小耳畸形-下颌发育不良综合征是一种罕见的先天性颅面畸形，其高度遗传异质性使定位新的致病基因成为该领域的研究焦点。申请人前期通过关联分析在散发病例中进行致病基因筛选，而所得候选基因与已报道散发病例的候选基因均缺乏足够的致病依据。家系是探索致病基因的可靠来源，但目前该病的家系鲜有报道。本项目已收集到4个家系，拟构建基于家系的候选基因筛选-斑马鱼-小鼠模型验证-致病机制探究的模式，为新的候选基因提供更强的致病依据。具体策略是：1）利用生物信息分析初步筛选出家系的候选基因；2）分别应用两套系统（Morpholino和CRISPR/Cas9）在斑马鱼中进行基因功能验证，高度可疑的致病基因在小鼠模型中再次验证；3）探索候选基因在软骨发育的三个关键过程中的具体作用。本项目旨在寻找出新的可靠的致病基因并探索其致病机制，为设计基因芯片辅助临床诊断及产前咨询奠定基础，同时为异常颅面发育的机制提供新的理解。

Otomandibular dysostosis is one of the rare congenital craniofacial anomalies with high genetic heterogeneity, strategy for discovering new pathogenic genes has been the research focus in this field. Our previous studies have identified several candidate genes in sporadic cases using association analysis. These genes, along with genes acquired in other sporadic cases-based researches, showed weak repeatability and lacked convincing evidence of pathogenicity. As a more reliable source of disease genes, pedigree study of otomandibular dysostosis was rarely reported. In this project, we have found four families of significance, and we aim to establish a gene screening method based on pedigree to identify new candidate genes and to provide a more reliable basis for pathogenicity. Our strategies are as follow: 1) Bioinformatic methods will be employed for preliminary screening of candidate genes for each pedigree. 2) Function verification of candidates will be performed in zebrafish model through two complementary approaches (Morpholino-mediated knockdown approach and CRISPR/Cas9-mediated knockout approach) respectively. After that, gene function will be further verified in mouse model. 3) We will further explore the role of candidates in three key steps of cartilage development. The project is planed to hunt for new and reliable pathogenic genes of otomandibular dysostosis and explore the pathogenic mechanism, which may lay the foundation for designing DNA microarray to assist clinical diagnosis and prenatal counseling, also shed light on the pathogenesis of craniofacial anomaly.