Molecular anatomy of mitochondrial disease

Finding new disease-genes by next generation sequencing

Next Generation Sequencing (NGS) technology offers rapid and relatively low-cost characterization of the genome of an individual organism, giving us a chance to make a substantial leap ahead in the molecular dissection of all mitochondrial disorders in humans. In collaboration with the Unit's Bioinformatics group, we have established and validated an NGS data-processing pipeline for the exome analysis of DNA samples from patients with respiratory chain diseases. This relies on a network of collaborations to provide DNA samples and fibroblast cell lines from patients, including those with the Carlo Besta Neurological Institute in Milan, with the diagnostic centre for mitochondrial disease at the University of Newcastle, and with other centres in the UK, such as the Institute of Neurology and the Hospital for Children at University College in London.

Once a new mutation has been identified, its pathogenicity is confirmed by applying several techniques:

  • Sanger sequencing is used to confirm the mutations found by NGS
  • Complementation studies in cells are used to re-express the wild-type gene and investigate its capacity to rescue the phenotype
  • In vitro import, mitochondrial fractionation and confocal immunofluorescence, including SYM (available in house) or STED super-resolution microscopy (available through a collaboration with the MRC Laboratory of Molecular Biology) are used to confirm the mitochondrial and sub-mitochondrial localization of the identified proteins