The mitochondrial genome is small, 16.5kb, and yet complex to study due to an abundance of mitochondria in any given cell or tissue. Mitochondrial DNA (mtDNA) mutations have been previously described in cancer, many of which were detected at low heteroplasmy. In this study we enriched the mitochondrial genome in primary pediatric tumors for detection of mtDNA variants. We completed mtDNA enrichment using REPLI-g, Agilent SureSelect, and long-range polymerase chain reaction (LRPCR) followed by next generation sequencing (NGS) on Illumina platforms. Primary tumor and germline genomic DNA from a variety of pediatric central nervous system (CNS) and extra-CNS solid tumors were analyzed by the three different methods. Although all three methods performed equally well for detecting variants at high heteroplasmy or homoplasmy, only LRPCR and SureSelect-based enrichment methods provided consistent results for variants that were present at less than five percent heteroplasmy. We then applied both LRPCR and SureSelect to three successive samples from a patient with multiply-recurrent gliofibroma and detected a low-level novel mutation as well as a change in heteroplasmy levels of a synonymous variant that was correlated with progression of disease. Implication: This study demonstrates that LRPCR and SureSelect enrichment, but not REPLI-g, followed by NGS are accurate methods for studying the mtDNA variations at low heteroplasmy, which may be applied to studying mtDNA mutations in cancer.

中文翻译：

---

用三种不同的富集方法检测儿童中枢神经系统和中枢神经系统外实体瘤中低水平异质性的线粒体 DNA 变异

线粒体基因组很小，只有 16.5kb，但由于任何给定细胞或组织中都有大量线粒体，因此研究起来很复杂。先前已在癌症中描述了线粒体 DNA (mtDNA) 突变，其中许多是在低异质性时检测到的。在这项研究中，我们丰富了原发性儿科肿瘤中的线粒体基因组，用于检测 mtDNA 变异。我们使用 REPLI-g、Agilent SureSelect 和长距离聚合酶链反应 (LRPCR) 完成了 mtDNA 富集，然后在 Illumina 平台上进行了下一代测序 (NGS)。通过三种不同的方法分析了来自各种小儿中枢神经系统 (CNS) 和 CNS 外实体瘤的原发肿瘤和种系基因组 DNA。尽管这三种方法在检测高异质性或同质性的变异方面表现同样出色，只有基于 LRPCR 和 SureSelect 的富集方法可为异质性低于 5% 的变异提供一致的结果。然后，我们将 LRPCR 和 SureSelect 应用于来自多发复发性胶质纤维瘤患者的三个连续样本，并检测到低水平的新突变以及与疾病进展相关的同义变异的异质性水平变化。意义：本研究表明，LRPCR 和 SureSelect 富集，而不是 REPLI-g，然后是 NGS，是研究低异质性 mtDNA 变异的准确方法，可用于研究癌症中的 mtDNA 突变。然后，我们将 LRPCR 和 SureSelect 应用于来自多发复发性胶质纤维瘤患者的三个连续样本，并检测到低水平的新突变以及与疾病进展相关的同义变异的异质性水平变化。意义：本研究表明，LRPCR 和 SureSelect 富集，而不是 REPLI-g，然后是 NGS，是研究低异质性 mtDNA 变异的准确方法，可用于研究癌症中的 mtDNA 突变。然后，我们将 LRPCR 和 SureSelect 应用于来自多发复发性胶质纤维瘤患者的三个连续样本，并检测到低水平的新突变以及与疾病进展相关的同义变异的异质性水平变化。意义：本研究表明，LRPCR 和 SureSelect 富集，而不是 REPLI-g，然后是 NGS，是研究低异质性 mtDNA 变异的准确方法，可用于研究癌症中的 mtDNA 突变。