Rucheton Benoit, Ader Flavie, Goudenege David, Filaut Sandrine, Legrand Laura, Bloch Adrien, MitoDiag Réseau, Fressart Véronique, Bonnefont-Rousselot Dominique, Mochel Fanny, Lamari Foudil, Richard Pascale, Procaccio Vincent, Bannwarth Sylvie
DMU BioGeM, AP-HP Sorbonne Université, Hôpitaux universitaires Pitié-Salpêtrière-Charles-Foix, Service de Biochimie métabolique, Paris, France.
Département de biochimie et génétique, MitoLab, UMR CNRS 6015-Inserm U1083, Université et CHU d'Angers, Angers, France.
Ann Biol Clin (Paris). 2021 Feb 1;79(1):28-40. doi: 10.1684/abc.2021.1621.
The molecular study of mitochondrial diseases, essential for diagnosis, is special due to the dual genetic origin of these pathologies: mitochondrial DNA and nuclear DNA. Complete mtDNA sequencing still remains the first line diagnostic test followed if negative, by resequencing panels of several hundred mitochondrially-encoded nuclear genes. This strategy, with an initial entire mtDNA sequencing, is currently justified by the presence of nuclear mitochondrial DNA sequences (NUMTs) in the nuclear genome. We designed a resequencing panel combining the mtDNA and 135 nuclear genes which was evaluated compared to the performances of the standard mtDNA sequencing. Method validation was performed on the reading depth and reproducibility of the results. Thirty patients were analyzed by both methods. We were able to demonstrate that NUMTs did not impact the mtDNA sequencing quality, as the identified variants and mutant loads were identical with the reference mtDNA sequencing method. Reading depths were higher than the recommendations of the MitoDiag French diagnostic network, for the entire mtDNA for muscle and for 70% of the mtDNA for blood. These results highlight the usefulness of combining both mtDNA and mitochondrially nuclear-encoded genes and thus obtain more complete results and faster turnaround time for mitochondrial disease patients.
线粒体疾病的分子研究对诊断至关重要，因其发病机制具有双重遗传起源，即线粒体DNA和核DNA，所以具有特殊性。完整的线粒体DNA测序仍是一线诊断检测方法，若结果为阴性，则接着对数百个线粒体编码的核基因进行重测序分析。目前，由于核基因组中存在核线粒体DNA序列（NUMTs），采用先进行完整线粒体DNA测序的策略是合理的。我们设计了一个包含线粒体DNA和135个核基因的重测序分析板，并与标准线粒体DNA测序的性能进行了比较评估。对结果的读取深度和可重复性进行了方法验证。用这两种方法对30名患者进行了分析。我们能够证明NUMTs不会影响线粒体DNA测序质量，因为所识别的变异和突变负荷与参考线粒体DNA测序方法相同。对于肌肉中的整个线粒体DNA以及血液中70%的线粒体DNA，读取深度高于法国线粒体疾病诊断网络MitoDiag的建议值。这些结果凸显了将线粒体DNA和线粒体核编码基因相结合的有用性，从而可为线粒体疾病患者获得更完整的结果和更短的周转时间。
