ARL15基因与2型糖尿病合并大血管病变遗传易感性关系及机制研究

Type 2 diabetes mellitus (T2DM) is a complex disease affected by multiple genes with epidemic proportions. Macroangiopathy (MA) is the most common complication of T2DM, and also accounts for the major mortality and morbidity in T2DM patients. Many studies have demonstrated that the decrease of adiponectin is highly associated with risk of T2DM and coronary heart disease. Recently the genome-wide association study (GWAS) and our preliminary case-control association study show that the ARL15 gene is highly associated with lower level of adiponectin and a higher risk of T2DM with MA. In this investigation, the following three aspects will be carried out, based on the study of GWAS and our previous case-control association: (1) scaling up our preliminary case-control association study and screening new risk SNPs or mutations in ARL15 gene to provide more genetic evidences; (2)sequencing ARL15 genes of T2DM patients with MA and with genetic background to obtain the rare variation or mutation, so as to provide targets for their fuction study; (3) using site-directed mutagenesis, report gene and gene chip technologies to explore the possible pathways which ARL15 may participate in to regulate the level of adiponectin. Furthermore, the T2DM with MA zebrafish model caused by ARL15 deficiency will be established to confirm the association results. Our work will confirm the correlation between ARL15 and T2DM with MA at the genetic and the functional levels. Our results will be helpful to reveal the physiological function of ARL15, to explore the role of ARL15 in the development of T2DM with MA, and further to provide potential targets for new early warning methods and therapy for T2DM with MA.

2型糖尿病(T2DM)是重要的多基因遗传病。大血管病变(MA)是其最常见并发症，也是患者致死主要原因。脂联素降低与T2DM和MA发病风险高度相关。最新国际上利用GWAS研究及我们前期病例对照研究均表明，ARL15基因多态与脂联素降低、T2DM合并MA易感性相关。本项目拟在国外GWAS和我们病例对照研究基础上深入开展如下研究：(1)扩大样本在朝鲜族人群进行ARL15基因病例对照研究，筛查新的风险SNP；(2)对T2DM合并MA患者进行ARL15基因全测序，获得罕见变异或突变，为基因功能研究提供靶点；(3)利用定点突变、报告基因、基因芯片等技术，初步探索ARL15基因在细胞水平功能及参与调控脂联素水平的通路；进而利用斑马鱼动物模型证实关联分析的结果。本研究将有助于揭示ARL15基因功能，证明ARL15与T2DM合并MA相关，为发展T2DM合并MA早期预警方法和治疗药物提供潜在的靶标。

2型糖尿病(T2DM)是重要的多基因遗传病。大血管病变(MA)是其最常见并发症，也是患者致死主要原因。最新国际上利用GWAS研究及我们前期病例对照研究均表明，ARL15基因多态与脂联素(PA)降低、T2DM合并MA易感性相关。本课题第一步进行ARL15基因SNP筛查与延边朝鲜族和汉族人群T2DM合并MA病例对照关联分析，ARL15基因全测序及其关联分析获得非常重要结果：发现新SNPIVS3+165061C>T与延边朝鲜族T2DM合并MA高度相关；群体关联分析显示rs26770-G、rs255758与PGC-1α rs7656250相互作用使ARL15上调，PA下调，引起血脂紊乱，罹患T2DM合并MA风险增加。第二部进行ARL15基因功能研究采用细胞生物学、免疫学和基因克隆等分子生物学技术，制备ARL15载体和ARL15-siRNA，转染到结肠癌细胞（HcT116、HT29、SW620）和人脐静脉血管内皮细（HUVEC），采用western blot 和RT-PCR技术检测ARL15蛋白和mRNA表达变化情况，研究发现当ARL15过表达和低表达后，脂肪代谢相关通路的蛋白水平无明显变化，而应用组蛋白去甲基化酶JIB-04后，ARL15水平明显下降，脂肪酸合成酶（FAS）、SREBP-1、GSK-3、AKT表达下降，该研究内容首次证明JIB-04可能通过抑制转录因子SREBP-1，下调FAS及ARL15水平。本研究结果将有助于揭示ARL15基因功能，对T2DM合并MA预防、临床诊断和发病机制研究以及治疗提供新的研究思路。