Mitochondrial diseases, due to nuclear or mitochondrial genome mutations causing mitochondrial dysfunction, have a wide range of clinical features involving neurologic, muscular, cardiac, hepatic, visual, and auditory symptoms. Making a diagnosis of a mitochondrial disease is often challenging since there is no gold standard and traditional testing methods have required tissue biopsy which presents technical challenges and most patients prefer a non-invasive approach. Since a diagnosis invariably involves finding a disease-causing DNA variant, new approaches such as next generation sequencing (NGS) have the potential to make it easier to make a diagnosis. We evaluated the ability of our traditional diagnostic pathway (metabolite analysis, tissue neuropathology and respiratory chain enzyme activity) in 390 patients. The traditional diagnostic pathway provided a diagnosis of mitochondrial disease in 115 patients (29.50%). Analysis of mtDNA, tissue neuropathology, skin electron microscopy, respiratory chain enzyme analysis using inhibitor assays, blue native polyacrylamide gel electrophoresis were all statistically significant in distinguishing patients between a mitochondrial and non-mitochondrial diagnosis. From these 390 patients who underwent traditional analysis, we recruited 116 patients for the NGS part of the study (36 patients who had a mitochondrial diagnosis (MITO) and 80 patients who had no diagnosis (No-Dx)). In the group of 36 MITO patients, nuclear whole exome sequencing (nWES) provided a second diagnosis in 2 cases who already had a pathogenic variant in mtDNA, and a revised diagnosis (GLUL) in one case that had abnormal pathology but no pathogenic mtDNA variant. In the 80 NO-Dx patients, nWES found non-mitochondrial diagnosis in 26 patients and a mitochondrial diagnosis in 1 patient. A genetic diagnosis was obtained in 53/116 (45.70%) cases that were recruited for NGS, but not in 11/116 (9.48%) of cases with abnormal mitochondrial neuropathology. Our results show that a non-invasive, bigenomic sequencing (BGS) approach (using both a nWES and optimized mtDNA analysis to include large deletions) should be the first step in investigating for mitochondrial diseases. There may still be a role for tissue biopsy in unsolved cases or when the diagnosis is still not clear for NGS studies.

线粒体疾病由于引起线粒体功能障碍的核或线粒体基因组突变，具有广泛的临床特征，涉及神经，肌肉，心脏，肝脏，视觉和听觉症状。诊断线粒体疾病通常是一项挑战，因为目前尚无黄金标准，传统的检测方法需要进行组织活检，这给技术带来了挑战，并且大多数患者更喜欢采用非侵入性方法。由于诊断总是涉及发现引起疾病的DNA变异，因此诸如下一代测序（NGS）之类的新方法有可能使诊断更加容易。我们评估了390名患者的传统诊断途径（代谢物分析，组织神经病理学和呼吸链酶活性）的能力。传统的诊断途径为115例患者（29.50％）提供了线粒体疾病的诊断。mtDNA的分析，组织神经病理学，皮肤电子显微镜，使用抑制剂测定的呼吸链酶分析，蓝色天然聚丙烯酰胺凝胶电泳在区分患者的线粒体诊断和非线粒体诊断方面均具有统计学意义。从这390位接受传统分析的患者中，我们招募了116位患者进行NGS研究（36位线粒体诊断（MITO）患者和80位无诊断（No-Dx）患者）。在36名MITO患者中，核全外显子组测序（nWES）对2名已经具有mtDNA致病性变异的病例进行了第二次诊断，并对诊断进行了修订（皮肤电子显微镜，使用抑制剂测定的呼吸链酶分析，蓝色天然聚丙烯酰胺凝胶电泳在区分患者的线粒体诊断和非线粒体诊断方面均具有统计学意义。从这390位接受传统分析的患者中，我们招募了116位患者进行NGS研究（36位线粒体诊断（MITO）患者和80位无诊断（No-Dx）患者）。在36名MITO患者中，核全外显子组测序（nWES）对2名已经具有mtDNA致病性变异的病例进行了第二次诊断，并对诊断进行了修订（皮肤电子显微镜，使用抑制剂测定的呼吸链酶分析，蓝色天然聚丙烯酰胺凝胶电泳在区分患者的线粒体诊断和非线粒体诊断方面均具有统计学意义。从这390位接受传统分析的患者中，我们招募了116位患者进行NGS研究（36位线粒体诊断（MITO）患者和80位无诊断（No-Dx）患者）。在36名MITO患者中，核全外显子组测序（nWES）对2名已经具有mtDNA致病性变异的病例进行了第二次诊断，并对诊断进行了修订（蓝色天然聚丙烯酰胺凝胶电泳在区分患者的线粒体诊断和非线粒体诊断方面均具有统计学意义。从这390位接受传统分析的患者中，我们招募了116位患者进行NGS研究（36位线粒体诊断（MITO）患者和80位无诊断（No-Dx）患者）。在36名MITO患者中，核全外显子组测序（nWES）对2名已经具有mtDNA致病性变异的病例进行了第二次诊断，并对诊断进行了修订（蓝色天然聚丙烯酰胺凝胶电泳在区分患者的线粒体诊断和非线粒体诊断方面均具有统计学意义。从这390位接受传统分析的患者中，我们招募了116位患者进行NGS研究（36位具有线粒体诊断（MITO）的患者和80位未诊断（No-Dx）的患者）。在36名MITO患者中，核全外显子组测序（nWES）对2名已经具有mtDNA致病性变异的病例进行了第二次诊断，并对诊断进行了修订（（GLUL）在一种病理异常但无致病性mtDNA变异的病例中。在80位NO-Dx患者中，nWES在26位患者中发现了非线粒体诊断，在1位患者中发现了线粒体。接受遗传学鉴定的53/116（45.70％）病例获得了基因诊断，但线粒体神经病理学异常的11/116（9.48％）没有获得遗传诊断。我们的结果表明，无创双基因测序（BGS）方法（同时使用nWES和优化的mtDNA分析以包括大缺失）应是研究线粒体疾病的第一步。在未解决的病例或NGS研究仍不清楚诊断时，组织活检仍可能起作用。