Denaturating high performance liquid chromatography, a rapid mutation screening technique for the entire mitochondrial genome.

Looking for mutations in the mitochondrial genome has always been a laborious work. There are already over 170 mutations described and more new pathogenic mutations are still being discovered. For many patients suspected of a mitochondrial disease, based on clinical and biochemical evidence, none of the classical mtDNA mutations can be detected. In these cases, analysis of the entire mitochondrial genome would be very useful. However, sequencing more than 26 kb of DNA is very laborious and expensive. In addition, in case of heteroplasmic mutations, heterogeneity up to 25-30% can be missed. Moreover, the mt genome is known to be very polymorphic and sequencing would also reveal all the homoplasmic polymorphisms of the patient. Because most pathogenic mtDNA mutations are heteroplasmic, denaturating high performance liquid chromatography (dHPLC) can be used as a very rapid, sensitive and cost-effective method to screen the mitochondrial genome. The technique is based on partially denaturating conditions, influenced by the size, the nucleotide composition, and the analysis temperatures of the fragment under investigation. To amplify the entire mtDNA, a set of 19 primers from Transgenomic Mitoscreen Assay Kit is used. After enzyme digestion, PCR fragments of 100-600 bp are created and run on the WAVE after heteroduplex formation. A group of patients in which one would expect to find a pathogenic mutation is currently under investigation. All fragments with an aberrant pattern will be sequenced. In a second analysis step, the DNA of the patient is mixed with the DNA of an unaffected maternal relative in order to stimulate the formation of heteroduplex fragments in case of a homoplasmic presence of a nucleotide change. For patients and their family it can be very important to know the underlying cause of their disease. With dHPLC, we now have a rapid method to reveal (new) pathogenic mutations in the mitochondrial genome.