野生型线粒体基因在线粒体病发生发展中的作用机制研究

A series of clinical abnormalities are caused by mitochondrial DNA (mtDNA) damage, and mtDNA mutation load or heteroplasmic ratio has long been considered to be important in this process. There is substantial evidence that total amount of wild-type mtDNAs may play more critical role than mutation loads do. To obtain a more accurate evaluation on importance of the percentage ratio of mutated to wild-type mtDNA, or merely wild-type mtDNA level, a cask principle on source of energy is proposed. Which is the shortest plate of mitochondrial respiration? It might be the left level of wild-type mtDNAs in patients who harbor mitochondrial cytopathies, even if the double contribution of nuclear and mitochondrial genome. To make sure that the wild-type mtDNA level is one of the determinants, a series of missense mutations, such as A3243G, A8344G, T8993G/C, G11778A, G3460A,T14484C, A1555G and a large fragment deletion called common deletion, either from peripheral leukocytes or cells from urinary sediment, are quantificationally measured. Both mutant and wild-type mtDNA copy numbers would be severe quantitatively correlated with clinical phenotype. We also correlated mtDNA genotype with non-neglectful biochemical phenotype, the mitochondrial respiratory chain enzyme activities in leukocytes from patients. Complex I, III, IV and V will be determined by a so-called CellSAIC microfluid technique developed by Merck-Millipore, or a Seahorse XF 24 analyzer. Platelet-mediated transformation of mtDNA-less human cells(rho0), with the prolonged viability of platelet mitochondria and the simplicity and efficiency of the mitochondria-transfer procedure, will be applied for patients with different mutation loads, and also for several unrelated normal human individuals after quantification of the amount of the wild-type mtDNA in platelets. A certain recovery in respiratory capacity is supposed to be observed among the platelet-derived rho0 cell transformants, especially in wild-type mtDNA-rich cell. Furthermore, we would like to introduce a system called clustered regularly interspaced short palindromic repeats, CRISPR/Cas, on mitochondrial genome to produce knock-out mice with mtDNA defects. If an overall recovery of mitochondrial function were checked out after the wild-type were knocked-in back, a new way of gene therapy for extra-nuclear genome would be find out. Then, the mitochondrial defects could be cured by 'add in', instead of 'move out'.

既往研究认为，线粒体病的临床表现取决于mtDNA的突变比例，然而大量体外实验和我们的病例研究发现，与野生型mtDNA拷贝数更相关。我们提出线粒体能量供应的"木桶"假说，即呼吸链水平的高低取决于短板- - 野生型mtDNA的水平。我们从4方面取证：以q-PCR方法定量患者外周血和尿液细胞中突变型和野生型mtDNA的拷贝数，对比临床表型，明确突变比例和野生型拷贝数孰轻孰重；以微流控(microfluid)技术定量检测微量白细胞中呼吸链酶复合体I、III~V的活性，确认生化表型是否与野生型更相关；以无核的血小板与无mtDNA的Rho0细胞融合的实验，将患者突变定量导入，了解野生型mtDNA的对呼吸链功能的恢复能力；最后将CRISPR/Cas转基因系统用于核外遗传物质mtDNA，快速便捷地建立动物模型，进一步研究线粒体病的发生发展机制。这是核外基因治疗的新思路- - 不必"去除"突变，只要"补充"正常"。

线粒体病的临床表型和基因型之间存在高度异质性，即同一种突变类型可以引起不同的临床表型，同一种临床表型也可以由不同的突变类型导致。这种高度异质性为线粒体病的诊断带来了很大的困难，仅仅依靠临床表现很难对线粒体病进行确诊及分型。本课题累计收集既往经PCR-RFLP检出阳性的患者共495例，年龄介于5个月至65岁，中位年龄11岁，男女比例1:1.21，以及跨越从新生儿到88岁不同年龄段的正常人400例作为对照组。与线粒体脑肌病相关的m.A3243G， m.A8344G，m.T8993G和m.G13513A；与LHON相关的m.G11778A，m.T14484C和m.G3460A；与AID相关的m.A1555G和m.C1494T 2个突变位点，利用不同荧光修饰基团分别标记各位点，实现多个mtDNA突变位点的同时检测（MMCA），再用实时荧光定量PCR Taqman探针方法定量检测线粒体A3243G点突变的突变率及野生型和突变型线粒体DNA拷贝数。标准品是将突变型探针和野生型质粒DNA按比例混合，突变比例为0~100%，共12组。所有上述克隆或突变检测均以测序方法确认。外周血白细胞内mtDNA拷贝数水平随着年龄的增加有降低趋势，主要在0~2岁和>50岁年龄段后拷贝数水平出现显著下降。我们分别建立了正常儿童和成人外周血单个白细胞内线粒体tDNA拷贝数的参考范围：儿童175~602 copies/cell（均值: 325 copies/cell）；成人164~500 copies/cell（均值: 287 copies/cell）。该参考范围的建立，有利于以线粒体DNA拷贝数对线粒体病患者的确诊和病情监测。项目从临床上最常见的mtDNA A3243G点突变所致的线粒体病出发，从线粒体病临床表型异质性和mtDNA拷贝数分子调控机制方面，探讨了mtDNA拷贝数作为线粒体病病情评估和疗效预后的标志物，并建立中国儿童和成人外周血的mtDNA拷贝数正常参考范围，有利于辅助临床对线粒体的诊断和治疗工作。