Functional Studies of Novel Genes Mutated in Primary Ciliary Dyskinesia II: Genotype to Phenotype

项目总结/摘要 在原发性纤毛运动障碍（PCD）中，在纤毛运动障碍的正确组装或功能中起作用的蛋白质突变， 纤毛导致纤毛搏动缺陷，这导致粘膜纤毛清除大大减少或缺失 （MCC）。缺乏有效的MCC会导致慢性肺部感染、支气管扩张、慢性鼻窦炎和耳炎 媒体尽管所有PCD受试者具有相似的临床表型，但该疾病的严重程度不同， 一些患者症状轻微，而另一些患者发展为严重的支气管扩张。因为基因突变 许多导致PCD的基因已被证明导致基本上不动的纤毛， 结果没有MCC，因此不清楚为什么存在如此广泛的临床表型。我们的假设是 在PCD患者中观察到的疾病严重程度的异质性大部分是遗传的结果， 异质性，不同基因突变导致不同水平的残留MCC， 不同的疾病严重程度。此外，我们假设同一基因的不同突变也可能导致 在不同程度的纤毛损伤，MCC，和疾病的严重程度。最后，我们建议， 疾病表型差异的机制基础将为发展 针对这种罕见疾病的新的个性化治疗方法。具体目标是： 1.为了研究CFAP 57和PCDP 1等基因和蛋白的表达和功能， 其突变最近被证明会导致PCD。 使用患者来源的人鼻上皮（HNE）细胞和/或诱导多能干（iPS）细胞，我们 将研究突变在蛋白质水平、纤毛功能（波形和搏动）水平上的影响 频率）和体外粘膜纤毛转运（MCT）。 2.研究患者来源的iPS细胞中的基因型/表型关系。 我们将研究不同基因（CFAP 57，PCDP 1，RSPH 1， CCDC 39和DNAI 1）和iPS中相同基因的不同突变（SPAG 1、CCDC 114、DNAH 5 PCD患者的细胞。 3.在Ccdc 39缺失的小鼠模型中研究疾病的发病机制。 我们将比较Ccdc 39的诱导性缺失与Dnaic 1的诱导性缺失的效果。 4.探讨基因型、MCC与临床表型的关系。 MCC将在具有RSPH 1突变的PCD受试者中测量，并与具有RSPH 1突变的PCD受试者进行比较。 其它基因中的突变（例如，DNAI1、DNAH5）。此外，MCC将在以下给药后测量： β-激动剂，以确定是否可以在RSPH 1受试者中刺激MCC。

Project Summary/Abstract In primary ciliary dyskinesia (PCD), mutations in proteins that play a role in the proper assembly or function of the cilia result in defective ciliary beating, which leads to greatly reduced or absent mucociliary clearance (MCC). The lack of effective MCC results in chronic lung infections, bronchiectasis, chronic sinusitis, and otitis media. Although all PCD subjects have a similar clinical phenotype, the disease is heterogeneous in severity, with some patients having mild symptoms, while others develop severe bronchiectasis. Because mutations in many of the genes that cause PCD have been shown to result in essentially immotile cilia and are expected to result in no MCC, it is not obvious why there exists such a wide range of clinical phenotypes. Our hypothesis is that much of the heterogeneity of disease severity observed in PCD patients is a result of genetic heterogeneity, and that mutations in different genes result in varying levels of residual MCC and consequently, varying severity of disease. Further, we hypothesize that different mutations in the same gene can also result in different levels of ciliary impairment, MCC, and disease severity. Finally, we propose that understanding the mechanistic basis for the differences in disease phenotype will provide targets and opportunities to develop new, personalized treatments for this rare disease. The specific aims are: 1. To investigate the expression and function of genes and proteins, including CFAP57 and PCDP1, mutations of which have been newly shown to cause PCD. Using patient derived human nasal epithelial (HNE) cells and/or induced pluripotent stem (iPS) cells, we will investigate the effect of the mutations at the level of the protein, ciliary function (waveform and beat frequency), and mucociliary transport (MCT) in vitro. 2. To investigate genotype/phenotype relationships in patient derived iPS cells. We will examine the functional consequences of mutations in different genes (CFAP57, PCDP1, RSPH1, CCDC39, and DNAI1) and of different mutations in the same gene (SPAG1, CCDC114, DNAH5) in iPS cells from PCD patients. 3. To investigate the pathogenesis of disease in a mouse model with a deletion of Ccdc39. We will compare the effects of an inducible deletion of Ccdc39 to an inducible of Dnaic1. 4. To investigate the relationship between genotype, MCC, and clinical phenotype in vivo. MCC will be measured in PCD subjects with RSPH1 mutations and compared to PCD subjects with mutations in other genes (e.g., DNAI1, DNAH5). In addition, MCC will be measured after administration of a beta-agonist to determine if MCC can be stimulated in the RSPH1 subjects.