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

测定纤毛运动和左右模式中的异向性患者基因

• 批准号: 8623176
• 负责人: Srinivas Chakra Chennubhotla
• 金额: $ 57.38万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8623176
• 关键词: Accounting; Affect; Algorithms; Biological Assay; Candidate Disease Gene; Cardiac Surgery procedures; Cilia; Classification; Code; Complement; Complex; Computational algorithm; Computer Vision Systems; Computer software; Congenital Abnormality; Congenital Heart Defects; Defect; Disease; Disease model; Embryo; Epithelial Cells; Etiology; Exhibits; Functional disorder; Gene Combinations; Gene Frequency; Genes; Genetic; Genetic Predisposition to Disease; Genotype; Handedness; Heart; Human; Incidence; Knock-out; Left; Link; Live Birth; Machine Learning; Modeling; Morbidity - disease rate; Motion; Mucous body substance; Mus; Mutagenesis; Mutant Strains Mice; Mutation; Organ; Outcome; Pathogenicity; Patients; Pattern; Phenotype; Primary Ciliary Dyskinesias; Production; Proteins; Randomized; Reagent; Regulation; Resources; Role; Sequence Analysis; Situs Inversus; Structure; Structure of respiratory epithelium; System; Technology; Testing; Time; Variant; Vesicle; Visceral; Visual Pattern Recognition; With laterality; Zebrafish; airway epithelium; cilium motility; congenital heart disorder; disease phenotype; exome sequencing; founder mutation; gene function; innovative technologies; insight; mortality; mouse model; mutant; new technology; novel; public health relevance; rare variant; research clinical testing; research study; respiratory; software development; tool

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.

描述（由申请人提供）：内脏异位是一种出生缺陷，涉及内脏器官的随机左右模式，通常与复杂先天性心脏病（CHD）相关，反映了左右模式在四腔心脏不对称形成中的重要性。异位症（HTX）患者有不明原因的较高发病率和死亡率，往往增加术后呼吸系统并发症。这可能反映了运动纤毛的共同需求，无论是在胚胎的左右模式，也在气道粘液清除。我们最近发现，42%的HTX合并CHD患者（HTX/CHD）具有与原发性纤毛运动障碍（PCD）相似的气道纤毛功能障碍（CD），原发性纤毛运动障碍（PCD）是一种隐性疾病，与气道中不动/运动障碍纤毛引起的粘液清除缺陷导致的偏侧缺陷和sinusoidal疾病相关。值得注意的是，外显子组测序显示HTX CD患者（HTX/CD）在已知引起PCD的基因和其他纤毛相关基因中富含新型/罕见编码变体（RCV）。在本申请中，我们将通过外显子组测序分析对39名HTX/CD患者中鉴定的53个纤毛候选基因进行功能检测。我们将评估基因敲除对气道纤毛运动的影响，使用一种新的检测方法与再纤毛人气道上皮细胞。为了分析左右模式所需的基因功能，将在斑马鱼胚胎中进行反义MO敲低以检查心脏和肠循环。将进一步测试在敲低后破坏气道纤毛运动并引起HTX的基因，以确定RCV是否是致病性的。具体来说，我们将研究是否RCV的表达可以拯救HTX表型引起的斑马鱼胚胎中的MO基因敲低。考虑到HTX/CD患者中鉴定的所有RCV都是杂合的，我们假设疾病的多基因模型，将通过检查在HTX-CD患者中观察到的具有双基因组合的双杂合小鼠和斑马鱼突变体的表型来测试。我们将通过检测双杂合突变体中能动纤毛功能和内脏器官位置来研究双基因相互作用的证据。这些实验将询问8种双基因组合，其利用从我们正在进行的小鼠诱变筛选中回收的9种新型小鼠突变体，以及使用现有突变系和通过TALENs基因破坏重新产生4种斑马鱼敲除系的斑马鱼中的7种其他双基因组合。最后，为了建立睫状体运动缺陷的基因型-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睫状体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是否与不同的睫状运动缺陷相关。这将为纤毛运动调节的结构-功能关系提供见解。该软件，将作为一个在线提供 工具，将具有用于患者气道纤毛运动DAA的临床评价的转化潜力。总之，这些研究将建立功能测定和软件，可以阐明CHD/HTX的遗传病因。