新疆维吾尔自治区Sonic hedgehog信号通路在非综合征型唇腭裂发病中的作用与机制研究

Cleft lip and cleft palate is the most common craniofacial birth defect. Until now, there are few studies on the causative genes and pathogenesis of non-syndromic cleft lip with or without cleft palate in Xinjiang Uygur Autonomous Region. Applicants found that Shh signaling pathway may be closely related to the cleft lip and palate through SNP detection of sporadic cases and the whole exome sequencing of the genetic families.Applicants will first attempt to cover 27 sites on the Shh signaling pathway by MassARRAY assay in a larger sample of sporadic cases, aiming to verify the correlation between the Shh signaling pathway and non-syndrome type cleft lip and palate. In addition, applicants intend to investigate the pathogenesis of the PTCH1 mutation identified in the hereditary family at the molecular, cellular function and zebrafish level. Finally, the applicant intends to establish a risk prediction model based on susceptibility sites and pathogenic mutations of the Shh signaling pathway to evaluate its efficacy in the prediction of cleft lip and palate. This study aims to clarify the role and mechanism of Shh signaling pathway in the pathogenesis of non-syndromic cleft lip and palate in Xinjiang Uygur Autonomous Region through the correlation among sporadic cases and risk prediction model based on the causative mutation, thus deepening the understanding for pathogenesis of cleft lip and palate, which is a beneficial exploration for the early diagnosis and prevention of the cleft lip and palate in the autonomous region.

唇腭裂是口腔颌面部最常见的先天性发育畸形，目前关于新疆维吾尔自治区非综合征型唇腭裂致病基因及机制的研究较少。申请者前期通过散发病例的SNP检测及针对遗传家系的全外显子组测序，发现Shh信号通路可能与唇腭裂的发生有密切关系。申请者拟首先在更大样本量的散发病例中，利用MassARRAY检测覆盖Shh信号通路上的27个位点，验证Shh信号通路与非综合征型唇腭裂之间的相关性。另外，申请者拟通过在分子机制、细胞功能及斑马鱼个体水平针对遗传家系鉴定出的PTCH1突变的致病机制进行研究。最后，申请者拟利用Shh信号通路中的易感位点及致病突变建立风险预测模型，评价其对唇腭裂发病预测的效能。本申请期望通过散发病例关联性及遗传家系突变致病机制这两个方面的研究，明确Shh信号通路在新疆维吾尔自治区非综合征型唇腭裂发病中的作用及机制，从而加深对唇腭裂致病机制的理解，为自治区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，发病率为1/700，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。虽然现在对唇腭裂的治疗越来越完善，但是对于唇腭裂的发病机制还尚不明确。本文从对可能引起唇腭裂的PTCH1基因的突变出发，使用人源口腔上皮细胞系GMSM-K并利用CRISPR/Cas9技术建立了PTCH1相关细胞系。实验发现，PTCH1敲除细胞系中细胞增殖减慢，其原因是细胞周期改变而不是细胞凋亡。通过转录组测序分析发现GMSM-K PTCH1敲除后差异基因主要富集于胞外基质且与癌症的发生有关联；通过样本对比分析，我们发现PTCH1突变V908G和D945X细胞系的基因表达谱与PTCH1敲除细胞系相似，均发现细胞外基质相关通路基因发生变化，包括PGDF通路和WNT/β-catenin通路。突变细胞系显示出功能丧失且具有潜在的致癌可能性。针对PTCH1基因及其突变的基础研究一方面有助于阐明唇腭裂的发病机制，另一方面也有利于针对携带PTCH1突变且具有潜在患恶性肿瘤的唇腭裂患者进行重点保护，因此具有一定临床应用价值。