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Biochemistry and Molecular Biology  |   March 2014
Frequency and Spectrum of Mitochondrial ND6 Mutations in 1218 Han Chinese Subjects With Leber's Hereditary Optic Neuropathy
Author Affiliations & Notes
  • Min Liang
    Institute of Genetics, Zhejiang University, Hangzhou, Zhejiang, China
    Attardi Institute of Mitochondrial Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Pingping Jiang
    Institute of Genetics, Zhejiang University, Hangzhou, Zhejiang, China
  • Feng Li
    Institute of Genetics, Zhejiang University, Hangzhou, Zhejiang, China
  • Juanjuan Zhang
    Institute of Genetics, Zhejiang University, Hangzhou, Zhejiang, China
  • Yanchun Ji
    Institute of Genetics, Zhejiang University, Hangzhou, Zhejiang, China
  • Yiqun He
    Attardi Institute of Mitochondrial Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang, China
    School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Meifen Xu
    Attardi Institute of Mitochondrial Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang, China
    School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Jinping Zhu
    Attardi Institute of Mitochondrial Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang, China
    School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Xiangjuan Meng
    Attardi Institute of Mitochondrial Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang, China
    School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Fuxin Zhao
    Attardi Institute of Mitochondrial Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang, China
    School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Yi Tong
    School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China
    Department of Ophthalmology, The First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, China
  • Xiaoling Liu
    School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Yanhong Sun
    Department of Ophthalmology, Dongfang Hospital, Beijing University of Chinese Medicine and Pharmacology, Beijing, China
  • Xiangtian Zhou
    Attardi Institute of Mitochondrial Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang, China
    School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Jun Qin Mo
    Division of Pathology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
  • Jia Qu
    Attardi Institute of Mitochondrial Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang, China
    School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Min-Xin Guan
    Institute of Genetics, Zhejiang University, Hangzhou, Zhejiang, China
    Attardi Institute of Mitochondrial Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang, China
    Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
  • Correspondence: Min-Xin Guan, Institute of Genetics, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, China; gminxin88@gmail.com
Investigative Ophthalmology & Visual Science March 2014, Vol.55, 1321-1331. doi:10.1167/iovs.13-13011
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      Min Liang, Pingping Jiang, Feng Li, Juanjuan Zhang, Yanchun Ji, Yiqun He, Meifen Xu, Jinping Zhu, Xiangjuan Meng, Fuxin Zhao, Yi Tong, Xiaoling Liu, Yanhong Sun, Xiangtian Zhou, Jun Qin Mo, Jia Qu, Min-Xin Guan; Frequency and Spectrum of Mitochondrial ND6 Mutations in 1218 Han Chinese Subjects With Leber's Hereditary Optic Neuropathy. Invest. Ophthalmol. Vis. Sci. 2014;55(3):1321-1331. doi: 10.1167/iovs.13-13011.

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Abstract

Purpose.: To investigate the molecular pathogenesis of Leber's hereditary optic neuropathy (LHON) in Chinese families.

Methods.: A cohort of 1218 Han Chinese subjects with LHON and 316 control subjects underwent the clinical and genetic evaluation and molecular analysis of mitochondrial (mt)DNA.

Results.: The age at onset of optic neuropathy in these subjects ranged from 5 to 55 years, with the average of 18 years. Mutational analysis of ND6 gene identified 92 (73 known and 19 novel) variants in these subjects. These variants included 29 (9 novel and 20 known) missense mutations and 63 silence variants. A total of 94 subjects carrying one of the known T14484C, T14502C, and G14459A mutations accounted for 7.7% cases of this cohort, particularly 4.4% for T14484C mutation. Furthermore, eight 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. Low penetrance of optic neuropathy in pedigrees carrying one of eight putative mutations indicated that the mutation(s) is necessary, but itself insufficient to produce a clinical phenotype. Mitochondrial DNAs in 98 probands carrying the ND6 mutation(s) were widely dispersed among 12 Eastern Asian subhaplogroups. In particular, the occurrences of haplogroups M9, M10, M11, and H2 in patients carrying the ND6 mutations were higher than those in controls.

Conclusions.: These data further support that the ND6 gene is the hot spot for mutations associated with LHON. Thus, our findings may provide valuable information for the further understanding of pathophysiology and management of LHON.

Introduction
Leber's hereditary optic neuropathy (LHON) is a maternally-inherited eye disease that generally affects children to young adults with the rapid, painless, bilateral loss of central vision. 14 The prevalence of LHON was 3.22 per 100,000 subjects in northeast England. 5 Maternal transmission of LHON indicated that mutations in mitochondrial DNA (mtDNA) are responsible for this disorder. 68 The mtDNA is a 16,569-nucleotide pair, double-stranded, circular molecule that codes for two ribosomal RNAs (rRNA), 22 transfer RNAs (tRNA), and 13 polypeptides of the mitochondrial energy generating process, oxidative phosphorylation (OXPHOS). Oxidative phosphorylation enzyme complexes consist of complex I (Nicotinamide adenine dinucleotide [NADH]:ubiquinone oxidoreductase), complex II (succinate:ubiquinone oxidoreductase), complex III (ubiquinol:ferrocytochrome c oxidoreductase), complex IV (ferrocytochrome c:oxygen oxidoreductase or cytochrome c oxidase), and complex V (ATP synthase). Three primary LHON-associated mtDNA mutations, can each cause LHON, ND1 G3460A, ND4 G11778A, and ND6 T14484C, which alter genes encoding the subunits of complex I. These three mutations account for approximately 90% of LHON pedigrees in some countries. 1012 However, these mtDNA mutations are only responsible for 38.3% cases in a cohort of 903 Chinese Han subjects with LHON. 13 In particular, the incidences of the T14484C mutation in a cohort of 903 Chinese subjects with LHON and two cohorts of white subjects with LHON were 3.3% and 14%, respectively, 1214 indicating the variation in the incidence and spectrum of mtDNA mutations among the different ethnic backgrounds. 
The mtDNA encoded 7 of 45 subunits of NADH dehydrogenase (complex I). Of these, the ND6 gene was proposed to be a hot spot for mutations associated with LHON. 15,16 The known ND6 LHON-associated mutations included the T14484C, G14459A, and T14502C mutations. 1618 However, the mutational spectrum of ND6 gene is poorly understood in the Chinese subjects with LHON. The aim of this study was to understand the mutational spectrum and incidence of the ND6 gene in Chinese populations with LHOH. For this purpose, we carried out a systematic and extended mutational screening of ND6 gene in a cohort of 1218 genetically unrelated Han Chinese subjects with LHON. This analysis identified 92 nucleotide changes including the T14484C mutation in ND6 gene in this cohort. These ND6 variants have been further evaluated by phylogenetic analysis, structure–function relation, and allelic frequency of these variants in a cohort of 316 Han Chinese controls from the same region. Furthermore, we performed the clinical and genetic analysis of probands carrying the known T14484C, T14502C mutations, and 14 subjects carrying one of eight putative LHON-associated ND6 mutations. Moreover, these mtDNAs were assigned to the Asian mitochondrial haplogroups by using the nomenclature of mitochondrial haplogroup. 
Methods
Subjects
A total of 1218 unrelated LHON subjects were recruited from the eye clinics of 29 provinces of China. This study was in compliance with the Declaration of Helsinki. Informed consent, blood samples, and clinical evaluations were obtained from all participating family members, under protocols approved by the Zhejiang University institute review board and the Wenzhou Medical College ethics committee. A comprehensive history and physical examination for these participating subjects were performed at length to identify both personal or family medical histories of visual impairment and other clinical abnormalities. The inclusion criteria of patients for this study, included acute or subacute visual loss in both eyes simultaneously or sequentially within 1 year, clinical evidence of relatively symmetric optic neuropathies with central visual loss, and age less than 60 years at onset of visual symptoms. A total of 316 Chinese control subjects used for screening for the presence of mtDNA variants were obtained from some regions. 
Ophthalmologic Examinations
The ophthalmic examinations of probands and other members of these families were conducted, including visual acuity, visual field examination (Humphrey Visual Field Analyzer II-i, SITA Standard; Carl Zeiss Meditec, Oberkochen, Germany), visual evoked potentials (VEP; RETI port gamma, flash VEP; Roland Consult, Brandenberg, Germany), and fundus photography (CR6-45NM fundus camera; Canon, Lake Success, NY). The degree of visual impairment was defined according to the visual acuity as follows: healthy greater than 0.3, mild equaled 0.3 to 0.1, moderate less than 0.1 to 0.05, severe less than 0.05 to 0.02, and profound less than 0.02. 
Mutational Analysis of Mitochondrial Genomes
Genomic DNA was isolated from whole blood of participants using Puregene DNA Isolation Kits (Gentra Systems, Minneapolis, MN). Subject's DNA fragments spanning the ND6 gene were amplified by PCR using oligodeoxynucleotides corresponding to mtDNA at positions 14260 to 14510. 19,20 Each fragment was purified and subsequently analyzed by direct sequencing in an ABI 3700 automated DNA sequencer using the Big Dye Terminator Cycle (Applied Biosystems, Foster City, CA) sequencing reaction kit. These sequence results were compared with the updated consensus Cambridge sequence (GenBank accession number: NC_012920). 9 The homoplasmy of the T14484C mutation in these subjects was determined as detailed previously. 20,21 The frequency of variants in the ND6 gene in 316 Chinese control subjects was determined by direct sequencing of PCR products as described above. 
For defining the mitochondrial haplogroups, fragments spanning the D-loop region were PCR-amplified by using oligodeoxynucleotides corresponding to mtDNA at positions 15811 to 775 and genomic DNAs from 20 probands carrying the T14502C mutation. The entire mitochondrial genomes of 14 probands carrying the T14484C mutations and 14 subjects with putative LHON-associated mtDNA mutations were PCR amplified in 24 overlapping fragments using sets of the light (L) strand and the heavy (H) strand oligonucleotide primers, as described previously. 19 Each fragment was purified and subsequently analyzed by direct sequencing in an ABI 3700 automated DNA sequencer using the BigDye Terminator Cycle sequencing reaction kit (Applied Biosystems). These sequence results were compared with the updated consensus Cambridge sequence (GenBank accession number: NC_012920). 9  
Structural Analysis
The secondary structure of human mitochondrial ND6 subunit and domains were predicted by the online software (available in the public domain at http://bp.nuap.nagoya-u.ac.jp/sosui/). 
Phylogenetic Analysis
A total of 17 vertebrates ND6 sequences were used in the interspecific analysis. These include: Bos Taurus, Cebus albifrons , Gorilla gorilla , Homo sapiens , Hylobates lar , Lemur catta , Macaca mulatta , Macaca sylvanus , Mus musculus , Nycticebus coucang , Pan paniscus , Pan troglodytes , Pongo pygmaeus , Pongo abelii , Papio hamadryas , Tarsius bancanus , and Xenopus laevis (Genbank). 22 The conservation index was calculated by comparing the human ND6 variants with other 16 vertebrates. 
Haplogroup Analysis
The mtDNA sequences of the 52 Chinese probands carrying the T14484C mutation, 12 subjects carrying the putative mtDNA mutations as well as 34 patients carrying the T14502C mutation were assigned to the Asian mitochondrial haplogroups by using the nomenclature of mitochondrial haplogroups. 23,24  
Results
Study Samples
The study samples with LHON consisted of 917 males and 301 females. All participants were Han Chinese subjects recruited from eye clinics of 29 provinces in China, as shown in Figure 1. Ophthalmologic evaluation showed that all affected subjects exhibited the variable severity and age at onset of optic neuropathy. Of these, 230 subjects exhibited profound visual impairment, 370 subjects had severe visual impairment, 319 individuals suffered from moderate visual impairment, and 299 subjects had mild visual impairment. The age at onset of optic neuropathy ranged from 5 to 55 years, with an average of 18 years. Comprehensive family medical histories of those probands showed no other clinical abnormalities, including diabetes, muscular diseases, hearing impairment, and neurological disorders. 
Figure 1
 
Geographic locations of 1218 Han Chinese subjects with LHON. The numbers in parentheses indicate 106 patients with NDG mutations.
Figure 1
 
Geographic locations of 1218 Han Chinese subjects with LHON. The numbers in parentheses indicate 106 patients with NDG mutations.
Mutational Analysis of Mitochondrial ND6 Gene
Deoxyribonucleic acid fragments spanning ND6 gene were PCR-amplified from genomic DNA of 1218 Chinese subjects with LHON and 316 Han Chinese control individuals. Each fragment was purified and subsequently analyzed by DNA sequencing. Comparison of the resultant sequences in affected subjects with the Cambridge consensus sequence identified 92 (73 known and 19 novel) nucleotide changes in the ND6 gene, 16 as shown in the Table 1. These variants included 29 (9 novel and 20 known) missense mutations, and 63 silent variants. The known mutations included the LHON-associated the G14459A, T14484C, and T14502C mutations in this gene. 13,1618,25 All the nucleotide changes were verified by sequence analysis of both strands and appeared to be homoplasmy. In the mutational screening, no nucleotide changes in this gene were detected in the 456 patients, while at least one ND6 variant was identified in 762 affected subjects. Among these, 94 individuals carried one of the known LHON-associated ND6 mutations: 1 subject with the G14459A mutation, 53 individuals carrying the T14484C mutation, 34 patients harboring the T14502C mutation, and 6 subjects carrying both T14484C and T14502C mutations. This translated to a frequency of 7.7% for these mutations (4.4% in only the T14484C mutation, 2.8% in only the T14502C mutation, 0.5% in both T14484C and T14502C mutations, and 0.08% in G14459A) in this cohort. 
Table 1
 
Variants in the ND6 Gene in 1218 Chinese Subjects With LHON
Table 1
 
Variants in the ND6 Gene in 1218 Chinese Subjects With LHON
Position Replacement Conservation Index, %* Number of Affected Subjects Percentage (number/1218) Number of Controls Percentage (number/316) Previously Reported
Known mutations
 14484 T-C (Met64Val) 78.6 59 4.8 0 0 Yes
 14502 T-C (Ile58Val) 71.4 40 3.3 6 1.9 Yes
 14459 G-A (Ala72Val) 100.0 1 0.1 0 0 Yes
Putative mutations
 14249 G-A (Ala142Val) 85.7 1 0.1 0 0 Yes
 14272 C-T (Leu134Phe) 85.7 1 0.1 0 0 Yes
 14617 A-T (Phe19Leu) 92.9 6 0.5 0 0 Yes
 14322 A-G (Phe118Leu) 78.6 1 0.1 0 0 No
 14463 T-C (Thr71Pro) 78.6 2 0.2 0 0 No
 14486 A-G (Met63Thr) 100.0 1 0.1 0 0 No
 14510 A-G (Val55Ala) 78.6 1 0.1 0 0 No
 14598 T-C (Ile26Val) 100.0 1 0.1 0 0 No
Other missense mutations
 14178 T-C (Ile166Val) 71.4 12 1.0 4 1.3 Yes
 14180 T-C (Tyr165Cys) 71.4 2 0.2 0 0 Yes
 14207 G-A (Thr156Ile) 57.1 1 0.1 0 0 Yes
 14256 T- C (Ile140Val) 78.6 1 0.1 1 0.3 Yes
 14259 G-A (Pro139Ser) 50.0 1 0.1 0 0 Yes
 14315 C-T (Ser120Asn) 42.9 1 0.1 0 0 Yes
 14318 T-C (Asn119Ser) 50.0 64 5.3 9 2.9 Yes
 14319 T-C (Asn119Asp) 50.0 3 0.3 0 0 Yes
 14325 T-C (Asn117Asp) 50.0 3 0.3 1 0.3 Yes
 14334 C-T (Val114Ile) 57.1 1 0.1 0 0 Yes
 14337 C-T (Val113Met) 78.6 5 0.4 1 0.3 Yes
 14340 C-T (Val112Met) 50.0 3 0.3 0 0 Yes
 14417 A-G (Val86Ala) 71.4 11 0.9 1 0.3 Yes
 14669 A-G (Met2Thr) 28.6 3 0.3 1 0.3 Yes
 14261 T-A (Asp138Val) 92.9 1 0.1 1 0.3 No
 14418 C-T (Val86Ile) 71.4 1 0.1 0 0 No
 14643 C-A (Gly11Cys) 14.3 2 0.2 0 0 No
 14664 C-T (Ala4Thr) 28.6 1 0.1 0 0 No
Silent mutations
 14149 C-T - 5 0.4 0 0 Yes
 14167 C-T 100 1 0.1 0 0 Yes
 14182 T-C 78.6 1 0.1 1 0.3 Yes
 14200 T-C 100 10 0.8 1 0.3 Yes
 14209 A-G 85.7 4 0.3 0 0 Yes
 14215 T-C 78.6 1 0.1 0 0 Yes
 14218 T-C 78.6 2 0.2 0 0 Yes
 14220 A-G 78.6 2 0.2 0 0 Yes
 14227 C-G 92.9 1 0.1 0 0 Yes
 14239 C-T 100 1 0.1 1 0.3 Yes
 14260 A-G 92.9 1 0.1 0 0 Yes
 14269 A-G 71.4 2 0.2 0 0 Yes
 14275 C-T 92.9 1 0.1 0 0 Yes
 14281 C-T 78.6 4 0.3 0 0 Yes
 14287 T-C 78.6 2 0.2 0 0 Yes
 14299 T-C 57.1 2 0.2 0 0 Yes
 14302 T-C 100 1 0.1 1 0.3 Yes
 14305 G-A 50 2 0.2 0 0 Yes
 14308 T-C 78.6 22 1.8 5 1.6 Yes
 14311 T-C 64.3 11 0.9 3 1 Yes
 14314 A-G 42.9 4 0.3 1 0.3 Yes
 14338 C-T 50 1 0.1 0 0 Yes
 14341 C-T 50 1 0.1 0 0 Yes
 14344 A-G 78.6 1 0.1 0 0 Yes
 14364 G-A 85.7 1 0.1 1 0.3 Yes
 14368 C-T 78.6 1 0.1 0 0 Yes
 14383 C-T 42.9 1 0.1 0 0 Yes
 14386 T-C 71.4 1 0.1 0 0 Yes
 14388 A-G 100 2 0.2 0 0 Yes
 14392 C-T 71.4 2 0.2 0 0 Yes
 14410 G-A 78.6 8 0.7 1 0.3 Yes
 14431 T-C 57.1 4 0.3 0 0 Yes
 14449 A-G 71.4 1 0.1 0 0 Yes
 14470 T-C 100 43 3.5 13 4.1 Yes
 14476 G-A 100 4 0.3 1 0.3 Yes
 14482 C-T 78.6 1 0.1 0 0 Yes
 14485 C-T 100 3 0.3 1 0.3 Yes
 14494 T-C 100 3 0.3 0 0 Yes
 14509 A-G 78.6 1 0.1 0 0 Yes
 14512 T-C 85.7 2 0.2 0 0 Yes
 14518 A-G 100 2 0.2 0 0 Yes
 14533 C-T 100 1 0.1 0 0 Yes
 14544 G-A 85.7 4 0.3 0 0 Yes
 14551 A-G 28.6 1 0.1 0 0 Yes
 14554 A-G 100 1 0.1 0 0 Yes
 14560 G-A 85.7 11 0.9 1 0.3 Yes
 14569 G-A 100 79 6.5 13 4.1 Yes
 14581 T-C 57.1 1 0.1 0 0 Yes
 14587 A-G 100 13 1.0 5 1.6 Yes
 14605 A-G 100 5 0.4 1 0.3 Yes
 14620 C-T 92.6 1 0.1 0 0 Yes
 14659 C-T 78.6 4 0.3 1 0.3 Yes
 14668 C-T 28.6 239 19.6 55 17.4 Yes
 14614 A-G 100 1 0.1 0 0 No
 14596 A-G 100 3 0.3 0 0 No
 14500 A-G 71.4 1 0.1 0 0 No
 14419 C-T 71.4 1 0.1 0 0 No
 14263 C-T 71.4 1 0.1 0 0 No
 14257 A-G 50 2 0.2 0 0 No
 14230 A-G 78.6 1 0.1 0 0 No
 14176 A-G 71.4 3 0.3 0 0 No
 14650 C-T 100 3 0.3 1 0.3 No
 14659 C-T 85.7 1 0.1 0 0 No
Evaluation of the ND6 Variants
These variants in ND6 were first evaluated by the phylogenetic analysis of these variants and amino acid sequences from other 16 vertebrates. The conservation index among these residues ranged from 14.3% to 100%, as shown in Table 1. Of these, conservation indexes of 13 variants, including 6 novel variants (A14322G, T14463C, A14486G, A14510G, T14598C, and T14261A), were greater than 78%, with potential functional significance. 26 However, conservation indexes of other 16 variants including 3 novel variants (C14418T, C14643A, and C14664T) and the known T14502C variant were less than 72%. These variants were then evaluated by examining the allelic frequency in a cohort of 316 Han Chinese controls. As shown in Table 1, 20 variants were absent in 316 Chinese controls, while the frequencies of 9 variants ranged from 0.3% to 2.9% in this control population. 
Furthermore, we analyzed the structural alteration of ND6 by these variants based on the predicated secondary structure. As shown in Supplementary Figure S1, ND6 polypeptide consists of transmembrane, outermembrane, and intermembrane domains. Sixteen variants (A14669G, C14664T, C14643A, A14617T, T14598C, A14510G, T14502C, A14486G, T14484C, T14463C, C14418T, A14417G, G14207A, T14180C, G14459A, and T14178C) localized at the transmembrane domain, and 13 variants (C14340T, C14337T, C14334T, T14325C, A14322G, T14319C, T14318C, C14315T, C14272T, T14261A, G14259A, T14256C, and G14249A) resided at the intermembrane domain. 
In addition to the known LHON-associated G14459A, T14484C, and T14502C mutations, eight missense mutations (three known [G14249A, C14272T, and A14617T], and five novel [A14322G, T14463C, A14486G, A14510G, T14598C]), which were absent in 316 Chinese controls and whose conservation indexes were greater than 78%, were the putative LHON-associated mutations. On the other hand, 18 other missense variants, which were present in the controls or lower conservation indexes, appeared to be the polymorphisms. 
Characterization of 14 Chinese Probands Carrying the Putative ND6 Mutation
Comprehensive medical histories of 14 probands carrying one of eight putative ND6 mutations and other members in these families showed no other clinical abnormalities, including diabetes, muscular diseases, hearing loss, and neurological disorders. As shown in Table 2 and Figure 2, these families exhibited a wide range of severity, age at onset, and penetrance of optic neuropathy. Of these, only one matrilineal relative per family in eight pedigrees suffered from optic neuropathy, while six pedigrees (WZ812, WZ1276, WZ385, WZ1185, WZ259, and WZ451) had a history of optic neuropathy. 
Figure 2
 
Fourteen Han Chinese pedigrees with LHON. Visually-impaired individuals indicated by filled symbols. Arrowhead denotes probands.
Figure 2
 
Fourteen Han Chinese pedigrees with LHON. Visually-impaired individuals indicated by filled symbols. Arrowhead denotes probands.
Table 2
 
Summary of the Clinical and Molecular Data for 14 Han Chinese Probands Carrying One of the Putative ND6 Mutations
Table 2
 
Summary of the Clinical and Molecular Data for 14 Han Chinese Probands Carrying One of the Putative ND6 Mutations
Putative ND6 Mutation Primary Mutation Proband Sex Age at Test, y Age at Onset, y Visual Acuity Right/Left Level of Visual Impairment Family History of Visual Loss mtDNA Haplogroup
G14249A G11778A WZ1276-III-2 M 21 21 CF/10cm/0.6 Profound Yes D5b1
C14272T - WZ400-III-6 M 16 14 0.01/0.06 Profound No H2
A14322G - WZ1246-III-3 F 21 19 0.04/0.04 Severe No H2
T14463C G11778A WZ385-IV-5 M 18 15 0.08/0.04 Severe Yes A
T14463C G11778A WZ1185-IV-4 M 12 12 0.01/0.01 Profound Yes D5a'b
A14486G - WZ812-IV-1 M 14 14 0.08/0.02 Profound Yes N9a3
A14510G - WZ860-III-3 M 17 16 0.2/0.25 Mild No H2
T14598C - WZ306-III-1 M 22 20 0.3/0.1 Mild No G2
A14617T G11778A WZ259-IV-2 M 18 17 0.1/0.1 Mild Yes D4b1
A14617T T14484C WZ338-IV-1 M 15 14 0.02/0.04 Profound No D4b1
A14617T - WZ419-IV-4 M 18 18 0.01/CF/10cm Profound No D4b1
A14617T - WZ451-IV-1 M 13 13 0.02/0.03 Profound Yes D4b1
A14617T T14484C WZ624-IV-3 M 24 23 0.1/0.1 Mild No D4b1
A14617T - WZ1124-III-1 M 21 15 0.1/0.6 Mild No D4b1
Table 3
 
Summary of the Clinical and Molecular Data for 40 Han Chinese Probands Carrying the T14502C Mutation
Table 3
 
Summary of the Clinical and Molecular Data for 40 Han Chinese Probands Carrying the T14502C Mutation
Proband Sex Age at Onset, y Visual Acuity Right/Left Level of Visual Impairment Primary mtDNA Mutation mtDNA Haplogroup Family History of Visual Loss
WZ465-III-1 F 15 0.05/0.1 Severe - M10a No
WZ589-IV-2 M 12 0.03/0.1 Profound - M10a No
WZ902-IV-1 F 9 0.4/0.4 Moderate - M10a No
WZ1017-III-2 M 14 0.6/0.2 Mild - M10a No
WZ1038-III-4 F 11 0.01/0.2 Profound - M10a No
WZ1063-IV-2 M 6 0.25/0.25 Mild - M10a No
WZ474-III-1 F 7 0.5/0.1 Mild - M10a No
WZ218-III-2 M 16 0.03/0.02 Severe G11778A M10a No
WZ418-IV-3 M 18 0.02/0.01 Severe G11778A M10a No
WZ195-III-14 F 13 0.1/0.05 Severe G11778A M10 Yes
WZ330-III-2 M 16 0.01/0.2 Profound G11778A H2 Yes
WZ452-IV-1 M 14 0.1/0.04 Severe G11778A M10a No
WZ878-III-3 M 35 0.03/0.01 Profound G11778A M8a2 Yes
WZ939-III-1 M 35 0.6/0.1 Mild G11778A M10a No
WZ956-IV-4 M 15 0.1/Fc/1m Profound G11778A M10a Yes
WZ1102-III-1 M 19 FC/1m/0.6 Profound G11778A H2 No
WZ1347-III-1 M 15 0.2/0.2 Mild G11778A M10a Yes
WZ1186-III-3 M 13 0.03/0.02 Severe G11778A M10a Yes
WZ1008-III-3 M 32 0.1/ Fc/1m Profound G11778A M10a No
WZ1239-III-6 M 17 Fc/1m/Fc/1m Profound G11778A B4a1 Yes
WZ125-III-1 M 16 0.02/0.03 Severe G11778A M10 Yes
WZ198-II-3 M 20 0.03/0.03 Severe G11778A M10 Yes
WZ995-IV-3 M 22 0.04/0.01 Severe G11778A M10 Yes
WZ1094-IV-2 F 14 0.1/0.1 Mild G11778A M10 Yes
WZ1244-III-4 M 16 0.03/0.03 Severe T14484C M10a No
WZ402-III-2* M 4 0.1/0.1 Mild - H2 No
WZ403-II-12* F 40 0.3/0.2 Mild - H2 No
WZ30-IV-8† M 18 0.02/0.03 Severe G3460A H2 Yes
WZ511-III-1*,‡ F 17 0.1/0.1 Mild T3866C M10a No
WZ512-III-1‡ F 5 FC/20cm/FC/30cm Profound T3866C R No
WZ50-III-3§ F 34 0.04/0.1 Severe G11778A M10a Yes
WZ404-II-7‖ F 38 0.04/0.04 Severe G11778A M10 Yes
WZ405-IV-9‖ M 24 0.01/0.3 Profound G11778A H2 Yes
WZ406-III-16‖ F 26 0.01/0.05 Profound G11778A M7c2 Yes
WZ407-III-10‖ M 19 0.08/0.1 Moderate G11778A M10a Yes
WZ60-III-16¶ M 24 0.02/FC/30cm Profound T14484C G3b1 Yes
WZ67-III-10¶ M 14 0.1/0.1 Mild T14484C M10a Yes
WZ73-III-1¶ F 11 0.8/0.2 Mild T14484C H2 Yes
WZ74-III-18¶ M 30 0.1/0.12 Mild T14484C M10a Yes
WZ75-II-1¶ M 16 0.1/0.02 Severe T14484C M10a Yes
The putative mutation(s) were first examined in all available members of these pedigrees. The mtDNA mutation(s) was presented in matrilineal relatives in each family in the homoplasmic form, but not in other members of every family (data not shown). Then, we performed the mutational screening of known LHON-associated mtDNA mutations in these probands. As shown in Table 2, four pedigrees (WZ1276, 385, 1185, and 259) carrying both G11778A and one putative mutation(s) (G14249A, T14463C, or A14617T), the pedigrees WZ338 and WZ624 harbored both T14484C and A14617T mutations, other eight pedigrees (WZ400, 1246, 812, 860, 306, 419, 451, and 1124) lacked three known LHON-associated mtDNA mutations. 
To assess the contribution that mtDNA variants make toward the variable penetrance and expressivity of optic neuropathy in these Chinese pedigrees, we analyzed entire mtDNA sequences in 14 Chinese probands. As shown in Supplementary Table S1, these probands exhibited distinct sets of mtDNA polymorphisms including 136 known and 16 novel variants, belonging to Eastern Asian haplogroups D4b, D5a, D5b, H2, G2, N9a, and A, 23,24 respectively. These variants in RNAs and polypeptides were further evaluated by phylogenetic analysis of these variants and sequences from other 16 vertebrates. The ND2 A4824G (T119A), ND5 A13651G (T439P), ND6 T14256C (I140V), tRNAGlu T14727C, and CYTB A14927G (T61A) variants showed evolutionary conservation in these species, while none of other variants showed evolutionary conservation. Interestingly, the A14927G mutation coexisted with the secondary LHON-associated tRNAThr A15951G mutation on haplogroup D4b1 among six probands carrying the A14671T mutation. 27  
Analysis of Entire mtDNA Sequences in Probands Carrying the T14484C Mutation
As shown in Supplementary Table S2, 55 Chinese individuals carrying the T14484C mutation consisted of 7 females and 48 males. The previous investigations examined the entire mtDNA sequences of 38 subjects carrying the T14484C mutation.22 In the present investigation, we determined the complete mtDNA sequence analysis of additional 14 probands carrying this mutation. As shown in Supplementary Table S3, these probands exhibited distinct sets of mtDNA polymorphisms including 200 known and 16 novel variants. We further performed the haplogroup analysis of mtDNAs carrying the T14484C mutation.23,24 As shown in Table 4, the mtDNAs from 52 Chinese families carrying the T14484C mutation were distributed among 10 different haplogroups. As shown in Table 4, the frequencies of mtDNA haplogroups B, D, G, M7, M8, M9, M10, M11, N, and R in 52 LHON families carrying the T14484C mutation were 11.5%, 21.2%, 5.8%, 3.8%, 19.2%, 7.7%, 11.5%, 3.8%, 7.7%, and 7.7%, respectively; while those of 316 Chinese controls were 18%, 22.8%, 4.1%, 6.6%, 8.9%, 1.6%, 1.3%, 0.6%, 8.2%, and 1.9%, respectively. 
Table 4
 
mtDNA Haplogroup From 98 Han Chinese LHON Probands Carrying ND6 Mutations and 316 Control Subjects
Table 4
 
mtDNA Haplogroup From 98 Han Chinese LHON Probands Carrying ND6 Mutations and 316 Control Subjects
Frequency of mtDNA Haplogroup, % Macrogroup M Macrogroup N F
D G M7 M8 M9 M10 M11 A B N R H2
All subjects with ND6 mutations, n = 98 17.3 4.1 3.1 10.2 3.1 31.6 2.0 1.0 7.1 5.1 6.1 9.2 -
Subjects with the T14484C mutation, n = 52 21.2 5.8 3.8 19.2 7.7 11.5 3.8 - 11.5 7.7 7.7 - -
Subjects with the T14502C mutation, n = 34 - - 2.9 - - 73.5 - - 2.9 - 2.9 17.6 -
Subjects with the putative ND6 mutations, n = 12 50 8.3 - - - - - 8.3 - 8.3 - 25.0 -
Control, n = 316 22.8 4.1 6.6 8.9 1.6 1.3 0.6 7.6 18.0 8.2 1.9 1.6 16.8
Evaluation of Chinese Subjects Carrying the T14502C Mutation
A total of 40 Chinese individuals carrying the T14502C mutation consisted of 13 females and 27 males. Mutational screening of other known LHON-associated mtDNA mutations showed that 9 subjects carried only the T14502C mutation, while 31 harbored the T14502C and one additional mutations (22 subjects carrying the G11778A mutation, 6 individuals harboring the T14484C mutation, 1 subject having the G3460A mutation, 28 and 2 probands carrying the T3866C mutation). 29 As shown in Table 3, ophthalmologic assessments of 40 probands showed that 12 individuals suffered from profound visual impairment, 14 subjects exhibited severe visual impairment, 1 proband had moderate visual impairment and 12 subjects exhibited mild visual impairment. The age of onset of visual loss in these subjects ranged from 4 to 40 years, with the average of 19.7 years. Of these, 16 probands (9 carrying only the T14502C mutation and 7 subjects carrying both T14502C and one primary mutation) did not have a history of optic neuropathy, while 24 subjects carrying both mtDNA mutations had a history of visual loss. In the previous study, we carried out the sequence analysis of entire mtDNA in 16 probands carrying the T14502C mutation. 18,25 For defining the mitochondrial haplogroups, we performed the sequence analysis of the PCR-amplified fragments spanning the D-loop region from 24 probands carrying the T14502C mutation. As shown in Supplementary Table S4, 47 variants were identified in the D-loop region. These included the haplogroup M10–specific variants T489C and T16311C, haplogroup H2–specific variants A73G and C16223T, and haplogroup B4–specific variants T16189C and T16217C. 23,24 As shown in Table 4, the frequencies of mtDNA haplogroups B, H2, M7, M10, and R in 30 LHON families carrying the T14502C mutation were 2.9%, 17.6%, 2.9%, 73.5%, and 2.9%, respectively. 
Discussion
Prevalence of the Known LHON-Associated ND6 Mutations
The large cohort of 1218 Chinese subjects with LHON consisted of 917 males and 301 females. A total of 59 genetically unrelated individuals carried the T14484C mutation, translated to 4.8% cases of this Chinese clinical population. In fact, the incidences of the T14484C mutation varied among different ethnic backgrounds. The incidences of the T14484C mutation were 3.3% in a cohort of 903 Chinese subjects with LHON, 8.8% in a cohort of Japanese subjects, and 4.3% in a cohort of Indian subjects, respectively. 11,13,30 However, the prevalence of T14484C mutation in the European populations were much higher than these in Asian countries. Indeed, the incidences of the T14484C mutation varied from 10% to 33% in several European cohorts. 3134 In this cohort, only one subject carried the G14459A mutation, as in the case of the European population. 17 Furthermore, the incidence of the T14502C mutation was 3.3% and 1.9% in this Chinese cohort of LHON and controls, respectively, while the low prevalence of this mutation occurs in the European population. 35 Here, 9 patients carrying only the T14502C mutation and 9 subjects carrying both T14502C and one primary mutations did not have a history of optic neuropathy, while 22 subjects carrying two mtDNA mutations had a history of visual loss. These data suggested that the T14502C mutation was itself insufficient to produce a clinical phenotype, but may act as a synergistic role to modulate the phenotypic manifestation of the primary LHON-associated mtDNA mutations. A total of 94 subjects carrying one of three known ND6 mutations accounted for 7.7% of this cohort of LHON patients (4.4% in only T14484C mutation, 2.8% in only the T14502C mutation, 0.5% in both T14484C and T14502C mutations, and 0.08% in G14459A mutation). However, the incidences of ND4 G11778A and ND1 G3460A mutations in a cohort of 903 Chinese subjects with LHON were 34.6% and 0.4%, respectively. 13  
Mutational Spectrum and Incidences of ND6 Gene
A total of 92 (73 known and 19 novel) variants in ND6 gene were identified in a cohort of 1218 Han Chinese subjects with LHON. Similar to other mtDNA variations, these variants can be grouped into three categories: neutral, adaptive, and deleterious. 26 To identify putative deleterious mutation, these variants were further evaluated using following criteria: (1) missense mutation, (2) conservation index greater than 75%, proposed by Ruiz-Pesini and Wallace, 26 (3) absent in the 316 Chinese controls, (4) potential structural and functional alterations, and (5) pedigree analysis. Of these, 63 variants were silent mutations, while 29 variants were missense mutations. Of the missense mutations, 20 variants were absent in 316 Chinese control subjects, while the frequencies of 9 variants ranged from 0.3% to 2.9% in the control populations. In particular, some of these variants occurring both control and patient populations were the haplogroup specific variants. 23 These included the C14149T variant of haplogroup B5, the T14138C variant of haplogroup CZ, and the C14337T variant of haplogroup M10. 23 Apparently, these missense variants and 63 silent mutations were adaptive or neutral but unlikely deleterious. Phylogenetic analysis showed that conservation indexes of 13 variants, including 6 novel variants (A14322G, T14463C, A14486G, A14510G, T14598C, and T14261A), were more than 78%. Despite their higher conservation index, three variants (T14256C, T14261A, and C14337T) were present in the controls. On the other hand, the conservation indexes for nine variants including the T14484C mutation were more than 78% but these variants were absent in 312 Chinese controls. Thus, these eight variants (G14249A, C14272T, A14322G, T14463C, A14486G, A14510G, T14598C, and A14617T) were fitted with criteria for the putative LHON-associated mtDNA mutations. The incidences of these mutations in this cohort were 0.1%, 0.1%, 0.1%, 0.2%, 0.1%, 0.1%, 0.1%, and 0.6%, respectively. 
The screening of three known mtDNA mutations in 14 subjects carried one of putative ND6 mutations show that four probands (WZ1276, 385, 1185, and 259) carrying both G11778A and one putative mutation(s) (G14249A, T14463C, or A14617T), the pedigrees WZ338 and WZ624 harbored both T14484C and A14617T mutations, the other eight probands (WZ400, 1246, 812, 860, 306, 419, 451, and 1124) lacked three known LHON-associated mtDNA mutations. Of these, four probands carrying one of the C14272T, A14322G, A14510G, and T14598C mutations, two probands carrying the only A14617T mutation, and two probands carrying both T14484C and A14617T mutations did not have a history of optic neuropathy. On the other hand, one proband harboring the T14598C mutation, one proband carrying the A14617T mutation, four probands carrying both G11778A and G14249A, T14463C, or A14617T mutations had a history of visual loss. These data suggested that the T14598C and A144617T variants may be the novel LHON mutations, and the other six mutations may act as secondary LHOH mutations. These mutations may cause a failure in the activity of NADH dehydrogenase, thereby leading to a deficient function of OXPHOS, a decrease in ATP synthesis, and an increasing generation of reactive oxygen species (ROS). 3638 The extremely low penetrance of optic neuropathy in these Chinese pedigrees carrying one of eight putative LHON mutations indicated that the mutation(s), similar to the G11778A and T14484C mutations, 21,39 is necessary but itself insufficient to produce a clinical phenotype. Therefore, the modifier factors such as nuclear and mitochondrial genetic modifiers, and environmental factors should modulate the phenotypic manifestation of these mtDNA mutations. 40  
The Phylogenetic and Haplogroup Analysis of Probands Carrying the ND6 Mutations
The average penetrance of optical neuropathy (affected matrilineal relatives/total matrilineal relatives) in 45 Chinese pedigrees carrying the T14484C mutations were 20.9%. In particular, 77 of 265 male matrilineal relatives of 45 pedigrees suffered from optical neuropathy. On the other hand, 50% males and approximately 10% females in Caucasians carrying the G3460A, G11778A, or T14484TC mutation indeed developed the optic neuropathy. 2,41,42 Here, mtDNAs in 52 LHON families carrying the T14484C mutation were widely dispersed among 10 common Eastern Asian subhaplogroups, while mtDNAs of European pedigrees carrying this mutation belonged to the common European haplogroups H, I, J, K, T, U, V, and L. 43,44 Indeed, the occurrences of mtDNA haplogroups G, M8, M9, M10, M11, and R in families carrying the T14484C mutation were higher than those in controls. Moreover, the frequencies of mtDNAs in haplogroups M10 and H2 in 34 Chinese families carrying the T14502C mutation as well as haplogroup H2 in 12 pedigrees carrying one of eight putative mutations were markedly higher than those in controls. Thus, the occurrences of haplogroups M9, M10, M11, and H2 in patients carrying the ND6 mutations were significantly higher than those in 316 Chinese controls and other Asian populations. 23,24 This discrepancy between the different ethnic backgrounds may be attributed to evolution. 45  
In summary, this is the first study to investigate the spectrum and incidence of mutations in ND6 genes in Chinese subjects with LHON. The three known ND6 mutations, T14484C, T14502C, and G14459A, in Chinese cohort accounted for 7.7% cases of 1218 Chinese subjects with LHON. Furthermore, the eight putative LHON-associated mtDNA mutations were the rare mutations, accounting for 1.1% cases in this Chinese cohort. A total of 106 subjects carrying one of the ND6 mutations accounted for 8.7% cases of 1218 Chinese subjects with LHON. These data further support the notion that the ND6 gene is the hot spot for mutations associated with LHON. Thus, our findings may provide valuable information for the further understanding of pathophysiology and management of LHON. 
Supplementary Materials
Acknowledgments
Supported by the National Key Technologies Research and Development Program Grant 2012BAI09B03 from the Ministry of Science and Technology of China (M-XG, PJ) and a Grant 81200724 from National Nature Science Foundation of China (JJZ), and Ministry of Education of Zhejiang Provincial Grant Y201017001 and Ministry of Science and Technology of Wenzhou City Grant Y20100272 (ML).  
Disclosure: M. Liang, None; P. Jiang, None; F. Li, None; J. Zhang, None; Y. Ji, None; Y. He, None; M. Xu, None; J. Zhu, None; X. Meng, None; F. Zhao, None; Y. Tong, None; X. Liu, None; Y. Sun, None; X. Zhou, None; J.Q. Mo, None; J. Qu, None; M.-X. Guan, None 
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Footnotes
 ML and PJ contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Figure 1
 
Geographic locations of 1218 Han Chinese subjects with LHON. The numbers in parentheses indicate 106 patients with NDG mutations.
Figure 1
 
Geographic locations of 1218 Han Chinese subjects with LHON. The numbers in parentheses indicate 106 patients with NDG mutations.
Figure 2
 
Fourteen Han Chinese pedigrees with LHON. Visually-impaired individuals indicated by filled symbols. Arrowhead denotes probands.
Figure 2
 
Fourteen Han Chinese pedigrees with LHON. Visually-impaired individuals indicated by filled symbols. Arrowhead denotes probands.
Table 1
 
Variants in the ND6 Gene in 1218 Chinese Subjects With LHON
Table 1
 
Variants in the ND6 Gene in 1218 Chinese Subjects With LHON
Position Replacement Conservation Index, %* Number of Affected Subjects Percentage (number/1218) Number of Controls Percentage (number/316) Previously Reported
Known mutations
 14484 T-C (Met64Val) 78.6 59 4.8 0 0 Yes
 14502 T-C (Ile58Val) 71.4 40 3.3 6 1.9 Yes
 14459 G-A (Ala72Val) 100.0 1 0.1 0 0 Yes
Putative mutations
 14249 G-A (Ala142Val) 85.7 1 0.1 0 0 Yes
 14272 C-T (Leu134Phe) 85.7 1 0.1 0 0 Yes
 14617 A-T (Phe19Leu) 92.9 6 0.5 0 0 Yes
 14322 A-G (Phe118Leu) 78.6 1 0.1 0 0 No
 14463 T-C (Thr71Pro) 78.6 2 0.2 0 0 No
 14486 A-G (Met63Thr) 100.0 1 0.1 0 0 No
 14510 A-G (Val55Ala) 78.6 1 0.1 0 0 No
 14598 T-C (Ile26Val) 100.0 1 0.1 0 0 No
Other missense mutations
 14178 T-C (Ile166Val) 71.4 12 1.0 4 1.3 Yes
 14180 T-C (Tyr165Cys) 71.4 2 0.2 0 0 Yes
 14207 G-A (Thr156Ile) 57.1 1 0.1 0 0 Yes
 14256 T- C (Ile140Val) 78.6 1 0.1 1 0.3 Yes
 14259 G-A (Pro139Ser) 50.0 1 0.1 0 0 Yes
 14315 C-T (Ser120Asn) 42.9 1 0.1 0 0 Yes
 14318 T-C (Asn119Ser) 50.0 64 5.3 9 2.9 Yes
 14319 T-C (Asn119Asp) 50.0 3 0.3 0 0 Yes
 14325 T-C (Asn117Asp) 50.0 3 0.3 1 0.3 Yes
 14334 C-T (Val114Ile) 57.1 1 0.1 0 0 Yes
 14337 C-T (Val113Met) 78.6 5 0.4 1 0.3 Yes
 14340 C-T (Val112Met) 50.0 3 0.3 0 0 Yes
 14417 A-G (Val86Ala) 71.4 11 0.9 1 0.3 Yes
 14669 A-G (Met2Thr) 28.6 3 0.3 1 0.3 Yes
 14261 T-A (Asp138Val) 92.9 1 0.1 1 0.3 No
 14418 C-T (Val86Ile) 71.4 1 0.1 0 0 No
 14643 C-A (Gly11Cys) 14.3 2 0.2 0 0 No
 14664 C-T (Ala4Thr) 28.6 1 0.1 0 0 No
Silent mutations
 14149 C-T - 5 0.4 0 0 Yes
 14167 C-T 100 1 0.1 0 0 Yes
 14182 T-C 78.6 1 0.1 1 0.3 Yes
 14200 T-C 100 10 0.8 1 0.3 Yes
 14209 A-G 85.7 4 0.3 0 0 Yes
 14215 T-C 78.6 1 0.1 0 0 Yes
 14218 T-C 78.6 2 0.2 0 0 Yes
 14220 A-G 78.6 2 0.2 0 0 Yes
 14227 C-G 92.9 1 0.1 0 0 Yes
 14239 C-T 100 1 0.1 1 0.3 Yes
 14260 A-G 92.9 1 0.1 0 0 Yes
 14269 A-G 71.4 2 0.2 0 0 Yes
 14275 C-T 92.9 1 0.1 0 0 Yes
 14281 C-T 78.6 4 0.3 0 0 Yes
 14287 T-C 78.6 2 0.2 0 0 Yes
 14299 T-C 57.1 2 0.2 0 0 Yes
 14302 T-C 100 1 0.1 1 0.3 Yes
 14305 G-A 50 2 0.2 0 0 Yes
 14308 T-C 78.6 22 1.8 5 1.6 Yes
 14311 T-C 64.3 11 0.9 3 1 Yes
 14314 A-G 42.9 4 0.3 1 0.3 Yes
 14338 C-T 50 1 0.1 0 0 Yes
 14341 C-T 50 1 0.1 0 0 Yes
 14344 A-G 78.6 1 0.1 0 0 Yes
 14364 G-A 85.7 1 0.1 1 0.3 Yes
 14368 C-T 78.6 1 0.1 0 0 Yes
 14383 C-T 42.9 1 0.1 0 0 Yes
 14386 T-C 71.4 1 0.1 0 0 Yes
 14388 A-G 100 2 0.2 0 0 Yes
 14392 C-T 71.4 2 0.2 0 0 Yes
 14410 G-A 78.6 8 0.7 1 0.3 Yes
 14431 T-C 57.1 4 0.3 0 0 Yes
 14449 A-G 71.4 1 0.1 0 0 Yes
 14470 T-C 100 43 3.5 13 4.1 Yes
 14476 G-A 100 4 0.3 1 0.3 Yes
 14482 C-T 78.6 1 0.1 0 0 Yes
 14485 C-T 100 3 0.3 1 0.3 Yes
 14494 T-C 100 3 0.3 0 0 Yes
 14509 A-G 78.6 1 0.1 0 0 Yes
 14512 T-C 85.7 2 0.2 0 0 Yes
 14518 A-G 100 2 0.2 0 0 Yes
 14533 C-T 100 1 0.1 0 0 Yes
 14544 G-A 85.7 4 0.3 0 0 Yes
 14551 A-G 28.6 1 0.1 0 0 Yes
 14554 A-G 100 1 0.1 0 0 Yes
 14560 G-A 85.7 11 0.9 1 0.3 Yes
 14569 G-A 100 79 6.5 13 4.1 Yes
 14581 T-C 57.1 1 0.1 0 0 Yes
 14587 A-G 100 13 1.0 5 1.6 Yes
 14605 A-G 100 5 0.4 1 0.3 Yes
 14620 C-T 92.6 1 0.1 0 0 Yes
 14659 C-T 78.6 4 0.3 1 0.3 Yes
 14668 C-T 28.6 239 19.6 55 17.4 Yes
 14614 A-G 100 1 0.1 0 0 No
 14596 A-G 100 3 0.3 0 0 No
 14500 A-G 71.4 1 0.1 0 0 No
 14419 C-T 71.4 1 0.1 0 0 No
 14263 C-T 71.4 1 0.1 0 0 No
 14257 A-G 50 2 0.2 0 0 No
 14230 A-G 78.6 1 0.1 0 0 No
 14176 A-G 71.4 3 0.3 0 0 No
 14650 C-T 100 3 0.3 1 0.3 No
 14659 C-T 85.7 1 0.1 0 0 No
Table 2
 
Summary of the Clinical and Molecular Data for 14 Han Chinese Probands Carrying One of the Putative ND6 Mutations
Table 2
 
Summary of the Clinical and Molecular Data for 14 Han Chinese Probands Carrying One of the Putative ND6 Mutations
Putative ND6 Mutation Primary Mutation Proband Sex Age at Test, y Age at Onset, y Visual Acuity Right/Left Level of Visual Impairment Family History of Visual Loss mtDNA Haplogroup
G14249A G11778A WZ1276-III-2 M 21 21 CF/10cm/0.6 Profound Yes D5b1
C14272T - WZ400-III-6 M 16 14 0.01/0.06 Profound No H2
A14322G - WZ1246-III-3 F 21 19 0.04/0.04 Severe No H2
T14463C G11778A WZ385-IV-5 M 18 15 0.08/0.04 Severe Yes A
T14463C G11778A WZ1185-IV-4 M 12 12 0.01/0.01 Profound Yes D5a'b
A14486G - WZ812-IV-1 M 14 14 0.08/0.02 Profound Yes N9a3
A14510G - WZ860-III-3 M 17 16 0.2/0.25 Mild No H2
T14598C - WZ306-III-1 M 22 20 0.3/0.1 Mild No G2
A14617T G11778A WZ259-IV-2 M 18 17 0.1/0.1 Mild Yes D4b1
A14617T T14484C WZ338-IV-1 M 15 14 0.02/0.04 Profound No D4b1
A14617T - WZ419-IV-4 M 18 18 0.01/CF/10cm Profound No D4b1
A14617T - WZ451-IV-1 M 13 13 0.02/0.03 Profound Yes D4b1
A14617T T14484C WZ624-IV-3 M 24 23 0.1/0.1 Mild No D4b1
A14617T - WZ1124-III-1 M 21 15 0.1/0.6 Mild No D4b1
Table 3
 
Summary of the Clinical and Molecular Data for 40 Han Chinese Probands Carrying the T14502C Mutation
Table 3
 
Summary of the Clinical and Molecular Data for 40 Han Chinese Probands Carrying the T14502C Mutation
Proband Sex Age at Onset, y Visual Acuity Right/Left Level of Visual Impairment Primary mtDNA Mutation mtDNA Haplogroup Family History of Visual Loss
WZ465-III-1 F 15 0.05/0.1 Severe - M10a No
WZ589-IV-2 M 12 0.03/0.1 Profound - M10a No
WZ902-IV-1 F 9 0.4/0.4 Moderate - M10a No
WZ1017-III-2 M 14 0.6/0.2 Mild - M10a No
WZ1038-III-4 F 11 0.01/0.2 Profound - M10a No
WZ1063-IV-2 M 6 0.25/0.25 Mild - M10a No
WZ474-III-1 F 7 0.5/0.1 Mild - M10a No
WZ218-III-2 M 16 0.03/0.02 Severe G11778A M10a No
WZ418-IV-3 M 18 0.02/0.01 Severe G11778A M10a No
WZ195-III-14 F 13 0.1/0.05 Severe G11778A M10 Yes
WZ330-III-2 M 16 0.01/0.2 Profound G11778A H2 Yes
WZ452-IV-1 M 14 0.1/0.04 Severe G11778A M10a No
WZ878-III-3 M 35 0.03/0.01 Profound G11778A M8a2 Yes
WZ939-III-1 M 35 0.6/0.1 Mild G11778A M10a No
WZ956-IV-4 M 15 0.1/Fc/1m Profound G11778A M10a Yes
WZ1102-III-1 M 19 FC/1m/0.6 Profound G11778A H2 No
WZ1347-III-1 M 15 0.2/0.2 Mild G11778A M10a Yes
WZ1186-III-3 M 13 0.03/0.02 Severe G11778A M10a Yes
WZ1008-III-3 M 32 0.1/ Fc/1m Profound G11778A M10a No
WZ1239-III-6 M 17 Fc/1m/Fc/1m Profound G11778A B4a1 Yes
WZ125-III-1 M 16 0.02/0.03 Severe G11778A M10 Yes
WZ198-II-3 M 20 0.03/0.03 Severe G11778A M10 Yes
WZ995-IV-3 M 22 0.04/0.01 Severe G11778A M10 Yes
WZ1094-IV-2 F 14 0.1/0.1 Mild G11778A M10 Yes
WZ1244-III-4 M 16 0.03/0.03 Severe T14484C M10a No
WZ402-III-2* M 4 0.1/0.1 Mild - H2 No
WZ403-II-12* F 40 0.3/0.2 Mild - H2 No
WZ30-IV-8† M 18 0.02/0.03 Severe G3460A H2 Yes
WZ511-III-1*,‡ F 17 0.1/0.1 Mild T3866C M10a No
WZ512-III-1‡ F 5 FC/20cm/FC/30cm Profound T3866C R No
WZ50-III-3§ F 34 0.04/0.1 Severe G11778A M10a Yes
WZ404-II-7‖ F 38 0.04/0.04 Severe G11778A M10 Yes
WZ405-IV-9‖ M 24 0.01/0.3 Profound G11778A H2 Yes
WZ406-III-16‖ F 26 0.01/0.05 Profound G11778A M7c2 Yes
WZ407-III-10‖ M 19 0.08/0.1 Moderate G11778A M10a Yes
WZ60-III-16¶ M 24 0.02/FC/30cm Profound T14484C G3b1 Yes
WZ67-III-10¶ M 14 0.1/0.1 Mild T14484C M10a Yes
WZ73-III-1¶ F 11 0.8/0.2 Mild T14484C H2 Yes
WZ74-III-18¶ M 30 0.1/0.12 Mild T14484C M10a Yes
WZ75-II-1¶ M 16 0.1/0.02 Severe T14484C M10a Yes
Table 4
 
mtDNA Haplogroup From 98 Han Chinese LHON Probands Carrying ND6 Mutations and 316 Control Subjects
Table 4
 
mtDNA Haplogroup From 98 Han Chinese LHON Probands Carrying ND6 Mutations and 316 Control Subjects
Frequency of mtDNA Haplogroup, % Macrogroup M Macrogroup N F
D G M7 M8 M9 M10 M11 A B N R H2
All subjects with ND6 mutations, n = 98 17.3 4.1 3.1 10.2 3.1 31.6 2.0 1.0 7.1 5.1 6.1 9.2 -
Subjects with the T14484C mutation, n = 52 21.2 5.8 3.8 19.2 7.7 11.5 3.8 - 11.5 7.7 7.7 - -
Subjects with the T14502C mutation, n = 34 - - 2.9 - - 73.5 - - 2.9 - 2.9 17.6 -
Subjects with the putative ND6 mutations, n = 12 50 8.3 - - - - - 8.3 - 8.3 - 25.0 -
Control, n = 316 22.8 4.1 6.6 8.9 1.6 1.3 0.6 7.6 18.0 8.2 1.9 1.6 16.8
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