Biochemical and molecular genetic investigations of patients with mitochondrial DNA disease

Abstract

Mitochondria are organelles present in all nucleated cells and are the only location of extra-chromosomal DNA within mammalian cells. They are responsible for the generation of ATP by oxidative phosphorylation (OXPHOS). An extensive range of molecular defects have been identified in the human mitochondrial genome, many associated with wellcharacterised, progressive neurological syndromes. Diseases due to mutations in the mitochondrial genome (mtDNA) are clinically, genetically and biochemically diverse. I have investigated the molecular genetic basis of mitochondrial disease in a cohort of patients with biochemical deficiencies, using a range of histochemical, biochemical, molecular genetic and cell biological techniques. Whole genome sequencing results revealed the identification of novel and recurrent pathogenic mutations in different subunits of complex I (m.11453G>A, m.11777C>A, m 13051G>A, m.13513G>A and m.14453G>A). To assign pathogenicity of these identified mutations, several approaches were carried out to distinguish between pathogenic and neutral mtDNA changes. Investigation of different tissues from patients and their relatives explored mtDNA heteroplasmy, and determined whether mutations had a risen sporadically or been maternally-inherited. Transmitochondrial cybrids were established as an in vitro model to assess the functional consequences of the m.11777C>A mutation, revealing significant defects in these cells supporting the role of this mutation in causing disease in patient. The m.11777C>A and m.13051G>A mutations were associated with increased reactive oxygen species (ROS) production as a consequence of the effect of these mutations on complex I function. Moreover, the expression of several mitochondrial complex I subunits was differentially affected in fibroblasts from patients based on western blotting analysis. In addition, 3 novel, pathogenic mt-tRNA mutations were identified; m.618T>G MTTF gene mutation, a m.12261T>C MTTS2 gene mutation and a m.12283G>A MTTL2 gene mutation. These mutations were shown to be pathogenic as they segregated with the biochemical defect within individual cytochrome c oxidase (COX)-deficient fibres. Interestingly, the m.12283G>A mutation was present at very low levels in mature muscle as compared to other mt-tRNA mutations, and exhibited an unusual pattern of segregation in single muscle fibres. The MitoChip (V2.0) is an oligonucleotide tiling array for the resequencing of the human mitochondrial genome, which has been proposed as an alternative diagnostic screening tool given it is rapid and relatively cheap. A comparative study between conventional dideoxy Sanger sequencing and MitoChip (V2.0) analysis revealed an inability of this technology to detect single nucleotide insertion and deletion mtDNA mutations.