Tissue distribution and expression of mitochondrial DNA mutations

Abstract

Human mitochondrial DNA (mtDNA) codes for 13 subunits of the respiratory chain enzyme complexes, in addition to two rRNAs and 22 tRNAs necessary for mitochondrial protein synthesis. Most pathogenic mtDNA mutations show heteroplasmy i.e. there is a mixture of mutant and wild-type mtDNA. When the proportion of mutant mtDNA exceeds a threshold level the effects of the mutation are expressed. Many pathogenic mtDNA point mutations such as the A8344G mutation, which causes myoclonus epilepsy and ragged-red fibers (MERRF), as well as mtDNA deletions, affect tRNAs resulting in impaired mitochondrial protein synthesis. Multiple mtDNA deletions have been described in syndromes, which are inherited as mendelian traits, such as autosomal dominant progressive external ophthalmoplegia (adPEO). However, an etiological association between the multiple mtDNA deletions and the disease manifestations in adPEO has not been proven. Multiple mtDNA deletions have also been described in muscle in other conditions including inclusion body myositis (IBM), which is an inflammatory myopathy.In this study the distribution of mtDNA deletions in adPEO and IBM was investigated at the cellular level in muscle and related to the activity of cytochrome c oxidase (COX), which is partially encoded by mtDNA. The techniques included enzyme histochemistry, in situ hybridization, and single fiber PCR analysis. In one patient with adPEO the organ distribution of mtDNA deletions was compared to morphological alterations and clinical symptoms. The multiple mtDNA deletion breakpoints were analyzed by cloning and sequencing of PCR amplified mtDNA fragments. We have also analyzed the threshold expression of the A8344G mutation at the cellular level in six individuals from two different families. This was performed by analysis of the proportions of mutant and wild-type mtDNA in single muscle fibers showing either normal or deficient enzyme histochemical COX activity. We have shown that mtDNA with multiple deletions in muscle tissue in adPEO and IBM have accumulated in muscle fiber segments with low COX activity. There were clonal expansions of mtDNA with deletions and reduced levels of wild-type mtDNA resulting in mitochondria with defective respiratory chain in these muscle fiber segments. Analysis of several hundred mtDNA deletions in patients with adPEO and IBM revealed more than 60 different deletions. The majority of the deletions breakpoints were flanked by direct nucleotide repeats and several of the deletions were identical in adPEO and IBM. These findings indicate that there may be a common mechanism for the generation of the mtDNA deletions in adPEO and IBM. In adPEO multiple mtDNA deletions had accumulated in brain and muscle tissues, which correlated to the clinical picture and morphological changes. This indicates that somatic multiple mtDNA deletions are associated with degenerative tissue changes and clinical manifestation in adPEO.Analysis of the A8344G mutation showed variability in the proportion of mutant mtDNA in single muscle fibers. In one of the individuals the proportion ranged from 0 to 80%. The threshold level for expression of enzyme histochemical COX deficiency in single muscle fibers varied from 95 to 98 % mutant mtDNA. Variation in threshold level and variation in distribution of mutant mtDNA in single fibers were important factors for expression of COX deficiency.