The clinical heterogeneity of mitochondrial disorders often makes it a challenge to establish a diagnosis in suspected mitochondrial patients. The classical approach to the diagnostic examination usually involves a muscle biopsy to evaluate mitochondrial function by measuring enzyme activities of the OXPHOS enzymes complex I to V, and to determine flux parameters such as oxygen consumption, the oxidation of various substrates, and ATP production. Based on the combination of clinical features and biochemical parameters, candidate genes are selected for subsequent molecular genetic studies in the conventional diagnostic work-flow. The procedures for diagnostic molecular genetic analysis of mitochondrial patients are rapidly evolving, due to the introduction of next generation sequencing (NGS) technologies in diagnostic laboratories. In addition to the application to mtDNAsequence analysis, NGS is used for the sequence analysis of multiple nuclear genes. The new technological possibilities for molecular genetic testing will result in a change in the diagnostic work- up of suspected mitochondrial patients. Because of the often poor genotype- phenotype correlation in mitochondrial disease, NGS analysis will often require functional follow up testing to confirm pathogenicity of genetic variants. This will create a demand for more specific diagnostic functional tests; not only for mitochondrial oxidative phosphorylation, but also for other mitochondrial processes like substrate transport, nucleotide homeostasis, mtDNA transcription/ translation, and so on. The development of a collection of sophisticated and preferably non- invasive diagnostic tests for functional follow- up of NGS molecular genetics results is the next challenge in the diagnostic examination of suspected mitochondrial patients.
