Use of next generation sequencing (NGS) in mitochondrial (mt) disorders: whole mitochondrial genome analysis

Mitochondrial (mt) diseases are a heterogeneous group of disorders, caused by both nuclear and mt genome mutations. A correct diagnosis is challenging, mainly because of the absence of clear phenotype-genotype correlations, the existence of heteroplasmy (presence of at least two different mt genotypes in the same cell) and the very large number of genes involved. Current traditional molecular diagnoses for disorders caused by a mt DNA defect, relies on the identification of (common) point mutations and large deletions with sequencing and Southern blot procedures. Complete mt genome sequencing, using Sanger nucleotide sequencing techniques, is reserved only for few well selected patients. The method is laborious, and not very sensitive to detect nucleotide variations below 15-20% heteroplasmy. Next Generation Sequencing (NGS) is a booming technology, promising to be an accurate and cost effective method to investigate considerable amounts of DNA, including the complete mt genome of patients. DNA samples of thirty patients with a (suspected) mt disorder were sequenced using the Ion PGM(TM) sequencer. Their mt genome has previously been characterized. A 100% coverage of the whole mt genome was obtained, 99.6% of the variants were concordant with Sanger sequencing. In depth sequencing allowed a sensitive detection of (pathogenic) heteroplasmic variations in a range of 4 to 79%, both in patients and controls. A sensitivity down to 4% is a major advantage in comparison with dideoxy nucleotide analysis. In addition, large multiple and single deletions, with visualization of their breakpoints, were identified. This study shows that NGS will be playing a major role in the molecular diagnostics of mt DNA disorders.