Pagani Ilaria S, Kok Chung H, Saunders Verity A, Van der Hoek Mark B, Heatley Susan L, Schwarer Anthony P, Hahn Christopher N, Hughes Timothy P, White Deborah L, Ross David M
Cancer Theme, South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia.
Cancer Theme, South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia; School of Medicine, Faculty of Health Sciences, University of Adelaide, Adelaide, South Australia, Australia.
J Mol Diagn. 2017 Sep;19(5):711-721. doi: 10.1016/j.jmoldx.2017.05.009. Epub 2017 Jul 18.
Somatic mitochondrial DNA (mtDNA) mutations have been identified in many human cancers, including leukemia. To identify somatic mutations, it is necessary to have a control tissue from the same individual for comparison. When patients with leukemia achieve remission, the remission peripheral blood may be a suitable and easily accessible control tissue, but this approach has not previously been applied to the study of mtDNA mutations. We have developed and validated a next-generation sequencing approach for the identification of leukemia-associated mtDNA mutations in 26 chronic myeloid leukemia patients at diagnosis using either nonhematopoietic or remission blood samples as the control. The entire mt genome was amplified by long-range PCR and sequenced using Illumina technology. Variant caller software was used to detect mtDNA somatic mutations, and an empirically determined threshold of 2% was applied to minimize false-positive results because of sequencing errors. Mutations were called against both nonhematopoietic and remission controls: the overall concordance between the two approaches was 81% (73/90 mutations). Some discordant results were because of the presence of somatic mutations in remission samples, because of either minimal residual disease or nonleukemic hematopoietic clones. This method could be applied to study somatic mtDNA mutations in leukemia patients who achieve minimal residual disease, and in patients with nonhematopoietic cancers who have a matched uninvolved tissue available.
体细胞线粒体DNA（mtDNA）突变已在包括白血病在内的多种人类癌症中被发现。为了鉴定体细胞突变，有必要获取同一个体的对照组织进行比较。当白血病患者达到缓解时，缓解期外周血可能是合适且易于获取的对照组织，但这种方法此前尚未应用于mtDNA突变的研究。我们开发并验证了一种新一代测序方法，用于在26例慢性髓性白血病患者诊断时，使用非造血或缓解期血液样本作为对照来鉴定白血病相关的mtDNA突变。通过长程PCR扩增整个mt基因组，并使用Illumina技术进行测序。变异检测软件用于检测mtDNA体细胞突变，由于测序错误，应用经验确定的2%阈值以尽量减少假阳性结果。针对非造血和缓解期对照检测突变：两种方法之间的总体一致性为81%（90个突变中的73个）。一些不一致的结果是由于缓解期样本中存在体细胞突变，这是由于微小残留病或非白血病造血克隆所致。这种方法可应用于研究达到微小残留病的白血病患者，以及有匹配的未受累组织的非造血癌症患者的体细胞mtDNA突变。
