Cardiac phenotyping of CHD candidate genes in Xenopus

描述(由申请人提供)：先天性心脏病(CHD)发生在1000名活产儿中约有8名，全球每年影响130万新生儿。虽然有证据表明CHD确实有遗传基础，但大多数CHD负担仍然无法从遗传学上解释。新的基因组学技术可以有效地识别冠心病患者基因组中的变异，但只有一小部分人有第二个无关的等位基因来验证它们是致病基因。因此，迫切需要开发功能分析来评估这些CHD候选基因。这些功能分析的主要目的有两个：1)提供支持候选基因致病的证据；2)确定候选基因对正常发育和疾病状态的作用机制。在这里，我们开发了非洲爪哇作为一个快速模型系统来测试CHD基因，并应用先进的光学成像方法来检测心脏表型。非洲爪哇是一种重要的先天性心脏病动物模型：可以很容易地操纵大量的胚胎，使用反义吗啉寡核苷酸可以下调蛋白质的表达，并且心脏很容易被可视化。为了扩大可以在非洲爪哇进行建模的CHD频谱，我们需要更好的微尺度心脏成像方法。在R21阶段，我们将测试两项技术，光学相干断层扫描(OCT)和我们新型的血红蛋白对比减影血管成像(HCSA)，以证明非洲爪哇的微尺度成像可以用于筛查CHD基因组。光学相干断层扫描是一种光学成像系统，可以以高采集速度捕获微观结构，从而能够对动态心脏结构进行高分辨率表型分析。血红蛋白对比减影血管造影术(HCSA)是一种非侵入性、非破坏性、定量的微血管造影术，在彩色成像过程中利用血红蛋白作为内源性血流造影剂，使我们能够描绘异常结构并量化生物力学表型。在R33阶段，我们的总体目标是应用这些方法来促进蝌蚪心脏详细的高分辨率结构表型，这些表型可以用于快速测试心脏表型的CHD候选基因。这将使我们能够识别CHD候选基因中以前在心脏发育中没有作用的心脏表型，并作为未来机制研究的跳板。

DESCRIPTION (provided by applicant): Congenital heart disease (CHD) occurs in approximately 8 out of 1000 live births and effects 1.3 million newborns per year worldwide. While there is evidence to indicate that CHD does have a genetic basis, most of CHD burden remains unexplained genetically. New genomics technologies can efficiently identify variations in the genomes of CHD patients, but only a small percentage have second unrelated alleles to validate them as disease causing. Therefore there is a pressing need to develop functional assays to evaluate these candidate genes for CHD. There are two main goals for these functional assays: 1) provide evidence supporting candidate genes as disease causing and 2) identify the mechanism for the candidate gene on normal development and the disease state. Here we develop Xenopus as a rapid model system for testing CHD genes and apply advanced optical imaging methods to detect cardiac phenotypes. Xenopus is as an important animal model of congenital heart disease: large numbers of embryos can be readily manipulated, protein expression can be knocked-down using antisense morpholino oligos, and the heart is easily visualized. To expand the CHD spectrum that can be modeling in Xenopus, we need better microscale cardiac imaging methods. During the R21 phase, we will test two technologies, optic coherence tomography (OCT) and our novel hemoglobin contrast subtraction angiography (HCSA) to demonstrate that microscale imaging of Xenopus can be used to screen CHD genomic hits. Optic coherence tomography is an optical imaging system that can capture microscopic structures at high acquisition speeds allowing high-resolution phenotyping of dynamic heart structures. Hemoglobin contrast subtraction angiography (HCSA) is a noninvasive, nondestructive, quantitative microangiographic method that exploits the hemoglobin as an endogenous flow contrast agent during color imaging enabling us to delineate abnormal structures as well as quantify biomechanical phenotypes. In the R33 phase, our overall goal is to apply these methods to facilitate detailed high-resolution structural phenotypin of tadpole hearts that can be used to quickly test CHD candidate genes for cardiac phenotypes. This will allow us to identify cardiac phenotypes in CHD candidate genes that have no previous role in cardiac development and serve as a springboard for future mechanistic studies.