The Role Of Mitochondrial ND4 And ND6 Mutations In Leber’s Hereditary Optic Neuropathy

Leber’s hereditary optic neuropathy (LHON) is a maternally inherited eye disease that generally affects young adults with the rapid, painless, bilateral loss of central vision. Mutations in mitochondrial DNA (mtDNA) are the molecular basis. Of this, MT-ND4 and MT-ND6 were proposed to be the hot spots for mutations associated with LHON.A total of 1218 genetically unrelated Han Chinese subjects with LHON were recruited from the eye clinics across the China. Mutational analysis of ND4 gene in 1218 Han Chinese subjects with LHON and 316 control subjects identified 149 variants. A total of 466 subjects carrying one of the known m.11253T>C, m.l 1778G>A and m.11696G>A mutations accounted for 38.26% cases of this cohort, particularly 36.12% for m.11778G>A mutation. In addition,10 pedigrees with 7 novel putative LHON-associated missense variants accounted for 0.82% cases of LHON subjects in this cohort.The five-generation family with ND4 m.11778G>A mutation including 111 members underwent Ophthalmological, genetic, and biochemical evaluations. Fund examination of 17 matrilineal relatives shown that 6 patients with optic discs pale or partial pale, while 13 carriers shown pink optic discs. Interestingly, four carriers (V-6, V-7, V-8 and V-9) exhibited pink optic discs but vascular tortuosity. OCT examination shown that affect patients showed significant decrease in all quadrants, while the unaffected carrier showed a statistically significant increase in the temporal quadrant. Interestingly, the Ⅳ-6 carriers showed significant decrease in temporal superior, and Ⅳ-8 showed significant decrease in nasal, nasal superior and temporal superior.Lymphoblastoid cell lines derived from five patients, five carriers, and four unrelated control subjects were immortalized by transformation with the Epstein-Barr virus. The mitochondrial membrane potentials were 61.15% in mutant cell lines and 66.46% in carrier cell lines compared with control cell lines. The levels of ATP production in mutant cell lines, in the presence of pyruvate and 2-deoxy-D-glucose with an average of 49.09% relative to the mean value measured in the control cell lines, while in the carrier cell lines, the levels of ATP production were 35.08% decreased. Moreover, the 85% and 56% increased the ROS production in mutant cell lines and the carrier cell lines, respectively.The protein expression of ND4, ND1, ND5, ND6, NDUFS1 and NDUFB8 in mutant cell lines and the carrier cell lines were significantly decreased relative to the control cell lines.Mutational analysis of ND6 gene identified 92 variants in these 1218 subjects, including 29 (9 novel and 20 known) missense mutations and 63 silence variants. A total of 94 subjects carrying one of the known m.14484T>C,m.14502T>C and m.14459G>A mutations accounted for 7.7% cases of this cohort, particularly 4.4% for T14484C mutation. Furthermore, twelve families with the putative LHON-associated ND6 mutations accounted for 1.1% case of this cohort. Thus,106 subjects carrying one of ND6 mutations accounted for 8.7% cases of this cohort. In particular, the occurrences of haplogroups M9, M10, M11, and H2 in patients carrying the ND6 mutations were higher than those in controls.In our study, the cybrids carrying only m.14502T>C mutation, only m.11778G>A mutation, both m.14502T>C and m.11778G>A mutations, and controls were constructed simultaneously. We showed that the Basal OCR, ATP-Linked OCR and Maximal OCR in cybrids carrying only m.14502T>C mutation were decreased 18.3%,15.2% and 33.7%, respectively. While the cybrids carrying both m.14502T>C and m.11778G>A mutations were significantly decreased compared with controls. The mitochondrial membrane potentials and the levels of ATP production in the cybrids with both m.14502T>C and m.11778G>A mutation were obviously decreased compared with control cell lines. Furthermore, the production of ROS in cybrids with two mutations were significantly higher than the cybrids with the only m.14502T>C mutation or only m.11778G>A mutation. These date further supported that the secondary mutation may worsen the mitochondrial dysfunction associated with the primary mutation.