Genetic and Functional Studies of Human Ciliary Syndromes

 描述(申请人提供)：尽管基因发现的速度很快，但我们对外显性和可变表现力的理解仍然有限。纤毛病变已成为研究这些现象的有用模型。对纤毛组成的了解的提高导致了许多疾病基因的识别，每个疾病基因都可以贡献因果和修饰等位基因。此外，对纤毛信号功能的认识使我们能够评估等位基因在体内的致病潜力。在上一个周期中，我们询问了鞭毛内转运(IFT)颗粒中的突变负载如何影响表型可变性。通过这项工作，我们识别了新的疾病位点；我们显示了CNV的显著贡献；我们还展示了IFT突变负载的增加如何随着表型严重程度的变化而变化。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，告知遗传缓冲机制，并帮助治疗学的发展。我们的最终目标是了解纤毛模块的差异是如何影响表现力的。然而，由于整个纤毛蛋白质组仍然在实验上难以处理，我们将基于从IFT颗粒获得的稳健的初步数据来锚定我们的工作，并扩展到其他两个已知的纤毛颗粒：过渡区复合体和BBSome。我们提出了三个目标。首先，通过对突变数据的系统功能分析，我们将询问三个复合体中突变的浓缩是否与内表型有关，以及特定亚颗粒之间是否存在遗传相互作用。其次，我们发现了致病性假阴性预测的来源来自进化保守，即人类突变的等位基因固定在不同的物种中。使用电子计算机和活体工具，我们发现这些现象可能是由顺式互补引起的，在这种情况下，同一基因中的共同进化的等位基因提供了保护作用。我们将询问这是否是一种针对有害等位基因的常见缓冲机制；以及我们是否可以通过研究致病蛋白及其模块内的共同进化来预测顺式互补位点。最后，我们最近展示了蛋白酶体增强可以修复核心BBSome组分的缺陷。然而，这种操作未能挽救IFT缺陷或由瞬时BBSome组分引起的缺陷，这表明蛋白质在模块中的位置可能对治疗有不同的反应。我们将通过在体内和体外抑制这三个复合体的每个成分来检验这一假设，并询问基因或化学蛋白酶体增强是否可以不同地挽救表型。总体而言，我们的研究将确定突变在模块内和相关模块之间的分布如何有助于变化的表现力；将询问未被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治疗干预的反应，从而为特定的基因类型量身定做未来的临床试验。

 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.