Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
Clin Chem. 2012 Sep;58(9):1322-31. doi: 10.1373/clinchem.2011.181438. Epub 2012 Jul 9.
Mitochondrial diseases are clinically and genetically heterogeneous, with variable penetrance, expressivity, and differing age of onset. Disease-causing point mutations and large deletions in the mitochondrial genome often exist in a heteroplasmic state. Current molecular analyses require multiple different and complementary methods for the detection and quantification of mitochondrial DNA (mtDNA) mutations. We developed a novel approach to analyze the mtDNA in 1 step.
The entire human mitochondrial genome was enriched by a single amplicon long-range PCR followed by massively parallel sequencing to simultaneously detect mtDNA point mutations and large deletions with heteroplasmic levels of the mutations and variants quantified. QC samples were designed and analyzed along with each sample. A total of 45 samples were analyzed for the evaluation of analytic sensitivity and specificity.
Our analysis demonstrated 100% diagnostic sensitivity and specificity of base calls compared to the results from Sanger sequencing. The deep coverage allowed the detection and quantification of heteroplasmy at every single nucleotide position of the 16 569-bp mitochondrial genome. Moreover, the method also detected large deletions with the breakpoints mapped.
This "deep" sequencing approach provides a 1-step comprehensive molecular analysis of the whole mitochondrial genome for patients in whom a mitochondrial disease is suspected.
线粒体疾病在临床上和遗传上具有异质性，其外显率和发病年龄存在可变性。线粒体基因组中的致病点突变和大片段缺失通常处于异质状态。目前的分子分析需要多种不同且互补的方法来检测和定量线粒体 DNA（mtDNA）突变。我们开发了一种新的一步法分析 mtDNA 的方法。
通过单个扩增子长距离 PCR 对整个人类线粒体基因组进行富集，然后进行大规模平行测序，以同时检测 mtDNA 点突变和大片段缺失，并定量突变和变体的异质比例。每个样本都设计和分析了质控样本。总共分析了 45 个样本，以评估分析的灵敏度和特异性。
与 Sanger 测序的结果相比，我们的分析显示碱基调用的诊断灵敏度和特异性达到 100%。深度覆盖允许在 16569bp 线粒体基因组的每个核苷酸位置检测和定量异质性。此外，该方法还检测到了断点映射的大片段缺失。
这种“深度”测序方法为疑似线粒体疾病的患者提供了一种一步式的全线粒体基因组综合分子分析。
