DHTKD1基因在能量代谢及神经发育中的作用机制研究

DHTKD1 gene is presumed to encode a nuclear-encoded mitochondrial precursor protein--dehydrogenase E1 and transketolase domain-containing 1, also known as "possible 2-oxoglutarate dehydrogenase E1 component." In recent years, we showed that DHTKD1 gene played an important role in the mitochondrial biosynthesis, energy metabolism and function maintaining. The DHTKD1 mutation can cause Charcot-Marie-Tooth disease (CMT2Q). The relevant research work has been published in the Am J Hum Genet. Meanwhile, the German scholars have reported that the DHTKD1 mutations can cause 2-Aminoadipic and 2-Oxoadipic Aciduria. However, all studies still remain at the cellular level, and the biological function for DHTKD1 gene is less clear to date. Therefore, we propose to generate a knock-in mouse model and a knock-out mouse model on the basis of the previous studies, respectively. Then, we intend to demonstrate the biological functions of Dhtkd1 gene from the whole animal level, to demonstrate the roles of the nonsense mutation and gene knock-out for energy metabolism and neurological development in mice, to identify the molecular mechanisms of the p.Tyr486* mutation in Dhtkd1 gene causing CMT2Q. We intend to demonstrate the role of nonsense mutation and gene knock-out in Dhtkd1 gene for the whole gene expression profiles and associated signaling pathway by using Affymetrix Mouse Gene 2.0 ST Array.

DHTKD1基因编码一种线粒体前体蛋白——脱氢酶E1和转酮酶结构域1，也称“可能的2-酮戊二酸脱氢酶E1的组件”。近年来，课题组研究显示该基因在线粒体的生物合成、能量代谢和功能维持中发挥重要的作用，该基因突变可导致腓骨肌萎缩症（CMT2Q），相关研究成果已发表在美国人类遗传学杂志（AJHG）等杂志上。同时，德国学者曾报道该基因突变可导致2-氨基己二酸和2-氧化己二酸尿症。然而，现有的研究尚停留在细胞水平，对DHTKD1生物学功能尚缺乏全面认识。为此，课题组拟在已有研究的基础上，分别建立Dhtkd1突变敲入和基因敲除2种小鼠模型，以便从整体动物水平明确Dhtkd1基因的生物学功能、阐明无义突变及基因敲除对小鼠能量代谢及神经发育的影响，揭示p.Tyr486*突变导致小鼠出现类似人类CMT2Q的分子机制；拟利用基因表达芯片阐明Dhtkd1无义突变及基因敲除对小鼠基因表达谱及相关信号通路的影响。

腓骨肌萎缩症（CMT）是外周神经系统最常见的遗传性神经系统疾病之一。CMT主要包括两种类型：其一为脱髓鞘的CMT1，其二为轴突型CMT2。迄今已有近30个基因被证实与CMT2相关，其中包括DHTKD1（脱氢酶E1和转酮酶结构域1）基因。前期研究证实DHTKD1外显子8的一个无义突变[(c.1455T>G（Tyr485*）]是导致CMT2Q（MIM 615025）的元凶。本研究的目的是在Dhtkd1突变敲入小鼠中再现CMT2Q的表型，以期从整体动物水平探讨该基因的生理功能和致病机制。课题组建立了Dhtkd1突变(Tyr486*)敲入小鼠模型，表型分析发现突变纯合子（mt/mt）小鼠肝脏和坐骨神经中Dhtkd1的表达水平明显低于野生型（wt）小鼠。mt/mt小鼠肝脏中ATP含量也明显低于wt小鼠。表型分析还发现异常的周围神经改变，包括轴突直径减少和过度髓鞘化。mt/mt小鼠还表现出明显的感觉受限，但运动能力未见异常。此外，突变敲入小鼠与野生型小鼠的运动与感觉神经传导速度未见明显差异。课题组观察到突变敲入小鼠肌肉中的线粒体积累、Dgkg的增加和Lpin2的降低。由此，课题组推测突变敲入小鼠正常的运动和感觉神经传导速度可能因线粒体的堆积和脂类代谢的增强而得到补偿。综上所述，此项研究表明Dhtkd1突变(Tyr486*)敲入小鼠部分再现了CMT2Q的表型，而线粒体堆积与脂类代谢改变部分补偿了突变敲入对小鼠神经传导速度的影响。