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 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.

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。

希特林缺陷病(Citrin Deficiency,CD)是SLC25A13基因突变影响希特林蛋白功能而形成的一种常染色体隐性遗传病。希特林缺陷导致的新生儿肝内胆汁淤积症(NICCD)是当前最主要的儿科CD表现型，其确诊依靠SLC25A13基因分析。但是，传统DNA分析法不能识别某些特定突变类型，尤其是大片段插入/缺失突变，而部分新突变的致病性及其分子机制也有待明确。经过四年研究，国家自然科学基金项目(No.81270957)取得一系列重要发现：首先，截止2016年底，课题组共确诊NICCD患者319人，建立了一个目前国际上最大的NICCD患者队列，为今后深入开展本病研究奠定了基础。其次，通过SLC25A13高频突变筛查、Sanger测序、定量PCR、家系SNP分析和cDNA克隆等技术，识别SLC25A13基因新突变14种(含大片段插入/缺失突变5种)，进一步丰富了我国NICCD患者SLC25A13基因突变谱，为患者诊断提供了可靠依据。第三，通过转录产物分子结构研究，在人外周血淋巴细胞中发现了26种新的SLC25A13替代剪接变异体，并进一步明确了部分新突变对SLC25A13基因转录表达的影响，为通过人外周血淋巴细胞SLC25A13转录子分析诊断NICCD提供了可靠理论依据和实践经验。第四，通过培养扩增人外周血淋巴细胞，分离富集线粒体并提取其蛋白后进行Western Blot检测，成功建立了一种更微创、更可行的Citrin蛋白分析方法，为NICCD分子诊断提供了新的蛋白质水平的研究工具。第五，构建了同源基因agc1敲除的、转染完整SLC25A13基因ORF突变体的酵母模型，并研究了部分SLC25A13新错义突变对希特林蛋白功能的影响，为确定其致病性提供了直接的功能学证据。最后，通过5' RACE 和报告基因技术，确定了SLC25A13转录起始位点和核心启动子区域，为今后确认调控区突变和确诊NICCD患者提供了重要实验依据。总之，本研究分别从DNA, mRNA和蛋白质水平识别了多种NICCD确诊的分子标志物，为阐明SLC25A13新突变的致病性并揭示其分子机制提供了实验依据，进一步深化了对NICCD实验室特征的科学认识，从而圆满完成了项目研究任务。

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