儿童哮喘易感基因ORMDL3的多个启动子及剪接异构体的鉴定

ORMDL3基因表达异常是儿童哮喘发病的危险因素，但该基因表达调控机制尚不明了。启动子和可变剪接在调控基因表达中起重要作用。生物信息学分析及我们前期研究发现，该基因有多个转录起始位点，表明该基因存在多个启动子及剪接异构体。我们假设ORMDL3表达受多个启动子和可变剪接调控。我们拟用快速末端扩增和引物延伸法寻找不同启动子的位置及多个剪接异构体；荧光素酶活性分析、补丁甲基化、甲基化特异性PCR、点突变和缺失突变，确定核心启动子的类型及关键转录因子结合位点；染色质免疫共沉淀、凝胶迁移实验、RNAi和过度表达有关转录因子证实对启动子的调节作用、对剪接异构体的影响；最后用荧光定量PCR和甲基化特异性PCR检测哮喘患者和健康对照组ORMDL3剪接异构体表达和启动子甲基化差异。这些研究将提供ORMDL3转录调控的关键信息，研究结果有望用于儿童哮喘基因组变异的临床分子诊断和新分子靶点的基因治疗。

Orosomucoid 1-like 3 (ORMDL3) gene dysregulated expression is a risk fact for child asthma, but the expression regulation mechanism is still unclear. Promoter and alternative splicing play important roles in regulating gene expression. Bioinformatics analysis and our preliminary study indicated that ORMDL3 has multiple promoters and alternative splicing variants. We hypothesis that ORMDL3 expression is regulated by multi-promoters and alternative splicing. We use 5'RACE, 3'RACE and primer extension analysis to define the alternative spliced variants; Luciferase activity assay, Patch methylation assay, Methylation-Specific PCR, point mutation and deletion mutation to define the core promoter type and key transcription factor binding sites; Chromatin immunoprecipitation assay, Electrophoresis mobilityshift and supershift assay, RNAi and over-expression to confirm the role of transcription factors on promoter activities and alternative splicing; Real-time PCR and Methylation-Specific PCR test the splicing variants expression differences and methylation differences between asthma children and the health. This research will find the important information of ORMDL3 transcriptional regulation, and the finding will be used to clinical molecular diagnosis and gene therapy of genome variation induced children asthma.