USE OF TRANSMITOCHONDRIAL CYBRIDS TO ASSIGN A COMPLEX-I DEFECT TO THEMITOCHONDRIAL DNA-ENCODED NADH DEHYDROGENASE SUBUNIT-6 GENE MUTATION AT NUCLEOTIDE PAIR-14459 THAT CAUSES LEBER HEREDITARY OPTIC NEUROPATHYAND DYSTONIA

A heteroplasmic G-to-A transition at nucleotide pair (np) 14459 within the mitochondrial DNA (mtDNA)encoded NADH dehydrogenase subunit 6 (ND 6) gene has been identified as the cause of Leber hereditary optic neu ropathy (LHON) and/or pediatric-onset dystonia in three unrelated fami lies. This ND6 np 14459 mutation changes a moderately conserved alanin e to a valine at amino acid position 72 of the ND6 protein. Enzymologi c analysis of mitochondrial NADH dehydrogenase (complex I) with submit ochondrial particles isolated from Epstein-Barr virus-transformed lymp hoblasts revealed a 60% reduction (P < 0.005) of complex I-specific ac tivity in patient cell lines compared with controls, with no differenc es in enzymatic activity for complexes II plus III, III, and IV. This biochemical defect was assigned to the ND6 np 14459 mutation by using transmitochondrial cybrids in which patient Epstein-Barr virus-transfo rmed lymphoblast cell lines were enucleated and the cytoplasts were fu sed to a mtDNA-deficient (rho(0)) lymphoblastoid recipient cell line. Cybrids harboring the np 14459 mutation exhibited a 39% reduction (P < 0.02) in complex I-specific activity relative to wild-type cybrid lin es but normal activity for the other complexes. Kinetic analysis of th e np 14459 mutant complex I revealed that the V-max of the enzyme was reduced while the K-m remained the same as that of wild type. Furtherm ore, specific activity was inhibited by increasing concentrations of t he reduced coenzyme Q analog decylubiquinol. These observations sugges t that the np 14459 mutation may alter the coenzyme Q-binding site of complex I.