Genetic and Functional Studies of Human Ciliary Syndromes

纤毛疾病是一组遗传性疾病，由重叠的结构和/或功能统一起来 纤毛和基底体的缺陷。对这些疾病的研究强调了基本的 发展和动态平衡机制，同时提供机会来开发计算和 发现新致病基因的功能工具；扩大已知基因的表型谱；以及 设计合理的治疗方案。在过去二十年取得显著进展的背景下， 挑战和机遇并存。具体地说，尽管一些纤毛疾病的诊断率 接近90%时，初级隐性基因座上的基因预测能力仍不足以 告知临床表现。这在一定程度上是因为我们对多效性的病理机制的理解 而可变的表现力仍然知之甚少。此外，到目前为止，几乎所有的睫状病发现工作都有： (A)侧重于编码突变和基因组重排；和(B)假设猪瘟的致病性 等位基因在所有细胞环境中都是恒定的。在这次更新中，我们渴望改善这些知识差距。 首先，我们将利用全基因组siRNA筛查报告异常的BBS4细胞 WNT信令。这项实验获得了81个基因，当被抑制时，会加剧睫状体细胞 表型，因此代表了事实上的候选包含因果或上位性突变 患有睫状结构疾病的患者。我们将通过对我们的表型多样化的患者进行测序来检验这一假设 使用最先进的活体工具对功能合成的候选基因和等位基因进行队列和测试。 关键的是，这81个基因中的大多数不是睫状器的组成部分，这提供了一个机会 在睫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 将探索一种新的等位基因致病现象的研究，在这种现象中，真正的致病误解 在一些剪接异构体中，变异对蛋白质功能有害，但在另一些剪接异构体中表现为良性。使用 结合体内互补测试和体外生化研究，我们会问有多常见 这些现象以及亚细胞定位或稳定性是否可能代表信息性的生化 这些现象的驱动因素。最后，通过全基因组筛选调控纤毛发生的miRNAs，我们 发现let-7b是一种纤毛长度调节因子。随后的转录分析确定了42个纤毛基因 差异表达与let-7b的剂量相关，并预测了let-7b在其3‘非编码区的位置。 我们将使用这个丰富的数据集来询问在已知睫状病基因的3‘UTRs中是否存在这样的miRNA结合位点 在这一组障碍中起到因果关系和修正作用。联合起来，我们的研究将告知基因 通过发现可能调节疾病表现性的位置来构建纤毛病疾病实体的架构； 通过探索致病变异的上下文依赖效应；通过询问对 一类在很大程度上未被探索的影响调控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.