constant in all cellular contexts. In this Renewal, we aspire to improve upon these knowledge gaps. First, we will take advantage of a genome-wide siRNA screen on cells ablated for BBS4 that report on aberrant Wnt signaling. This experiment harvested 81 genes that, when suppressed, exacerbate ciliary cellular phenotypes and therefore represent de facto candidates for harboring either causal or epistatic mutations in patients with ciliary disease. We will test this hypothesis by sequencing our phenotypically diverse patient cohort and testing functionally resultant candidate genes and alleles using state-of-the-art in vivo tools. Critically, the majority of these 81 genes are not components of the ciliary apparatus, offering the opportunity to forge new biological links between ciliary dysfunction and other cellular processes. In parallel, we will pursue studies that will explore a new phenomenon of allele pathogenicity, in which bona fide pathogenic missense variants behave as deleterious to protein function in some splice isoforms but benign in others. Using a combination of in vivo complementation testing and in vitro biochemical studies, we will ask how common these phenomena are and whether subcellular localization or stability might represent informative biochemical drivers of these phenomena. Finally, through a genome-wide screen for miRNAs that regulate ciliogenesis, we discovered let-7b as a regulator of ciliary length. Subsequent transcriptomic analysis identified 42 ciliary genes with differential expression correlated to let-7b dosage, and which harbor predicted let-7b sites in their 3’ UTR. We will use this rich dataset to ask whether such miRNA binding sites in 3’ UTRs of known ciliopathy genes contribute to causality and modification in this group of disorders. Together, our studies will inform the genetic architecture of the ciliopathy disease entity by discovering sites that likely modulate the expressivity of disease; by exploring the context-dependent effect of pathogenic variation; and by interrogating the contribution to disease of a largely unexplored class of variants impacting regulatory mechanisms.