Diagnoses and molecular bases of mitochondrial respiratory chain disorders

背景技术背景：先天性和原发性乳酸性酸中毒是最常见的先天性代谢缺陷之一，其中只有30%的人有其确切的原因确定。目的：应用蓝色非变性聚丙烯酰胺凝胶电泳（BN-PAGE）结合常规酶法对线粒体呼吸链疾病（MRCD）进行快速、准确的诊断。方法：个体呼吸链复合物和线粒体基质标记酶柠檬酸合酶的活性在肝脏、心脏和肌肉匀浆和从培养的成纤维细胞分离的线粒体组分中测量。将从皮肤成纤维细胞分离的组织匀浆或线粒体溶解在正十二烷基麦芽糖苷中，并使用对复合物I至IV亚基特异的单克隆抗体进行4-13% BN-PAGE和蛋白质印迹。定量聚合酶链反应测定组织内线粒体DNA和核DNA拷贝数。结果：267例候选患者中有110例被诊断为MRCD。最常见的是复合物I缺乏症，其中许多患者有组织特异性类型的缺陷。110例患者中有20例存在线粒体DNA致病突变，这意味着大多数儿童期发病的MRCD是核起源的。线粒体DNA突变患者的症状比可疑核突变患者轻。MtDNA缺失综合征（MDS）是多发性MRCD的常见病因。10个家系中12例诊断为肝源性MDS，5例诊断为肌病性MDS。在12例肝性MDS中，发现4个家系的6例患者存在DGUOK、POLG和MPV 17核基因突变。结论：我们必须怀疑几乎每一种疾病都可能是MRCD。BN-PAGE是一个有用的指南，以迅速和正确的诊断，并为呼吸链疾病分类的未来分子分析。

BACKGROUND : Congenital and primary lactic acidosis is one of the most frequent inborn errors of metabolism, of whom only 30% have had its precise cause identified. Our aim is to make a prompt and correct diagnosis of mitochondrial respiratory chain disorders (MRCD), using Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) combined with conventional enzyme assay. METHODS : Activities of the individual respiratory chain complexes and the mitochondrial matrix marker enzyme citrate synthase were measured in liver, heart and muscle homogenates and mitochondrial fractions isolated from cultured fibroblasts. Tissue homogenates or mitochondri isolated from skin fibroblasts were solubilised in n-dodecyl-maltoside and subjected to 4-13% BN-PAGE and western blotting using monoclonal antibodies specific for Complex I to IV subunits. Mitochondrial DNA and nuclear DNA copy numbers within tissues were determined by quantitative polymerase chain reaction. RESULTS : One hundred and ten patients were diagnosed to have MRCD out of 267 candidate patients. Most frequent was complex I deficiency, of whom many patients had tissue-specific type deficiency. Twenty patients out of 110 had mitochondrial DNA pathogenic mutations, which meant the majority of childhood-onset MRCD was nuclear origin. Patients with mtDNA mutations had milder symptoms than those suspected to have nuclear mutation. MtDNA depletion syndrome (MDS) was a prevalent cause of multiple MRCD. Twelve patients in 10 families were diagnosed to have hepatic MDS, and 5 patients were diagnosed to have myopathic MDS. Out of 12 hepatic MDS, we discovered nuclear genes mutations of DGUOK, POLG and MPV17 in 6 atients from 4 families. CONCLUSION : We must have a suspicion that almost every disease may be a MRCD. BN-PAGE is a useful guide to prompt and correct diagnosis, and future molecular analysis for categorizing respiratory chain disorders.