Genetic and Functional Studies of Human Ciliary Syndromes

 描述（由申请人提供）：尽管基因发现的快速加速，但我们对突变率和可变表达率的理解仍然有限。纤毛病变已成为一个有用的模型来研究这些现象。睫状体组成的知识的提高导致了许多疾病位点的鉴定，每个位点都可以贡献致病和修饰等位基因。此外，纤毛的信号传导功能的升值允许评估等位基因的体内致病潜力。在前一个循环中，我们询问了鞭毛内转运（IFT）颗粒中的突变负荷如何影响表型变异。通过这项工作，我们确定了新的疾病位点;我们显示了CNVs的显着贡献;我们显示了IFT突变负荷增加如何与表型严重程度相联系。在这里，我们将扩展我们的研究，以了解遗传疾病的模块化方法如何提高遗传数据的预测能力，为遗传缓冲机制提供信息，并帮助开发治疗方法。我们的最终目标是了解睫状体模块的变化如何影响表达能力。然而，由于整个睫状体蛋白质组在实验上仍然难以处理，我们将锚我们的工作从IFT颗粒获得的稳健的初步数据，并扩展到其他两个已知的睫状体颗粒：过渡区复合物和BBSome。我们提出三个目标。首先，通过对突变数据的系统功能分析，我们将询问三种复合物中突变的富集是否与内表型有关，以及特定亚颗粒之间是否存在遗传相互作用。其次，我们发现致病性假阴性预测的来源来自进化保守，其中人类突变等位基因在不同物种中是固定的。使用计算机和体内工具，我们发现这些现象可以由顺式互补引起，其中同一基因中的共同进化等位基因提供保护作用。我们会问，这是否是一个共同的缓冲机制，对有害的等位基因，以及我们是否可以预测顺式互补网站通过研究共同进化的致病蛋白质和它们的模块。最后，我们最近表明，蛋白酶体扩增可以挽救核心BBSome组件缺陷。然而，这样的操作未能挽救IFT缺陷，或由瞬时BBSome组分诱导的缺陷，这表明模块内蛋白质的位置可能对治疗剂具有不同的反应。我们将通过抑制体内和体外三种复合物的每一种成分来测试这一假设，并询问遗传或化学蛋白酶体增强是否可以不同地拯救表型。总体而言，我们的研究将确定模块内和相关模块之间的突变分布如何有助于可变表达;将询问未充分研究的顺式修饰现象，这可以告知人类单倍型的致病潜力;并将开始探索功能复合体中突变的不同位置如何影响对治疗干预的反应，从而使未来的临床试验适应于特定的基因型

 DESCRIPTION (provided by applicant): Despite the rapid acceleration of gene discovery, our understanding of penetrance and variable expressivity remains limited. The ciliopathies have emerged as a useful model to study these phenomena. An improved knowledge of ciliary composition has led to the identification of numerous disease loci, each of which can contribute causal and modifying alleles. Moreover, an appreciation of the signaling functions of the cilium has allowed the assessment of the in vivo pathogenic potential of alleles. In the previous cycle, we asked how mutational load in the intraflagellar transport (IFT) particle can influence phenotypic variability. Through this work, we identified new disease loci; we showed a significant contribution of CNVs; and we showed how increased IFT mutational load tracks with phenotypic severity. Here, we will extend our studies to understand how a modular approach to genetic disease can improve the predictive power of genetic data, inform genetic buffering mechanisms, and assist the development of therapeutics. Our ultimate goal is to understand how variation across the ciliary module affects expressivity. However, because the entire ciliary proteome remains experimentally intractable, we will anchor our work on robust Preliminary Data gained from the IFT particle and extend to the other two known ciliary particles: the transition zone complex and the BBSome. We propose three Aims. First, through the systematic functional analysis of mutational data, we will ask whether enrichment for mutations in the three complexes tracks with endophenotypes and whether there are genetic interactions between specific subparticles. Second, we have found a source of false negative prediction of pathogenicity to derive from evolutionary conservation, where a human mutant allele is fixed in a different species. Using in silico and in vivo tools, we found that these phenomena can be caused by cis complementation, where a co- evolving allele in the same gene offers a protective effect. We will ask whether this is a common buffering mechanism against deleterious alleles; and whether we can predict cis complementing sites by studying co- evolution within disease-causing proteins and their modules. Finally, we showed recently that proteasome augmentation can rescue core BBSome component defects. However, such manipulations failed to rescue IFT defects, or defects induced by transient BBSome components, suggesting that the position of proteins within modules might have a differential response to therapeutics. We will test this hypothesis by suppressing each component of the three complexes in vivo and in vitro and ask whether genetic or chemical proteasome augmentation can rescue phenotypes differentially. Overall, our studies will determine how the distribution of mutations within modules and between associated modules contribute to variable expressivity; will interrogate the understudied phenomenon of cis-modification, which can inform the pathogenic potential of haplotypes in humans; and will begin to explore how the differential position of mutations in functional complexes can influence response to therapeutic interventions and thus tailor future clinical trials to specific genotypes.