2型糖尿病相关的线粒体tRNA突变的致病机理研究

Type 2 diabetes mellitus (T2DM [MIM125853]) is one of the major public health problems worldwide, particularly affecting approximately 10% of Chinese adult population. According to International Diabetes Federation (IDF), there are more than 96 million Chinese adults living with diabetes ranging from 20 to 79 years old in 2014. However, 53.2% of the people with diabetes are undiagnosed. In 2014, diabetes caused 1.2 million deaths in China. .T2DM is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency, which could be caused by single gene or multifactorial conditions resulting from interactions between environmental and inherited factors. Of inherited factors, a maternal excess in the transmission of T2DM was implicated in many epidemiologic studies, suggesting that mutation(s) in mitochondrial DNA (mtDNA) is one of the molecular bases for this disorder. These diabetes-associated mtDNA mutations, which are often present in heteroplasmy (mixture of wild type and mutated mtDNA), exhibited a remarkable variability of clinical manifestation..In present study, we performed a clinical and genetic evaluation and mutational screening of 22 mitochondrial tRNA genes in two Han Chinese families with maternally transmitted diabetes mellitus and built the experimental system both in vitro and ex vivo to: .1) analyze the correlation between mitochondrial tRNA mutations and T2DM;.2) construct the immortalized lymphoblastoid cell lines as well as cybrid cell lines carrying mitochondrial tRNA mutations derived from the family members with maternally transmitted diabetes mellitus as precious genetic resources;.3) evaluate the tRNA mutations and the possible function of base modification related with these mutations in T2DM;.4) demonstrate whether the transfer of human mitochondrial leucyl-tRNA synthetase into the cybrid cells carrying the mutations could improve the efficiency of aminoacylation and stability of mitochondrial tRNAs and then increase the rates of mitochondrial translation and respiration, consequently correcting the mitochondrial dysfunction and provide new insights into the molecular mechanism of maternally inherited diseases and a step toward therapeutic interventions for these disorders.

2014年，20-79岁的中国人群中，糖尿病患者超过9600万，诊断率仅为46.8％。同年超过120万人死于糖尿病及其并发症。.部分2型糖尿病呈现母系遗传规律, 因此人线粒体DNA突变可能是2型糖尿病发病的分子基础之一。本项目拟对携带A14692G突变的两个2型糖尿病家系和36个正常对照为研究对象，建立体外研究模型和细胞研究模型：.1）筛选线粒体tRNA中与2型糖尿病相关的新突变位点，并绘制线粒体tRNA基因在中国2型糖尿病人群中的突变频谱，为临床诊断提供新的靶点，为干预和遗传咨询等提供科学理论依据；.2）构建新突变位点的永生淋巴细胞系，保存珍贵的遗传资源；.3）评估线粒体tRNA 突变及涉及的碱基修饰在疾病中的作用，揭示线粒体相关的2型糖尿病致病的分子机制。.4）探索细胞内高表达氨基酰-tRNA合成酶对tRNA突变的拯救功能，为疾病治疗、药物研发等提供新的思路和参考依据。

部分2型糖尿病/耳聋等疾病呈现母系遗传规律, 因此人线粒体DNA突变可能是2型糖尿病/耳聋等疾病发病的分子基础之一。.本项目首先对携带A14692G突变的3个2型糖尿病/耳聋家系和正常对照为研究对象，建立了体外研究模型和细胞研究模型，从分子遗传学、生物化学及细胞生物学的角度，首次将线粒体tRNA修饰缺陷与2型糖尿病/耳聋建立起联系，分析了线粒体A14692G突变对tRNAGlu及线粒体功能缺陷的影响；.此外，在对2070例母系遗传相关疾病以及512例健康对照者的线粒体基因组进行了系统的突变筛查研究，根据突变位点保守性、是否影响tRNA结构功能稳定性，同时结合生化与细胞实验，鉴定出47个与母系遗传疾病相关的致病性突变位点，代表性位点之一的T3253C明显降低了稳态下tRNALeu(UUR)的含量；同时作为氨基酰-tRNA合成酶可能的识别位点，突变还导致了Leu-tRNALeu水平的降低；.首次构建携带线粒体tRNA基因突变（m.15927 G>A）的人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells，HUVEC）转线粒体细胞系作为新的细胞模型进行研究。.综上所述，项目研究揭示了2型糖尿病/耳聋等线粒体相关疾病模型中tRNA突变致病的分子机制，并为线粒体基因突变致病的组织特异性研究提供了新的思路；为临床诊断提供新的靶点，为干预和遗传咨询等提供科学理论依据。.