Role of BN-PAGE in the diagnosis of mitochondrial DNA depletion, a frequent cause of OXPHOS dysfunction.

Aims: The group of mtDNA depletion (MIM 251880), a prevalent cause of mitochondrial disease, is a clinically and genetically heterogeneous group of autosomal recessive diseases characterized by reduced mtDNA copy number. Responsible genes are involved either in mtDNA replication or in recycling of the nucleotides in the mitochondria. The aim was to study the role of BN-PAGE in the diagnosis.
Methods: Tissue samples from 434 clinically suspected patients were analyzed using spectrophotometrical analysis and Blue Native PolyAcrylamide Gel Electrophoresis (BN-PAGE) followed by activity staining in the gel. The amount of mtDNA was measured using real-time PCR. In the patients in whom mtDNA depletion was found, DNA analysis of thymidine kinase 2 (TK2), deoxyguanosine kinase (DGUOK) and DNA polymerase gamma (POLG) was performed.
Results: In 91 patients a decreased enzyme activity in one or more OXPHOS complexes was detected using spectrophotometrical analysis. In 44 patients the presence of V subcomplexes was detected following BN-PAGE evaluation. Extensive testing of the mtDNA revealed 25 patients in whom a mtDNA defect was identified. In nine of them mtDNA depletion was found. Mutations were detected in the POLG (7), DGUOK (1) and TK2 gene (1).
Conclusions: Our study shows that mtDNA depletion is a prevalent cause of OXPHOS dysfunction. Application of BN-PAGE proves to be a powerful tool in selecting the patients for genetic evaluation of the mtDNA.