人肝细胞citrin蛋白编码基因SLC25A13转录调控机制研究

The human SLC25A13 gene encodes citrin, the liver-type mitochondrial aspartate/glutamate carrier (AGC2), and SLC25A13 mutations cause Citrin deficiency (CD). CD has proven a disease entity with high prevalence in South China. SLC25A13 transcriptional regulation mechanism is a necessary research base for research pathogenic mechanisms and molecular diagnostics of CD, however, the research in this region is still blank. Preliminary analysis showed that the SLC25A13 promoter region is TATA-less and GC-riched, lacking a consensus transcriptional start site (TSS). There were inhibitors and other important regulatory elements in the sequence of gene 5’ regulatory region, meanwhile, the SLC25A13 knockout LO2 cell model have been successfully constructed. Based on these results, we will further identify the core promoter, the important regulatory factors and miRNA related to SLC25A13 regulation, and then study the molecular mechanisms of them and related regulatory factors, such as cytoplasmic Ca2+, how to regulate the SLC25A13 gene transcription. At the same time, the 5' flanking regulation sequence function analysis model will be build to evaluate the risk of mutations in this region, which will provide new experimental evidences for the diagnosis of CD patients. This study will promote the CD pathogenic research and clinical molecular diagnostics, having great theoretical value and scientific significance.

人SLC25A13基因突变导致其编码的肝细胞线粒体内膜天冬氨酸/谷氨酸载体蛋白citrin功能受损，引发一类遗传代谢疾病--Citrin缺陷病(CD)。我国南方地区是CD高发区。SLC25A13基因转录调控机制研究是CD发病机制研究和分子诊断的必要基础，但该方向目前尚未有系统深入研究。我们前期已证实该基因启动子为一类无TATA box、富含GC且转录起始位点不固定的组成型启动子，其5′调控序列中存在抑制子等重要调控元件，且已成功构建该基因敲除正常肝细胞LO2细胞模型。此基础上，本项目拟进一步确定SLC25A13核心启动子、重要调控因子和参与调控的miRNA，及其与胞质Ca(2+)等相关诱导调节因子调节SLC25A13基因表达的分子机制，同时构建该基因5′侧翼序列突变功能分析模型，为疑难CD患者确诊提供新的实验依据。本研究将推动CD发病机制研究和临床分子诊断，具有重要理论价值和科学意义。

人SLC25A13基因突变使其编码蛋白citrin天冬氨酸-谷氨酸载体(AGC)功能受损，导致Citrin缺陷病(CD), 我国南方地区是CD高发区。5'侧翼序列缺失分析证实该基因启动子为无TATA box、富含GC且转录起始位点不固定的组成型启动子，核心启动子区位于基因5′端-221至-126(以ATG前第一个碱基为-1)，且5' UTR序列对维持基因的正常表达水平非常重要；利用pGL3质粒建立了启动子区突变功能分析模型。课题组构建了数个SLC25A13基因敲除L02单克隆细胞株，mRNA-Seq分析发现较正常细胞有127个差异表达基因，富集通路分析发现影响药物反应调节通路，值得进一步结合小鼠模型进行研究。已构建slc25a13-KO小鼠和gpd2-KO小鼠，双基因敲除小鼠正在构建中(CD模型小鼠需要双敲除)。广东省3299例人群筛查发现SLC25A13起始密码子变异c.2T>C携带率为1/22，远高于最常见I型致病突变携带率(1/65)，且无纯合子发现；在461例确诊CD病人中携带率也很低，也无纯合子发现。据此推测此变异可能具有胚胎致死活性，而非正常SNP。构建不同表达质粒转染细胞实验证实c.2T>C变异体可表达p.Met1_Phe34del变异citirn蛋白(暂时命名为AGC3)，激光共聚焦显微镜成像技术证实其可定位于线粒体，但表达量显著低于正常citrin；回补实验证明AGC3不具备完全citrin功能。同时课题组构建成功slc25a13敲除但表达人AGC3的人源化小鼠，其杂合子自交后代出现纯合子且生长发育正常，说明单AGC3蛋白对小鼠不致死。下一步gpd2-KO小鼠与人源化小鼠杂交实验正在进行。项目因中途调整研究内容和小鼠构建周期长等原因，暂时未能证实人SLC25A13基因c.2T>C变异的生物学活性，但已有成果为后续研究打下坚实基础。