BRWD1经BRG1调控Notch通路在原发性纤毛运动障碍左右非对称发育中的作用

Our previous study have confirm that BRWD1/WDR9 plays a key role in left-right asymmetry of primary ciliary dyskinesia(PCD),but the mechanism and pathway are not clear. There are several studies suggest that BRWD1 interacts with BRG1, which is a kind of key protein subunit within transcription factor of the SWI/SNF complex, and play the role of a transcriptional regulatory factor together. As is known to all, the SWI/SNF complex presents a crucial status in the integration process of cardiac structure and function, and the key protein subunit BRG1 is closely associated with the activation of Notch signaling pathway. Studies have confirmed that the depletion of key protein in the SWI/SNF complex does result in the damage of asymmetric development, and the specific mechanism lies on that the impact on the activation of Notch signaling pathway and the expression of downstream key target genes (Nodal). So we hypothesize that BRWD1 participate in asymmetric development by the regulation of Notch signaling pathway and expression of downstream target genes (Nodal) through BRG1. This study is proposed to observe the change of BRG1, Notch signaling pathway and Nodal through PCD patients with BRWD1/WDR9 variants, zebrafish model and mice fibroblast cells in vitro experimental. The purpose of this research is to investigate whether BRWD1 may cause asymmetric development by BRG1, as it can regulate Notch signaling pathway and the expression of downstream target genes (Nodal). It also aims to provide guidance to the scientific issue about the specific mechanism of situs inversus viscerum regulated by BRWD1, namely the cilia and left-right asymmetry decision. Contemporarily, this study can supply possible new targets for the pathogenesis of PCD research, the subsequent clinical diagnosis and treatment of PCD.

我们前期研究发现BRWD1在原发性纤毛运动障碍(PCD)左右非对称发育(L-R asymmetry)中起关键作用，但确切机制不明。研究提示BRWD1可与SWI/SNF复合物中关键亚基BRG1相互作用共同起转录调控因子的作用。SWI/SNF在心脏发育过程中相当关键，且其亚基BRG1多与Notch通路激活相关。有研究者证实BRG1缺失确实会致左右非对称发育破坏，具体机制可能在于影响Notch通路激活及下游关键Nodal的表达。故我们推测BRWD1经BRG1调控Notch通路及下游靶基因Nodal表达从而参与左右非对称发育。本研究拟通过BRWD1突变PCD患者、斑马鱼以及体外细胞实验观察BRG1、Notch通路和Nodal的变化，探讨BRWD1是否经BRG1调控Notch通路及下游靶基因Nodal参与左右非对称发育，为解析BRWD1调控内脏反位具体机制提供指导，并为后续PCD诊疗提供可能新靶点。

项目背景：原发性纤毛运动障碍 ( primary ciliary dyskinesia， PCD)是一类由于纤毛异常所致的罕见常染色体隐性遗传病，已鉴定的PCD致病基因大约50余个，但既往几乎均为欧美国家所克隆，国内研究匮乏。本课题组采用全外显子测序技术对PCD的家系进行基因分析，前期研究中我们使用全外显子测序发现一例内脏反位近亲家系存在BRWD1纯合突变，我们结合文献和相关研究发现BRWD1在PCD左右非对称发育中起关键作用。.研究内容：通过对PCD和常见呼吸系统遗传性疾病的家系进行已知突变基因筛查、新致病基因的探索，并构建了brwd1敲除斑马鱼及Brwd1点突变小鼠功能验证等方法，进一步研究BRWD1基因突变对纤毛发育异常及左右不对称发育的影响。.主要结果：本课题在通过扩大遗传病家系筛查，利用免疫荧光、高速视频成像、透射电镜等先进技术进行含纤毛器官的研究，在国际上首次发现及证明BRWD1是导致PCD合并精子鞭毛多形态异常（MMAF）的致病基因；此外我们还成功构建了brwd1敲除斑马鱼和Brwd1点突变小鼠，并观察到brwd1-/-斑马鱼精子超微结构异常及心脏构型障碍。在呼吸系统遗传性近亲家系的筛查中，除了我们的BRWD1的常规筛查外，还意外发现其他的重大呼吸系统遗传疾病的新基因及新位点。如锁定CARD10纯合突变（p.R420C），通过生物信息学、高通量细胞因子、流式细胞学免疫功能检测等实验结果在国际上首次揭示了CARD10基因纯合突变与原发性免疫缺陷病相关。通过2个特发性肺纤维化家系中新变异位点的鉴定及变异的功能性评估，发现SFTPA2不同位点突变均可破坏SFTPA2蛋白的稳定性，进一步诱导内质网应激及细胞调亡，最终导致间质性肺病。我们还报道了国内首例Traboulsi综合征患者，揭示其致病基因ASPH与马凡综合征的可能联系。.科学意义：本研究不仅证实了BRWD1是PCD的新致病基因，揭示了BRWD1对纤毛发育异常及左右不对成发育的重要影响，并且还首次发现CARD10基因纯合突变与原发性免疫缺陷病相关，并且扩大了ASPH、SFTPA2基因致病突变位点库，同时为上述家系患者的遗传诊断及家系遗传咨询提供了非常重要的作用。该研究有助于深入探讨纤毛调控机制及与人类疾病关系，对呼吸系统遗传性疾病的发病机制研究及后续临床上诊治均具有重要的指导意义。

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