Assaying Heterotaxy Patient Genes in Cilia Motility and Left-Right Patterning

描述（由申请人提供）：异位是一种涉及内脏器官随机左右排列的出生缺陷，通常与复杂先天性心脏病（CHD）有关，反映了左右排列在四腔心脏不对称形成中的重要性。异位（HTX）患者有不明原因的较高发病率和死亡率，通常伴有术后呼吸并发症。这可能反映了纤毛运动的共同需求，无论是在胚胎的左右模式和气道粘液清除。我们最近发现42%的HTX合并CHD （HTX/CHD）患者有与原发性纤毛运动障碍（PCD）相似的气道纤毛功能障碍（CD），这是一种隐性疾病，与侧侧缺陷和肺疾病相关，由气道内纤毛不动/运动障碍引起的粘液清除缺陷。值得注意的是，外显子组测序显示HTX患者的CD （HTX/CD）在已知引起PCD的基因和其他纤毛相关基因中富含新颖/罕见编码变体（RCV）。在本应用中，我们将通过外显子组测序分析对39例HTX/CD患者中鉴定的53个纤毛候选基因进行功能分析。我们将评估基因敲低对气道纤毛运动性的影响，使用一种新的实验与人类气道上皮细胞进行调节。为了测定左右模式蛋白所需的基因功能，将在斑马鱼胚胎中进行反义MO敲除，以检查心脏和肠道循环。将进一步检测显示破坏气道纤毛运动并在敲除后引起HTX的基因，以确定rcv是否具有致病性。具体来说，我们将研究RCV的表达是否可以挽救斑马鱼胚胎中MO基因敲除引发的HTX表型。鉴于在HTX/CD患者中发现的所有rcv都是杂合的，我们假设存在一种多基因疾病模型，将通过检测HTX-CD患者中观察到的双杂合小鼠和斑马鱼突变体的双基因组合表型来验证这一模型。我们将通过分析双杂合突变体的运动纤毛功能和内脏器官位置来研究基因相互作用的证据。这些实验将利用我们正在进行的小鼠突变筛选中恢复的9种新的小鼠突变体来研究8种基因组合，以及利用现有突变系和通过TALENs基因破坏重新生产的4种斑马鱼敲除系来研究斑马鱼中的7种其他基因组合。最后，为了建立纤毛运动缺陷的基因型-表型相关性，我们将使用计算机视觉和机器学习算法进行视觉模式识别的计算方法开发用于纤毛运动缺陷定量分类的软件。使用该软件，我们将确定不同的rcv是否与不同的纤毛运动缺陷相关。这将为纤毛运动调节中的结构-功能关系提供见解。这个软件，将作为一个在线提供

DESCRIPTION (provided by applicant): Heterotaxy, a birth defect involving randomized left-right patterning of visceral organs, is frequently associated with complex congenital heart disease (CHD), a reflection of the importance of left-right patterning in formation of asymmetries in the four-chamber heart. Heterotaxy (HTX) patients have unexplained higher morbidity and mortality, often with increased postsurgical respiratory complications. This may reflect the common requirement for motile cilia, both in embryonic left-right patterning and also mucus clearance in the airway. We recently showed 42% of HTX patients with CHD (HTX/CHD) have airway ciliary dysfunction (CD) similar to that of primary ciliary dyskinesia (PCD), a recessive disorder associated with laterality defects and sinopulmonary disease due to mucus clearance defects caused by immotile/dyskinetic cilia in the airway. Significantly, exome sequencing showed HTX patients with CD (HTX/CD) are enriched for novel/rare coding variants (RCV) in genes known to cause PCD and other cilia related genes. In this application, we will functionally assay 53 cilia candidate genes identified in 39 HTX/CD patients by exome sequencing analysis. We will assess the effects of gene knockdown on airway cilia motility using a novel assay with reciliating human airway epithelial cells. To assay gene function required for left-right patternin, antisense MO knockdown in zebrafish embryos will be carried out to examine heart and gut looping. Genes shown to disrupt airway cilia motility and cause HTX after knockdown will be further tested to determine whether the RCVs are pathogenic. Specifically we will examine whether expression of the RCV can rescue the HTX phenotype elicited by MO gene knockdown in the zebrafish embryo. Given all of the RCVs identified in HTX/CD patients were heterozygous, we hypothesize a multigenic model of disease, which will be tested by examining the phenotypes of double heterozygous mouse and zebrafish mutants with two-gene combinations observed in the HTX-CD patients. We will examine for evidence of digenic interactions by assaying motile cilia function and visceral organ situs in the double heterozygous mutants. These experiments will interrogate 8 digenic combinations that make use of 9 novel mouse mutants recovered from our ongoing mouse mutagenesis screen, and 7 other digenic combinations in zebrafish using existing mutant lines and de novo production of 4 zebrafish knockout lines by TALENs gene disruption. Finally, to establish genotype-phenotype correlation in ciliary motion defects, we will develop software for quantitative classification of ciliary motin defects using a computational approach with computer vision and machine learning algorithms for visual pattern recognition. Using this software, we will determine whether different RCVs are associated with different ciliary motion defects. This will provide insights into structure-functio relationships in the regulation of cilia motility. This software, to be made available as an online tool, will have translational potential for clinical evaluation of patient airway ciliary motion daa. Together, these studies will establish functional assays and software that can elucidate the genetic etiology of CHD/HTX.