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

描述(申请人提供)：异位畸形是一种出生缺陷，涉及随机的左右内脏器官模式，经常与复杂的先天性心脏病(CHD)相关，反映了左右模式在四心腔心脏不对称形成中的重要性。异位症(HTX)患者具有无法解释的较高的发病率和死亡率，通常伴有术后呼吸道并发症的增加。这可能反映了对活动纤毛的普遍要求，无论是在胚胎的左右模式中，还是在呼吸道的粘液清除中。我们最近发现，42%的HTX合并CHD(HTX/CHD)患者的呼吸道纤毛功能障碍(CD)类似于原发性睫状体运动障碍(PCD)，PCD是一种隐性疾病，与偏侧畸形和鼻肺疾病相关，原因是呼吸道纤毛运动不全/运动障碍导致粘液清除缺陷。值得注意的是，外显组测序显示，患有CD的HTX患者(HTX/CD)在已知的导致PCD和其他纤毛相关基因的基因中富含新的/罕见的编码变体(RCV)。在这一应用中，我们将通过外显子序列分析，对39例HTX/CD患者中53个纤毛候选基因进行功能性分析。我们将评估基因敲除对呼吸道纤毛运动的影响，使用一种新的人呼吸道上皮细胞检测方法。为了测试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。将进一步测试被证明扰乱呼吸道纤毛运动并在击倒后导致HTX的基因，以确定RCVS是否是致病的。具体地说，我们将研究RCV的表达是否可以挽救斑马鱼胚胎中MO基因敲除所引发的HTX表型。鉴于在HTX/CD患者中发现的所有RCVS都是杂合的，我们假设了一个多基因疾病模型，该模型将通过在HTX-CD患者中观察到的双杂合小鼠和斑马鱼突变的表型来检验。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检验双基因交互作用的证据。这些实验将询问8个双基因组合，这些组合利用了从我们正在进行的小鼠突变筛选中恢复的9个新的小鼠突变，以及使用现有突变系在斑马鱼中的其他7个双基因组合，以及通过TALENS基因破坏从头产生4个斑马鱼基因敲除系。最后，为了建立睫状肌运动缺陷的基因型-表型相关性，我们将利用计算机视觉和机器学习视觉模式识别算法开发用于睫状肌运动缺陷定量分类的软件。使用该软件，我们将确定不同的RCVS是否与不同的睫状体运动缺陷有关。这将为纤毛运动调节中的结构-功能关系提供洞察力。此软件将以在线方式提供 工具，将具有翻译潜力的临床评估患者的呼吸道纤毛运动DAA。总而言之，这些研究将建立功能分析和软件，可以阐明CHD/HTX的遗传病因。

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.