NICCD患儿SLC25A13基因新突变对基因表达、调控和Citrin蛋白AGC功能的影响

Citrin缺陷病(CD)是SLC25A13基因突变影响Citrin蛋白AGC功能而形成的遗传代谢病。NICCD是当前儿科主要的CD表型，而SLC25A13突变分析是公认的本病确诊依据。但是，传统DNA分析法不能识别某些特定突变类型，部分新突变的致病性及其分子机制也有待明确。在课题组长期NICCD分子诊断研究基础上,本项目以实时定量RT-PCR和Western blot等技术分析疑诊NICCD患儿SLC25A13转录和翻译产物，明确其诊断；构建含新突变的ORF序列酵母表达体系，分析突变重组体AGC功能，评价新突变致病性；用报道基因、EMSA和cDNA测序等方法研究新突变对基因调控和表达产物的影响，探讨新突变致病分子机制。研究发现将突破传统DNA分析法技术局限，为NICCD确诊开辟新的研究途径，为阐明SLC25A13新突变的致病性并揭示其分子机制提供实验依据，因此具有重要科学意义和理论价值。

Citrin deficiency (CD),an inherited metabolic disease due to impaired Aspartate/Glutamate Carrier (AGC) function of Citrin protein,is caused by mutations in SLC25A13 gene. Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency (NICCD) is the major pediatric CD phenotype currently,and SLC25A13 mutation analysis remains well-recognized evidence for its definitive diagnosis. However, some specific mutations could not be identified just by conventional DNA analysis, while pathogenicity of some novel mutations and the corresponding molecular mechanisms still remain to be elucidated. Based on our long-term investigative experience of NICCD molecular diagnosis, this project was designed to analyse SLC25A13 transcriptional and translational products in suspected NICCD patients by means of such tools as real-time quantitative RT-PCR and Western blot, so as to confirm their diagnoses; To construct yeast expression systems of SLC25A13 ORF sequences that contain novel mutations and investigate AGC function of the mutant recombinants, so as to evaluate pathogenicities of the novel mutations; To explore effect of novel mutations on gene regulation and expression products by methods including reporter gene, Electrophoretic Mobility Shift Assay (EMSA) and cDNA sequencing, so as to research pathogenic mechanisms of the novel mutations. The research findings will break through the technical limitations of conventional DNA analysis, open up new investigative routes for definitive diagnosis of NICCD, and provide laboratory evidences for clarification of pathogenicities of the novel SLC25A13 mutations and elucidation of their corresponding molecular mechanisms, therefore possessing essential scientific significance and academic value.