食管闭锁（EA）是新生儿期最常见的先天性食管发育畸形。胚胎期食管发育分化异常是EA发生的重要原因，机制不明。GLI3基因是Hedgehog信号通路上的关键抑制性转录因子，与食管正常发育分化密切相关，动物实验已发现GLI3基因变异可导致食管发育分化异常。申请人前期研究首次在EA患儿中检测出GLI3基因点突变，并发现c.332T>C（p.M111T）新突变位点。本项目拟建立GLI3基因M111T突变体重组真核细胞表达载体，运用荧光素酶报告系统，免疫荧光，染色质免疫沉淀法及组蛋白去乙酰化酶活性实验等检测分析M111T突变对GLI3转录抑制功能的影响；应用凝胶迁移实验，荧光定量PCR，免疫印迹，细胞增殖及凋亡实验等观察M111T突变对GLI3下游靶基因功能的影响。初步揭示GLI3基因突变对食管发育分化的影响及分子机制，进一步阐明EA发生的分子遗传机制，为EA遗传咨询，产前筛查及早期预防提供依据。

Esophageal atresia (EA) is one of the most common congenital esophageal anomalies in newborns. Abnormalities in the esophageal development result in EA. As a key repressive transcriptional factor, GLI3 plays a critical role in Hedgehog signaling pathway which is associated with the esophageal development. Multiple animal studies showed genetic variant of GLI3 lead to the pathogenic esophageal development. Our previous studies have reported a novel point mutation c.332T>C (p.M111T) in GLI3 gene in EA patients. Here, we further investigated mutant GLI3 functions. Eukaryotic expression plasmids of mutant GLI3 gene were generated and identified. Mutant GLI3 protein transcriptional activities were detected by luciferase activity assays, Immunofluorescence, chromatin immunoprecipitation, and histone deacetylase enzymatic assays respectively. GLI3 target gene activities were investigated by electrophoretic mobility shift assay, real-time PCR, western blot, MTT and TUNEL assays respectively. This study will explore the role of the novel GLI3 mutation in EA and elucidate the pathogenesis of EA with the goal of creating improved precautions, genetic counseling and prenatal screenings for EA.