Genetic and Functional Studies of Human Ciliary Syndromes

描述（由申请人提供）：纤毛病是一种新兴的临床重叠疾病，其特征包括视网膜变性，肾囊性疾病以及中枢和周围神经系统缺陷。不同临床表型的统一，如肾病、Bardet-Biedl、Meckel-Gruber和Jeune综合征，是由共同的细胞基础（初级纤毛缺陷）和遗传重叠驱动的，其中相同基因的突变既可以引起离散的表型，也可以促进修饰突变，调节原发性遗传病变的外显率和表达性。这些观察结果导致的想法，总突变负荷在初级纤毛是一个强大的表型决定因素。此外，强大的，生理相关的体外和体内检测报告细胞器输出为我们提供了研究人类第二位点修饰剂的性质和特性的机会。在第一个资助期间，我们将纤毛病作为一个统一的实体，确定了几个致病和修饰基因和等位基因，并开发了评估其影响的检测方法。我们现在建议扩展这项工作，并首次评估一个离散的、生化表征的大分子系统纤毛功能系统的总突变负荷。我们将1)对所有已知的鞭梢内转运基因在严重程度谱上的大量患者进行测序，并询问表型严重程度如何与遗传病变相跟踪；2)我们将在纤毛细胞中使用体内互补和体外蛋白质稳定性和定位分析相结合的方法对所有发现的等位基因进行功能分析；最后，我们将开始探讨这些等位基因在哺乳动物中的意义。作为我们初步数据的一部分，我们已经在IFT139中发现了一个错义等位基因P209L，该等位基因在遗传上足以引起囊性肾病，但也与其他纤毛病基因中的等位基因相互作用，作为潜在的囊性肾病修饰因子。因此，我们将设计一只敲入小鼠，并询问a)该突变的纯合性是否足以导致肾囊肿的形成；b)在遗传敏感背景下引入209L是否会加重或增强囊性肾病。我们的研究将产生更多导致睫状体疾病的基因座，并扩大我们对第二位点修饰因子的知识库。此外，我们预计，上位的遗传属性的理解有可能告知广泛的疾病和提高遗传信息的临床应用。

DESCRIPTION (provided by applicant): The ciliopathies are an emerging group of clinically overlapping disorders, hallmarked by features that include retinal degeneration, renal cystic disease, and central and peripheral nervous system defects. The unification of diverse clinical phenotypes such as nephronophthisis, Bardet-Biedl, Meckel-Gruber and Jeune syndromes is driven by both common cellular basis (defects at the primary cilium) and genetic overlap, where mutations in the same genes can both cause discrete phenotypes and contribute modifying mutations that modulate the penetrance and expressivity of primary genetic lesions. These observations have led to the idea that the total mutational load in the primary cilium is a strong phenotypic determinant. Moreover, robust, physiologically relevant in vitro and in vivo assays that report on organelle output affords us the opportunity to study the nature and properties of second-site modifiers in humans. During the first funding period, we established the ciliopathies as a unified entity, identified several causal and modifying genes and alleles, and developed assays to evaluate their effect. We now propose to expand on this work and, for the first time, assess the total mutational load in a discrete, biochemically characterized macromolecular system ciliary functional system. We will 1) sequence a large cohort of patients across the severity spectrum for all known intraflagellar transport genes and ask how phenotypic severity might track with genetic lesions; 2) we will functionally assay all discovered alleles using a combination of in vivo complementation and in vitro protein stability and localization assays in ciliated cells; 3) finally, we will begin to interrogate the significance of such alleles in mammals. As part of our Preliminary Data, we have identified a missense allele, P209L, in IFT139 that appears genetically sufficient to cause cystic renal disease but also interacts with alleles in other ciliopathy genes as a potential cystogenic modifier. We will therefore engineer a knock-in mouse and ask a) whether homozygosity for this mutation is sufficient for renal cyst formation; and b) whether introduction of 209L in a genetically sensitized background can exacerbate or potentiate cystic renal disease. Our studies will yield additional loci that cause ciliary disease and expand our knowledge base of second-site modifiers. Moreover, we anticipate that understanding of the genetic attributes of epistasis has the potential to inform a broad range of disorders and improve the clinical utility of genetic information. PUBLIC HEALTH RELEVANCE: Although individually rare, the ciliopathies collectively represent a significant health burden and their genetic dissection will expedite prognosis, management, and treatment. Moreover, this group of disorders is emerging as an exceptionally useful system to study the relationship between genetic load in a functional system and clinical variability, offering a unique opportunity to understand pathology and disease progression at the level of the individual patient.