1例Alagille综合征患儿JAG1基因筛查及突变功能分析
JAG1 gene screening and mutation function analysis for an Alagille syndrome patient

目的 ·对1例Alagille综合征患儿进行JAG1基因筛查及突变功能分析。 方法 ·收集患儿及其父母的临床资料及辅助检查结果，抽提DNA进行JAG1基因突变筛查。构建JAG1野生型及突变型表达质粒，转染NIH-3T3细胞，通过real-time RT-PCR、Western blotting分析突变体mRNA和蛋白的表达量；糖苷内切酶H消化实验分析蛋白糖基化结构；RBP-Jκ荧光素酶报告基因检测突变体蛋白对Notch信号通路转录因子RBP-Jκ的激活作用。 结果 ·在患儿及其父亲中发现1个未报道过的 JAG1错义突变c.1655C>T （p.Pro552Leu），该突变在患儿母亲及对照健康儿童中未发现。与野生型相比，突变型JAG1在mRNA、蛋白表达量上无明显改变，翻译后糖苷化修饰正常，但突变蛋白对Notch信号通路转录因子RBP-Jκ的激活作用减弱。 结论 ·该患儿携带的JAG1错义突变使JAG1蛋白功能受损，可能是Alagille综合征的致病原因。
： Objective · To perform JAG1 gene screening and mutation function analysis for an Alagille syndrome patient. Methods · Clinical data and auxiliary examination results of the patient and his parents were collected. Genomic DNA was extracted for JAG1 mutation screening. Vectors with wild-type and mutant JAG1 expressions were constructed and transfected into NIH-3T3 cells. mRNA and protein expression levels were analyzed with real-time RT-PCR and Western blotting. Endoglycosidase H digestion experiment was performed to analyze the glycosylational structure. The effect of JAG1 protein on activating the transcription factor RBP-Jκ in Notch signaling pathway was detected with RBP-Jκ luciferase reporter gene assay. Results · A novel missense mutation c.1655C>T (p.Pro552Leu) was detected in the patient and his father, but was not found in his mother or healthy controls. Wild-type and mutant JAG1 had no difference in mRNA or protein expression levels. Post-translational glycosylational structure of the mutant JAG1 was the same as wild-type JAG1. However, the effect of activating the transcription factor RBP-Jκ in Notch signaling pathway was reduced in mutant JAG1 than in wild-type JAG1. Conclusion · The JAG1 missense mutation carried by the patient results in impaired function of JAG1, which may be the cause of Alagille syndrome