Diagnoses and molecular bases of mitochondrial respiratory chain disorders

背景：先天性和原发性乳酸中毒是最常见的先天性代谢疾病之一，其中只有30%的人明确了病因。我们的目的是用蓝色聚丙烯酰胺凝胶电泳法(BN-PAGE)结合常规的酶分析方法，对线粒体呼吸链疾病(MRCD)做出快速、正确的诊断。方法：分离培养成纤维细胞，测定肝、心、肌肉组织匀浆和线粒体部分的呼吸链复合体和线粒体基质标记酶柠檬酸合成酶的活性。从皮肤成纤维细胞中分离出组织匀浆或有丝分裂原，用N-十二烷基麦芽糖苷溶解，用4-13%BN-PAGE和针对复合体I-IV亚基的单抗进行免疫印迹。用定量聚合酶链式反应检测组织内线粒体DNA和核DNA拷贝数。结果：在267例候选患者中，110例被诊断为MRCD。最常见的是复杂I缺乏症，其中许多患者患有组织特异型缺乏症。在110名患者中，有20名患者存在线粒体DNA致病突变，这意味着大多数儿童期起病的MRCD是核来源的。MtDNA突变患者的症状比怀疑有核突变的患者要轻。线粒体DNA耗竭综合征(MDS)是多发MRCD的常见原因。10个家系12例诊断为肝性MDS，5例诊断为肌病MDS。在12例肝性MDS中，我们在4个家系的6例患者中发现了DGUOK、POLG和MPV17的核基因突变。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，以及时和正确的诊断，并在未来的分子分析分类呼吸链疾病。

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.