Cytochrome c oxidase deficiency in Leigh's syndrome: genetic evidence for a nuclear DNA-encoded mutation

Although an apparently generalized defect of cytochrome c oxidase (COX) occurs in many patients with subacute necrotizing encephalomyelopathy (Leigh's syndrome), the mode of inheritance in this disorder is not known. We transformed COX-deficient fibroblasts from a child with Leigh's syndrome with simian virus 40 to obtain cells with an infinite life span. These cells were still COX-deficient, grew normally in HAT medium, and were ouabain-sensitive. We fused these cells with a HAT-sensitive, ouabain-resistant variant of HeLa cells (HeLacot) and isolated surviving hybrid clones in ouabain-containing HAT medium. Prolonged cultivation of the hybrids was accompanied by preferential loss of HeLacot mitochondrial DNA (mtDNA), as determined by mtDNA restriction patterns of parental and hybrid cell DNA with the restriction endonuclease HaeII. COX activity was normal or higher than normal in hybrids, including the progeny of cell clones that had lost almost all the HeLacot mtDNA. These data demonstrate that COX deficiency in this Leigh's syndrome patient's cells was corrected by a nuclear DNA-encoded factor from the HeLacot parent and ruled out an mtDNA mutation as the basis for COX deficiency. This system can be used to determine whether different generalized mitochondrial disorders are due to mutations of nuclear or mtDNA.