Defective respiratory capacity and mitochondrial protein synthesis in transformant cybrids harboring the tRNA(Leu(UUR)) mutation associated with maternally inherited myopathy and cardiomyopathy.

Abstract

We studied the physiometabolic effects of a mitochondrial DNA (mtDNA) heteroplasmic point mutation, the A Gtransition associated with maternally inherited myopathy and cardiomyopathy. To eliminate the possible influence of the autochthonous nuclear gene set, we fused myoblast-derived cytoplasts of a patient with a human tumoral cell line deprived of mtDNA (Rho0). The presence and amount of the mutant G3260 vs the wild-type A326" were measured by solid phase minisequencing. We observed a marked reduction of the percentage of mutant mtDNA in the culture system compared with that measured in the donor's muscle biopsy, suggesting the presence of negative selection against the mutation. Furthermore, stable mitotic segregation of the two mtDNA populations was observed in 18 of 19 transformant clones, suggesting the presence of intraorganelle and possibly intracellular homoplasmy in the precursor cells of the donor. Several indexes of mtDNA-related respiratory capacity, including oxygen consumption, complex I-and complex IV-specific activities, and lactate production, were markedly abnormal in the clones containing a high proportion of mutant mtDNA, as compared with those containing homoplasmic wild-type mtDNA, possibly because of impaired mitochondrial protein synthesis. We conclude that (a) the A -G31 transition is indeed responsible for the mitochondrial disorder identified in the donor patient, and (b) transformant cybrid system gives direct evidence of the mitochondrial origin of a genetic disorder and should be adopted for the evaluation of the pathogenic potential of the mtDNA mutations. (J. Clin.

Pathological mechanisms underlying single large‐scale mitochondrial DNA deletions

“…For instance, in class II deletions, where only complex I and tRNAs genes are lost, the threshold for complex IV is substantially higher. Interestingly, this threshold of approximately 90% is similar to those recorded in other mtDNA defects caused by pathogenic variants in mt‐tRNA genes,37, 38, 39, 40 suggesting that complex IV deficiency in this class of deletions occurs solely due to a translational defect following removal of tRNA gene sequences. These findings are in line with a report by Hammans and colleagues, who also observed a relationship between the location of mtDNA deletion and the biochemical defect, and therefore the determinant role of mtDNA‐encoded COX genes when removed by the deletion 26.…”

Impairment of tRNA processing by point mutations in mitochondrial tRNALeu(UUR) associated with mitochondrial diseases

“…However, when comparing an in vitro analysis of mitochondrial tRNA processing to data obtained in vivo, one is inevitably faced with the complexities of the mitochondrial genetic system: a mixture of wild-type and mutant genomes (heteroplasmy) and variable nuclear genetic background. These problems can be partially avoided by the use of transmitochondrial cybrids [6 8], and this technology has helped to establish the causal relationship of certain (mt)tRNA L''"/cUR/ mutations (3243, 3256, 3260, 3271) and the pathological mitochondrial phenotype [8][9][10][11]14].…”

“…3' pre-tRNase, precursor-tRNA 3'-endonuclease; MELAS, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes; (nat), mitochondrial; mtDNA, mitochondrial DNA; mtRNase P, mitochondrial RNase P; nRNase P, nuclear RNase P; PEG, polyethylene glycol tein synthesis and consequently a decrease in oxidative phosphorylation capacity [7][8][9][10][11][12][13][14]. More specifically, in one case a decreased aminoacylation of the affected tRNA was found [15], and in two other cases the specific accumulation of the tRNA precursor [8,16].…”

Neurological presentations of mitochondrial diseases

“…This system allows one to test the intrinsic pathogenicity of a given mtDNA mutation independently of the autochthonous nuclear background (Mariotti et al 1994). The reliability of this system is further improved when the mutation is heteroplasmic, because the effects of the mutant mtDNA species can then be compared with those produced by a haploidentical wild-type species, derived from the same individual.…”