Biochemical Features of mtDNA 14484 (ND6/M64V) Point Mutation Associated with Leber’s Hereditary Optic Neuropathy V. Carelli, MD,* A. Ghelli, PhD,† L. Bucchi, DSc,* P. Montagna, MD,* A. De Negri, MD,‡ V. Leuzzi, MD,§ C. Carducci, DSc,  G. Lenaz, MD,† E. Lugaresi, MD,* and M. Degli Esposti, DSc¶ We report the effect on complex I function of the 14484 Leber’s hereditary optic neuropathy (LHON) mutation affecting the ND6 subunit gene. The same gene was also reported to carry another mutation, at position 14459, associated with the LHON/dystonia phenotype that induces a reduction of complex I–specific activity and increases the sensitivity to the product decylubiquinol. Given the proximity of both mutations in the ND6 gene, we tested the specific activity of complex I and its sensitivity to myxothiazol and nonylbenzoquinol, both inhibitors at the ubiquinol product site, in platelet submitochondrial particles from nine 14484 homoplasmic individuals, 8 Italians with Caucasian mtDNA hap- logroup J (adjunctive 4216 and 13708 mutations), and 1 Tunisian with an African mtDNA haplogroup. The specific activity of complex I was not affected by the 14484 mutation, but the sensitivity to both inhibitors was significantly increased compared with control subjects regardless of the presence of haplogroup J polymorphisms. Analysis of 70 different amino acid sequences of the ND6 subunit indicated that the 14484 mutation affects an amino acid belonging to its most conserved region, which shows local similarities with cytochrome b regions interacting with ubiquinone or ubiquinol in complex III. Our results suggest that both 14484 and 14459 mutations may affect amino acids forming the interaction site of ubiquinol product, and the 14484 mutation produces a biochemical defect resembling in part that already reported for the common 11778/ND4 LHON mutation. Carelli V, Ghelli A, Bucchi L, Montagna P, De Negri A, Leuzzi V, Carducci C, Lenaz G, Lugaresi E, Degli Esposti M. Biochemical features of mtDNA 14484 (ND6/M64V) point mutation associated with Leber’s hereditary optic neuropathy. Ann Neurol 1999;45:320 –328 Leber’s hereditary optic neuropathy (LHON) is a ge- netic optic neuropathy mostly affecting young males; it is inherited strictly along the maternal line. 1–3 Most cases worldwide are associated with one of the three mtDNA point mutations that are considered patho- genic at positions 11778, 3460, and 14484. 1–4 These mutations affect the ND4, ND1, and ND6 subunit genes of complex I, respectively. The 14484 mutation presents at least two peculiari- ties. From a clinical point of view, it is associated with a very high recurrence of spontaneous recovery of vi- sual acuity if the disease onset is before the age of 20. 1,3–7 Moreover, in caucasian subjects it is strongly associated with a specific mtDNA lineage, the haplo- group J, which is defined by the presence of some an- cient polymorphisms including those at positions 4216 and 13708 in the ND1 and ND5 subunits of complex I, that had previously been held to be “secondary LHON mutations.” 8 –14 Thus, the 14484 mutation is considered the least deleterious of the LHON patho- genic mutations and it was proposed that the nucleo- tide changes characteristic of haplogroup J could en- hance its penetrance in the European cases. 12,13 However, cases of LHON/14484 patients with non- European mtDNA background were recently reported with the same clinical characteristics, thereby confirm- ing the intrinsic pathogenicity of this mutation. 15,16 Given that the pathogenic mutations in LHON pa- tients involve complex I subunits, it is natural to as- sume that they affect some functional properties of this energy-conserving enzyme complex. Complex I mal- function, however, can be subtle and thus difficult to detect by following only the NADH-ubiquinone (Q) reductase activity of the enzyme, as in the case of the most frequent LHON mutation at position 11778. 17–22 On the other hand, the 3460 mutation From the *Istituto di Clinica Neurologica and †Dipartimento di Biochimica, Universita ` di Bologna, Bologna; ‡Istituto San Raffaele del Monte Tabor, Roma; and §Dipartimento di Scienze Neuro- logiche e Psichiatriche dell’ Eta ` Evolutiva, and  Dipartimento di Medicina Sperimentale, Universita ` di Roma “La Sapienza,” Rome, Italy; and ¶Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia. Received May 18, 1998, and in revised form Oct 5. Accepted for publication Oct 13, 1998. Address correspondence to Dr Carelli, University of Southern Cal- ifornia School of Medicine, Doheny Eye Institute, 1355 San Pablo Street, DVRC 311, Los Angeles, CA 90033. 320 Copyright © 1999 by the American Neurological Association