TFAM与线粒体呼吸链联合缺陷病发病机制的研究

Mitochondrial disease is a group of genetically metabolic disorders which have some difficulty in the clinical diagnosis and therapy. In our previous research, we studied characters of mitochondrial respiratory chain deficiencies in Chinese children. This research supplements the information for clinic and genetics in Chinese mitochondrial disorders. Results showed that combined complex deficiencies of mitochondrial respiratory chain is one of common biochemical phenotypes in Chinese children with mitochondrial disease and has complicated genotypes. Based on our and other researches, the mitochondrial transcription factor A (TFAM) is responsible for replication and repair of mitochondrial DNA, and maintains mitochondrial structure and function. We speculate that TFAM may result in mitochondrial DNA mutation and/or the disorders of transcription and protein expression, which is probably a main cause of mitochondrial combined respiratory chain deficiencies. The research will be performed as follows：① to analyze the clinical manifestation of mitochondrial combined respiratory chain deficiencies, and the difference of respiratory chain enzyme activities, genotype, mitochondrial copy number, and the metabolism in different tissues (buccal epithelial cells, fibroblast, muscle and leucocytes). ② to analyze the correlation between TFAM genetype and mitochondrial DNA mRNA, respiratory chain complexes activities and mitochondrial copy number in combined respiratory chain deficiencies. ③ to analyze the expression of the autophagic marker LC3, Beclin, P62 and hexokinase in the patients with TFAM mutations. ④ to explore the role TFAM plays in combined respiratory chain deficiencies by transforming TFAM gene into yeast and by siRNA interference. We hope to provide some helpful information used in understanding the mechanism of combined respiratory chain deficiencies, and in improving the clinical diagnosis and therapy, genetic counseling and prenatal diagnosis.

线粒体病是一种临床较难诊治的遗传代谢病。经前期研究我们初步掌握了我国儿童线粒体病的部分临床和分子遗传特征，发现有复杂基因型的呼吸链联合缺陷是该病的主要类型之一。基于线粒体转录因子A（TFAM）是线粒体基因复制和转录中关键而特殊的因子，并维持线粒体基因结构和功能稳定，故推测TFAM通过影响线粒体基因的复制和/或转录，导致线粒体基因突变和/或转录异常，可能是线粒体呼吸链联合缺陷病的发病机制之一。本课题拟研究：①呼吸链联合缺陷病患儿的临床特征，及不同组织的呼吸链酶活性，线粒体基因型，拷贝数等的差异；②不同TFAM基因型与线粒体基因mRNA水平、拷贝数和呼吸链酶活性的相关性；③自噬相关蛋白LC3、Beclin、P62蛋白和己糖激酶等在TFAM基因突变患儿中的表达；④通过siRNA干扰和酵母细胞转化方法来探索研究TFAM在该病发病中的作用。为提高临床诊治、提供高质量的遗传咨询和产前诊断提供理论依据。

线粒体呼吸链联合缺陷病是一种临床较难诊治的遗传代谢病。由于该病机制不清，病情复杂，加之对我国儿童线粒体呼吸链联合缺陷病知之甚少，故而该病以往不被认识，临床常被误诊为其他疾病，延误治疗。通过此项研究发现：① 线粒体呼吸链联合缺陷病多在婴儿期发病，以神经系统受累为主，同时还表现为神经、肌肉、肝、肾、心肌等多系统损害；首发症状多表现为运动倒退、智力运动发育落后、无力等。目前共发现4种联合缺陷类型，分别是I 和 II、I 和 IV、I 和 V、IV 和 V 缺陷；线粒体呼吸链复合物联合缺陷病在不同组织中的酶活性不尽相同。约61.9%的外周血白细胞线粒体呼吸链复合物酶活性缺陷病患儿的皮肤成纤维细胞酶复合物活性同样降低，90.5%患儿的肌肉细胞酶活性降低。② 41.5%的线粒体呼吸链酶复合物联合缺陷患儿携带线粒体基因突变，ND2 4833A>G突变频率最高。均属H2a2a单倍型。携带线粒体基因突变和未携带线粒体基因突变患儿的线粒体基因拷贝数均高于正常对照（P<0.05）。③ 所有患儿未发现TFAM基因突变，但TFAM mRNA表达和ROS水平高于正常对照（P<0.05）。携带线粒体基因突变和未携带线粒体基因突变患儿间的TFAM mRNA表达水平无统计学差异（P>0.05）。④ 线粒体呼吸链联合缺陷患儿的精氨酸、瓜氨酸、蛋氨酸、苯丙氨酸、色氨酸和酪氨酸低于正常对照（P<0.05）；血乙酰肉碱、肉豆蔻二酰肉碱、羟肉豆蔻二酰肉碱、羟棕榈烯酰肉碱水平增高；游离肉碱降低；尿有机酸发现乳酸、丙酮酸、苯乳酸、4羟基苯乳酸等有机酸升高，差异均具有统计学意义（P<0.05）。⑤ 通过过表达和shRNA干扰转导成纤维细胞研究显示TFAM基因是线粒体呼吸链联合缺陷病的重要调节基因，通过上调TFAM表达对线粒体基因拷贝数、线粒体呼吸链酶活性、体内氧自由基生成等有重要调控作用。但不是线粒体呼吸链联合缺陷病的直接病因。⑥ 线粒体呼吸链复合物活性联合缺陷是一些遗传代谢病患儿的继发临床表现之一。