ECM29基因突变在非综合征型唇腭裂发病中的作用与机制研究

Cleft lip with or without palate (CL/P) is the most common craniofacial congenital disease, imposing serious influence on the appearance and function of the affected individuals. In our preliminary work, we conducted whole-exome sequencing in a hereditary family affected with non-syndromic CL/P, and identified a novel mutation located at the coding region of the ECM29 gene, which is potentially causative for the disease. However, the pathogenicity of ECM29 for CL/P requires further demonstration. We hereby propose verification work in the following three aspects. First, on the molecular cell level, we plan to conduct ECM29 overexpression and expression suppression in human embryonic cleft shelf mesenchymal cells. The change of cell migration and the influence of cytoskeleton will then be evaluated. Second, on the in-vitro organ level, we intend to isolate and culture palate shelves from mice embryos, and test whether ECM29 expression affect palatal fusion. Finally, on the animal model level, we plan to perform Ecm29 rescue experiments in the zebrafish, to see if the mutant contribute to abnormal craniofacial growth and development. Apart from verification work described above, we also plan to perform Sanger sequencing among sporadic cases to assess the genetic contribution of ECM29 to the CL/P pathogenesis. Through this application project, we hope to clarify the role of this novel CL/P causing gene and its pathogenic cellular and molecular mechanisms, so as to contribute to the early diagnosis and prevention of the disease.

唇腭裂是口腔颌面部最常见的先天性发育畸形，严重影响患者的外貌与口腔功能。针对唇腭裂新致病基因的筛选和鉴定，有助于加深致病机制的理解，丰富分子诊断的依据。申请人前期针对一个非综合征型唇腭裂遗传家系进行全外显子组测序，鉴定了位于ECM29基因编码区的一个潜在致病突变。本申请拟在分子细胞层面，对ECM29作为唇腭裂致病基因进行验证，即通过人胚胎腭板间充质细胞中ECM29的过表达与表达抑制，检测细胞迁移运动能力的变化及其和蛋白酶体相互作用的关联。在离体器官层面，通过对离体腭板进行处理，观测ECM29蛋白对腭板融合的影响。模式动物层面，在斑马鱼体内利用ECM29突变质粒挽救实验，验证ECM29突变是否造成颅颌面生长发育异常。同时申请者拟在大样本散发型病例中评估ECM29的遗传贡献度。本课题希望明确新型唇腭裂致病基因ECM29的功能与其致病的细胞分子机制，为未来唇腭裂的早期诊断和防治做出有益的探索。

唇腭裂是颌面部常见的发育性疾病，特征是唇、腭部出现先天性裂隙，对患者的面部发育、呼吸、吞咽和发音产生较为严重的影响。前期研究中我们针对一个非综合征型唇腭裂遗传家系进行了全外显子组测序，鉴定了位于ECM29基因编码区的一个潜在致病突变。本课题首先在分子细胞层面，对ECM29作为唇腭裂潜在致病基因进行验证，即通过人胚胎腭板间充质HEPM细胞中ECM29的过表达与敲低，检测细胞迁移运动能力的变化及其对蛋白酶体的影响，发现ECM29对细胞增殖和迁移能力是正相关的，而T644S突变丧失了这种增强的能力。其次在离体器官层面，通过对离体腭板进行培养和慢病毒敲低ECM29处理，观察发现敲低ECM29后腭板不能正常融合。另外在模式动物层面，利用斑马鱼体内ECM29基因的敲低实验，证明ECM29表达抑制能够造成颅颌面生长发育异常。这些都证明了ECM29对于颅颌面发育，尤其是唇腭部的正常发育中起到重要的作用。同时申请者在散发型病例中对ECM29基因进行Sanger测序，评估了其对散发型唇腭裂发病的遗传贡献度，为未来临床转化应用提供理论数据。本课题一定程度上明确了新型唇腭裂潜在致病基因ECM29的功能与其致病的细胞分子机制，为未来唇腭裂的早期诊断和防治做出有益的探索。