A generalized defect of complex IV (cytochrome c oxidase, COX) is frequently found in subacute necrotizing encephalomyelopathy (Leigh's syndrome), the most common mitochondrial disorder in infancy. We previously demonstrated the nuclear origin of the COX defect in one case, by fusing nuclear DNA-less cytoplasts derived from normal fibroblasts with mitochondrial DNA (mtDNA)-less transformant fibroblasts derived from a patient with COX-defective [COX((-))] Leigh's syndrome. The resulting cybrid line showed a specific and severe COX((-)) phenotype. Conversely, in the present study, we demonstrate that a COX((+))) phenotype could be restored in hybrids obtained by fusing COX((-)) transformant fibroblasts of seven additional Leigh's syndrome patients with mtDNA-less, COX((-)) tumor-derived rho°cells. Both these results are explained by the presence of a mutation in a nuclear gene. In a second set of experiments, in order to demonstrate whether COX(-) Leigh's syndrome is due to a defect in the same gene, or in different genes, we tested several hybrids derived by fusing our original COX((-)) cell line with each of the remaining seven cell lines. COX activity was evaluated in situ by histochemical techniques and in cell extracts by a spectrophotometric assay. No COX complementers were found among the resulting hybrid lines. This result demonstrates that all our cases were genetically homogeneous, and suggests that a major nuclear disease locus is associated with several, perhaps most, of the cases of infantile COX((-)) Leigh's syndrome. This information should make it easier to identify the gene responsible.
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