Data_Sheet_1_Whole Exome Sequencing Is the Preferred Strategy to Identify the Genetic Defect in Patients With a Probable or Possible Mitochondrial Cause.pdf

Mitochondrial disorders, characterized by clinical symptoms and/or OXPHOS deficiencies, are caused by pathogenic variants in mitochondrial genes. However, pathogenic variants in some of these genes can lead to clinical manifestations which overlap with other neuromuscular diseases, which can be caused by pathogenic variants in non-mitochondrial genes as well. Mitochondrial pathogenic variants can be found in the mitochondrial DNA (mtDNA) or in any of the 1,500 nuclear genes with a mitochondrial function. We have performed a two-step next-generation sequencing approach in a cohort of 117 patients, mostly children, in whom a mitochondrial disease-cause could likely or possibly explain the phenotype. A total of 86 patients had a mitochondrial disorder, according to established clinical and biochemical criteria. The other 31 patients had neuromuscular symptoms, where in a minority a mitochondrial genetic cause is present, but a non-mitochondrial genetic cause is more likely. All patients were screened for pathogenic variants in the mtDNA and, if excluded, analyzed by whole exome sequencing (WES). Variants were filtered for being pathogenic and compatible with an autosomal or X-linked recessive mode of inheritance in families with multiple affected siblings and/or consanguineous parents. Non-consanguineous families with a single patient were additionally screened for autosomal and X-linked dominant mutations in a predefined gene-set. We identified causative pathogenic variants in the mtDNA in 20% of the patient-cohort, and in nuclear genes in 49%, implying an overall yield of 68%. We identified pathogenic variants in mitochondrial and non-mitochondrial genes in both groups with, obviously, a higher number of mitochondrial genes affected in mitochondrial disease patients. Furthermore, we show that 31% of the disease-causing genes in the mitochondrial patient group were not included in the MitoCarta database, and therefore would have been missed with MitoCarta based gene-panels. We conclude that WES is preferable to panel-based approaches for both groups of patients, as the mitochondrial gene-list is not complete and mitochondrial symptoms can be secondary. Also, clinically and genetically heterogeneous disorders would require sequential use of multiple different gene panels. We conclude that WES is a comprehensive and unbiased approach to establish a genetic diagnosis in these patients, able to resolve multi-genic disease-causes.

以临床症状及/或氧化磷酸化（oxidative phosphorylation, OXPHOS）缺陷为特征的线粒体疾病，由线粒体基因的致病变异引发。然而，部分此类基因的致病变异所导致的临床表现，可与其他神经肌肉疾病出现重叠——此类神经肌肉疾病既可由线粒体基因的致病变异引发，也可由非线粒体基因的致病变异导致。

线粒体致病变异既可存在于线粒体DNA（mitochondrial DNA, mtDNA）中，也可分布于全部1500个具备线粒体功能的核基因内。我们针对117名以儿童为主的患者队列，采用了两步法二代测序（next-generation sequencing, NGS）技术，该队列患者的表型大概率或可能由线粒体疾病病因所致。根据既定临床与生化诊断标准，共计86名患者被确诊为线粒体疾病；其余31名患者表现为神经肌肉症状，其中仅少数存在线粒体遗传病因，而非线粒体遗传病因的可能性更高。

所有患者均接受了线粒体DNA（mtDNA）致病变异筛查，若筛查结果为阴性，则进一步接受全外显子组测序（whole exome sequencing, WES）。针对存在多个受累同胞和/或近亲婚配父母的家系，我们对检测到的变异进行过滤，筛选出符合常染色体或X连锁隐性遗传模式的致病变异；对于仅存在单个受累患者的非近亲婚配家系，我们额外在预设基因集内筛查了常染色体及X连锁显性突变。

我们在20%的患者队列中检出了线粒体DNA（mtDNA）的致病变异，在核基因中的检出率为49%，整体诊断成功率达68%。我们在两组患者中均检出了线粒体基因与非线粒体基因的致病变异，显然线粒体疾病患者队列中受影响的线粒体基因数量更多。此外，我们发现线粒体疾病患者队列中有31%的致病基因未纳入MitoCarta数据库，因此基于MitoCarta的基因面板检测会漏诊此类变异。

我们认为，针对这两类患者，全外显子组测序（WES）均优于基因面板检测：一方面线粒体基因列表尚未完整，另一方面线粒体症状可作为继发表现出现。此外，临床表现与遗传异质性较高的疾病需要依次使用多种不同的基因面板。综上，全外显子组测序（WES）是一种针对此类患者建立遗传诊断的全面且无偏倚的方法，能够明确多基因致病的疾病病因。