Background: 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.
Methods: 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.
Results: 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.
Conclusions: 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.