纤毛功能障碍对内脏异位合并先天性心脏病预后的影响及相关致病基因研究

Congenital heart disease (CHD) patients with heterotaxy show high postsurgical morbidity/mortality, with some developing respiratory complications. While this is often attributed to the CHD, airway clearance and left-right patterning both require motile cilia function. Recent studies have revealed an association among heterotaxy, congenital heart disease, and primary ciliary dyskinesia. Thus airway ciliary dysfunction (CD) similar to that of primary ciliary dyskinesia (PCD) may have relevance for increased respiratory complications in heterotaxy patients. We will compare mortality and respiratory complications in CHD patients heterotaxy with that in CHD patients without heterotaxy to explore the relation between heterotaxy and postsurgical mortality, as well as respiratory complications. We will recruit CHD patients with heterotaxy and also without heterotaxy as control to assess the incidence of CD by analysis of measurement of nasal NO and airway ciliary motion, which is evaluated by using standardized computational quantitative approach. Outcome studies will be conducted to determine if heterotaxy patients with CD undergoing cardiac surgery may suffer more respiratory complications and worse outcome. We will undertake exome sequencing to determine if mutations in cilia related genes are specifically associated with heterotaxy patients with CD to further verify the unifying role of cilia defects in CD and worse outcome. We will determine if there are genotype-phenotype correlations between different types of cilia structural defects and ciliary motion defects. New candidate genes will be functionally assessed for their role in motile cilia function with gene knockdown in zebrafish embryos with antisense morpholino knockdown to assay for left-right patterning defects. These studies have the potential to transform patient care and provide the basis for future clinical trials to evaluate the efficacy of applying pre and postoperative pulmonary therapy to improve outcome for patients undergoing high-risk cardiac surgeries.

先心病合并内脏异位患者的术后呼吸道并发症和死亡率较高。研究提示，内脏异位、先心病和原发性纤毛不动症之间存在密切关系。我们假设，纤毛运动障碍可能是导致先心病合并内脏异位患者术后呼吸道并发症高发的重要原因。本研究内容包括：1.通过临床病例对照研究，观察先心病合并内脏异位患者呼吸道并发症和预后情况；2.测定先心病合并内脏异位患者鼻窦一氧化氮浓度、气道纤毛运动，观察纤毛功能障碍的发生率；3.利用二代测序技术检测纤毛功能相关基因的突变情况；4.对发现有基因突变的纤毛进行基因表达、形态运动和功能分析，并分析基因型与纤毛运动表型之间的关系；5.针对新发现的致病突变，通过吗啡林技术在斑马鱼胚胎中下调，观察其在左右模式缺陷中的作用。本研究结果不仅可阐明纤毛功能障碍对先心病合并内脏异位患者呼吸道并发症的影响，而且可揭示纤毛功能障碍在内脏异位中的作用机制，有助于改善先心病的临床预后。

先心病合并内脏异位患者的术后呼吸道并发症和死亡率较高，研究提示，内脏异位、先心病和原发性纤毛运动障碍之间存在密切关系。本研究内容包括：1. 通过临床病例对照研究，观察先心病合并内脏异位患者呼吸道并发症和预后情况；2. 测定先心病合并内脏异位患者鼻窦一氧化氮浓度、呼吸道纤毛运动，观察纤毛功能障碍的发生率；3. 利用二代测序技术检测纤毛功能相关基因的突变情况；4. 对发现有基因突变的纤毛进行基因表达、形态运动和功能分析，并分析基因型与纤毛运动表型之间的关系；5. 针对新发现的致病突变，通过吗啡啉技术在斑马鱼胚胎中下调基因，通过rescue方法观察致病突变在左右模式缺陷中的作用。本研究发现46.3%的内脏异位合并先心病患儿存在纤毛功能障碍，且伴有鼻窦一氧化氮的降低；而纤毛功能障碍的内脏异位合并先心病患儿手术后呼吸道并发症增加，基因检测结果提示，原发性纤毛功能障碍致病基因在纤毛功能障碍的内脏异位合并先心病患儿中有着较高的突变率，基因型与纤毛运动表型之间关联度良好。同时，根据ZIC3的新发突变位点，我们利用细胞及斑马鱼平台进行功能验证实验，证实了其致病性。本课题研究从临床问题出发，以临床问题为出发点，采用先进技术和跨学科的思路进行研究，通过病例对照研究，分析合并内脏异位的先心病患者的纤毛功能障碍，以及与呼吸道并发症的关系。从临床角度明确合并内脏异位的先心病患儿术后是否存在高呼吸道并发症和不良预后，然后通过高通量测序技术筛选、发现相关致病基因突变，并通过体外纤毛细胞的培养观察和斑马鱼模式动物研究，明确基因突变与纤毛运动及左右发育模式的关系及具体机制，充分证明了研究的假说：纤毛运动障碍是导致先心病合并内脏异位患者术后呼吸道并发症高发的重要原因，研究不仅从纤毛的角度创新性地解释了内脏异位的发病机理，而且也为内脏异位合并先心病患儿的围术期临床诊治提供理论依据，具有很好的临床转化意义。