Relationship of Genotype to Phenotype in Fibroblast-derived Transmitochondrial Cell Lines Carrying the 3243 Mutation Associated with the Melas Encephalomyopathy: Shift towards Mutant Genotype and Role of mtDNA Copy Number

Abstract

Transmitochondrial cell lines were isolated by fusing mtDNA-less rho degrees 206 cells with enucleated fibroblasts derived from four members of a pedigree carrying in their muscle varying proportions of the mutation at position 3243 in the tRNA(Leu(UUR)) gene associated with the MELAS encephalomyopathy. The mitochondrial transformants derived from an asymptomatic individual were all homoplasmic for wild-type mtDNA. The proportion of wild-type transformants derived from clinically affected members of the pedigree appeared to decrease in correspondence with an increase in severity of the clinical symptoms of the cell donor. Furthermore, the average proportion of wild-type mtDNA in the transformants derived from each member of the pedigree was very similar to that found in mtDNA from the fibroblasts of that individual, suggesting that the distribution of genotypes in the transformants reflected fairly closely that in the fibroblasts. The genotype and phenotype of ten transformants derived from one severely affected individual were investigated during continuous culture up to 17-24 weeks after the transformation step. Six heteroplasmic clones showed a progressive increase in the proportion of mutant mtDNA, whereas the mitochondrial genotype remained constant in four clones apparently homoplasmic for wild-type mtDNA or nearly homoplasmic for mutant mtDNA. An analysis of the rate of repopulation of rho degrees 206 cells with fibroblast-derived mtDNA revealed a large variability among different transformants, with the full re-establishment of the control ratio of mtDNA to nuclear DNA being observed between approximately 6 weeks and more than 22 weeks after the transformation step. An increase in rate of O2 consumption generally accompanied the increase in mtDNA copy number of the transformants, pointing to the important role of the mtDNA copy number in determining the phenotype of a cell. The observation that a very small amount of wild-type mtDNA (2 to 5% of the control level), coexisting with strongly predominant mutant mtDNA, conferred upon the transformants a substantial respiratory capacity (50% or more) and the evidence of proportionality between O2 consumption rate and mtDNA copy number, which occurred at widely different mutant to wild-type mtDNA ratios, strongly suggest a contribution of the mutant mtDNA to the cell respiratory competence.

Aging-dependent Functional Alterations of Mitochondrial DNA (mtDNA) from Human Fibroblasts Transferred into mtDNA-less Cells

“…On the other hand, the constancy with the donor age in the extent of variation observed in O 2 consumption rate within the population of transformants derived from each individual suggested that age alone was not a major factor in the observed variability. In view of the observed highly significant positive correlation between O 2 consumption rate and mtDNA content in the total transformant population, it is very likely that the heterogeneity in respiration capacity detected among the o cell transformants derived from the same individual reflected in part the very slow and variable process of repopulation of o cells with fibroblast-derived mtDNA, which has been described recently (43). However, it is also reasonable to assume that possible differences in nuclear gene content and/or activity among the recipient o cells (36) played a significant role in the observed variability in respiratory capacity among individual donor-derived transformants.…”

“…mtDNA Content and Growth Rate of Transformant Clones-It has been shown that the repopulation of o cells with exogenous mtDNA by mitochondria-mediated transformation can be slow and proceed at a variable rate, depending upon the donor cell type (43), the recipient cells, and other factors. It was, therefore, conceivable that a lower than normal mtDNA content of the transformants could have affected their respiration capacity, being responsible for the interclonal variability within the clone distribution from the same individual.…”

Relative fitness of carriers of the mitochondrial DNA mutation 3243A > G

“…For disease-causing mutations it is common that only a portion of the mtDNA molecules harbours the mutation, leading to heteroplasmy. Experiments with transmitochondrial cells have shown that the proportion of 3243A > G mutant genomes must exceed approximately 90% before normal respiratory chain function is impaired, 14 indicating that cells harbouring 3243A > G are faced with negative selection at the cellular level if the heteroplasmy is higher than this threshold. At the host level, factors that would lead to negative selection include female infertility, miscarriages, female mortality before or during reproductive years, and psychosocial constraints imposed by the disease.…”

MRI features of spongiform leukoencephalopathy following heroin inhalation

“…High percentage levels of mutated mtDNA are associated with a biochemical defect at the cellular level and severe disease in patients. However, it is not clear whether the pathology is primarily due to high amounts of mutated mtDNA, 3 low amounts of wild-type mtDNA, 4 or a combination of both. To develop our understanding of this issue, we followed mtDNA disease progression in seven subjects, correlating the absolute amount of mutated and wild-type mtDNA with the histochemical defect.…”