Genotypic stability, segregation and selection in heteroplasmic human celllines containing np 3243 mutant mtDNA

The mitochondrial genotype of heteroplasmic human cell lines containing the pathological np 3243 mtDNA mutation, plus or minus its suppressor at np 12 300, has been followed over long periods in culture. Cell lines containing various different proportions of mutant mtDNA remained generally at a consi stent, average heteroplasmy value over at least 30 wk of culture in nonsele ctive media and exhibited minimal mitotic segregation, with a segregation n umber comparable with mtDNA copy number (greater than or equal to 1000). Gr owth in selective medium of cells at 99% np 3243 mutant rntDNA did, however , allow the isolation of clones with lower levels of the mutation, against a background of massive cell death, hs a rare event, cell lines exhibited a sudden and dramatic diversification of heteroplasmy levels, accompanied by a shift in the average heteroplasmy level over a short period (<8 wk), ind icating selection. One such episode Tvas associated with a gain of chromoso me 9. Analysis of respiratory phenotype and mitochondrial genotype of cell clones from such cultures revealed that stable heteroplasmy values were gen erally reestablished within a few weeks, in a reproducible but clone-specif ic fashion. This occurred independently of any straightforward phenotypic s election at the individual cell-clone level. Our findings are consistent wi th several alternate views of mtDNA organization in mammalian cells One mod el that is supported by our data is that mtDNA is found in nucleoids contai ning many copies of the genome, which can themselves be heteroplasmic, and which are faithfully replicated. We interpret di diversification and shifts of heteroplasmy level as resulting from a reorganization of such nucleoids , under nuclear genetic control. Abrupt remodeling of nucleoids in vivo wou ld have major implications for understanding the developmental consequences of heteroplasmy, including mitochondrial disease phenotype and progression .