July 2015
Volume 56, Issue 8
Free
Genetics  |   July 2015
Prevalence of Mitochondrial ND4 Mutations in 1281 Han Chinese Subjects With Leber's Hereditary Optic Neuropathy
Author Affiliations & Notes
  • Pingping Jiang
    Institute of Genetics, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
    Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China
  • Min Liang
    Institute of Genetics, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
    Attardi Institute of Mitochondrial Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Juanjuan Zhang
    Institute of Genetics, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
    School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Yinglong Gao
    Attardi Institute of Mitochondrial Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Zheyun He
    Attardi Institute of Mitochondrial Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Han Yu
    Attardi Institute of Mitochondrial Biomedicine, 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
  • Yanchun Ji
    Institute of Genetics, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
  • Xiaoling Liu
    Attardi Institute of Mitochondrial Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang, China
    School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • Minglian Zhang
    Department of Ophthalmology, Xingtai Eye Hospital, Xingtai, Hebei, China
  • Qun Fu
    Department of Ophthalmology, The Third Affiliated Hospital, Xinxiang Medical College, Xinxiang, Henan, China
  • Yi Tong
    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
  • Taosheng Huang
    Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
  • 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, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
    Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou, China
    Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States
    School of Public Health, Zhejiang University, Hangzhou, Zhejiang, China
  • Correspondence: Min-Xin Guan, Institute of Genetics, School of Medicine, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, China; gminxin88@zju.edu.cn
  • Footnotes
     PJ and ML contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science July 2015, Vol.56, 4778-4788. doi:10.1167/iovs.14-16158
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      Pingping Jiang, Min Liang, Juanjuan Zhang, Yinglong Gao, Zheyun He, Han Yu, Fuxin Zhao, Yanchun Ji, Xiaoling Liu, Minglian Zhang, Qun Fu, Yi Tong, Yanhong Sun, Xiangtian Zhou, Taosheng Huang, Jia Qu, Min-Xin Guan; Prevalence of Mitochondrial ND4 Mutations in 1281 Han Chinese Subjects With Leber's Hereditary Optic Neuropathy. Invest. Ophthalmol. Vis. Sci. 2015;56(8):4778-4788. doi: 10.1167/iovs.14-16158.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To investigate the prevalence and spectrum of mitochondrial ND4 mutations in subjects with Leber's hereditary optic neuropathy (LHON).

Methods: A cohort of 1281 Chinese Han probands and 478 control subjects underwent clinical and genetic evaluation, and sequence analysis of mitochondrial (mt) DNA, as well as enzymatic assay of NADH:ubiquinone oxidoreductase.

Results: In this cohort, 503 probands had a family history of optic neuropathy and 778 subjects were sporadic cases. Mutational analysis of ND4 gene identified 149 (102 known and 47 novel) variants. The prevalence of known m.11778G>A mutation was 35.36%. Furthermore, we identified the known m.11696G>A and m.11253T>C mutations and five novel putative LHON-associated mutations. These mutations accounted for 2.74% of cases of LHON subjects. By enzymatic assay, we showed a mild decrease in the activity of NADH:ubiquinone oxidoreductase in mutant cell lines carrying only one putative mtDNA mutation. The low penetrance of optic neuropathy and mild biochemical defects in these pedigrees carrying only m.11696G>A mutation and one putative LHON-associated mutation suggested that the mutation(s) is(are) necessary but is(are) itself(themselves) insufficient to produce a visual failure. Moreover, mtDNAs in 169 probands carrying the LHON-associated mutation(s) were widely dispersed among 13 Eastern Asian haplogroups. In particular, the frequencies of haplogroups D, M8, M10, M11, and H in probands carrying the LHON-associated mtDNA mutation(s) were higher than those in Chinese controls.

Conclusions: These results suggested that the ND4 gene is the hot spot for mutations associated with LHON. Thus, these findings may provide valuable information for the further understanding of pathogenic mechanism of LHON.

Leber's hereditary optic neuropathy (LHON) is one of the most common inherited optic neuropathies, affecting 3.22 per 100,000 subjects in the North East of England.1,2 This disease generally affects from children to young adults with the rapid, painless, bilateral loss of central vision.35 While LHON is traditionally considered to be familial, many individuals represent isolated cases.6 In familial cases of LHON, all affected individuals only present in the maternal lineage, indicating the involvement of mutations in mitochondrial DNA (mtDNA).7,8 More than 17 mtDNA missense mutations have been identified that contribute to LHON, though to varying degrees.9 These mtDNA mutations include the primary mutations, which can each cause LHON, and the secondary mutations, which may interact with the primary mutations to increase the probability of clinical expression.1,5 Three common primary LHON-associated mtDNA mutations are the ND1 m.3460G>A, ND4 m.11778G>A, and ND6 m.14484T>C mutations.1,8 These mutations are responsible for approximately 90% of LHON pedigrees in some countries, but for only 38.3% of cases in a cohort of 903 Chinese Han subjects with LHON.1013 In particular, ND4 m.11778G>A mutation is the most prevalent LHON-associated mtDNA mutation worldwide.1316 The LHON-associated mtDNA mutation(s) often occur in nearly homoplasmy or homoplasmy. Typical features in LHON pedigrees carrying the mtDNA mutation(s) are incomplete penetrance and male bias among the affected subjects, reflecting the complex etiology of this disease.1,16 
Seven mitochondrial genes encoding essential subunits of NADH dehydrogenase (complex I) are proposed to be the hot spots for mutations associated with LHON.1719 Our previous investigation20 has shown the mutational incidence and spectrum of the ND6 gene in a large cohort of Chinese subjects with LHON. In the present investigation, we carried out the pedigree evaluation and mutational analysis of ND4 gene in a cohort of 1281 genetically unrelated Han Chinese subjects with LHON. Pedigree analysis showed that 503 probands had a family history of optic neuropathy and 778 subjects were sporadic cases. Mutational analysis of the ND4 gene identified 149 nucleotide changes including the known m.11778G>A mutation in this cohort. These variants included 32 (14 novel and 18 known) missense and 117 silent variants. 
To identify putative deleterious mutations, these variants were further evaluated by using the following criteria: (1) missense mutation; (2) conservation index (CI) from other 16 vertebrates>75%; (3) absence in the 478 Chinese controls; (4) potential structural and functional alterations; (5) pedigree analysis; and (6) biochemical assays. Furthermore, we performed the clinical and genetic evaluation, and entire mtDNA sequence analysis of 10 subjects carrying one of seven putative LHON-associated variants. Functional significance of these mutations was assessed for enzymatic activities of electron transport chain complexes in cell lines derived from probands carrying one of these mtDNA mutations. In addition, we carried out the genetic and mtDNA analysis of 41 probands carrying the m.11696G>A mutation and 120 probands carrying the m.11778G>A mutation. Moreover, the mtDNAs of 169 probands were assigned to the Asian mtDNA haplogroups by using the nomenclature of mtDNA haplogroups. 
Subjects and Methods
Subjects
A total of 1281 genetically unrelated Han Chinese subjects with LHON were recruited from the eye clinics across 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 Cincinnati Children's Hospital Medical Center Institute Review Board and Zhejiang University and Wenzhou Medical University Ethic Committees. A comprehensive history and physical examination for these participating subjects were performed to identify both personal or family medical histories of visual impairment and other clinical abnormalities. The inclusion criteria of patients for this study were 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 symptom. A total of 478 Han Chinese control subjects used for screening for the presence of mtDNA variants were from the same regions. 
Ophthalmologic Examinations
The ophthalmic examinations of probands, other members of these families, and 478 control subjects were conducted, including visual acuity, visual field examination (Humphrey Visual Field Analyzer IIi, SITA Standard; Carl Zeiss Meditec, Oberkochen, Germany), visual evoked potentials (VEPs; RETI port gamma, flash VEP; Roland Consult, Brandenberg, Germany), and fundus photography (CR6-45NM fundus camera; Canon, Lake Success, NY, USA). The severity of visual impairment was defined according to the visual acuity as follows: normal, >0.3; mild, =0.3 to 0.1; moderate, <0.1 to 0.05; severe, <0.05 to 0.02; and profound, <0.02. 
Mutational Analysis of mtDNAs
Genomic DNA was isolated from whole blood of 1281 probands and 478 control subjects by using Puregene DNA Isolation Kits (Gentra Systems, Minneapolis, MN, USA). Subjects' DNA fragments spanning the ND4 gene were amplified by PCR using oligodeoxynucleotides corresponding to mtDNA at positions 10760 to 12137.21,22 Each fragment was purified and subsequently analyzed, as detailed elsewhere.23 These sequence results were compared with the updated consensus Cambridge sequence (GenBank accession number: NC_012920).21 The homoplasmy of the m.11778G>A mutation in these subjects was determined as detailed previously.23 The allelic frequencies of variants in the ND4 gene in 478 Chinese control subjects were determined by direct sequencing of PCR products as described above. For defining the mtDNA haplogroups, fragments spanning the D-loop regions were PCR amplified by using oligodeoxynucleotides corresponding to mtDNA at positions 15811 to 775 and genomic DNAs from 41 probands carrying the m.11696G>A variant, 120 subjects carrying m.11778G>A mutation, and 478 control subjects. The entire mitochondrial genomes of 10 subjects harboring one of the putative LHON-associated mtDNA mutations were PCR amplified in 24 overlapping fragments by using sets of oligonucleotide primers, and their sequences were determined, as described previously.22 These sequence results were compared with the updated consensus Cambridge sequence (GenBank accession number: NC_012920).21 
Structural Analysis
The secondary structure of human mitochondrial ND4 subunit and domains were predicted by using online software (provided in the public domain by SOSUI at http://bp.nuap.nagoya-u.ac.jp/sosui/). 
Phylogenetic Analysis
A total of 17 vertebrate ND4 sequences were used in the interspecies 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).20 The CI was calculated by comparing the human ND4 variants with other 16 vertebrates. 
Cell Culture
Lymphoblastoid cell lines were immortalized by transformation with the Epstein-Barr virus, as described previously.24 Cell lines derived from nine probands (WZ4-IV-2 carrying only m.11778G>A mutation,25 WZ6-III-1 harboring only m.14484T>C mutation,20 WZ1183-IV-1, WZ410-III-1, WZ317-III-3, WZ1165-IV-1, WZ1168-III-3, WZ1123-IV-1, and WZ987-III-2) and three genetically unrelated control individuals (A40, A41, and A42) were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum. 
Enzymatic Assays
The enzymatic activities were assayed by following the modified protocol by Birch-Machin and Turnbull.26,27 Citrate synthase activity was analyzed by the reduction of 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) at 412 nm in the assay buffer containing 0.1 mM DTNB, 50 μM acetyl coenzyme A, and 250 μM oxaloacetate. Complex I activity was determined with 2 μg/mL by following the decrease in the absorbance due to the NADH oxidation at 340 nm in assay buffer. The activity of complex II was analyzed by tracking the secondary reduction of 2,6-dichlorophenolindophenol (DCPIP) by decylubiquinone (DB) at 600 nm in the assay buffer. Complex III activity was determined in the presence of 2 μg/mL antimycin A by measuring the reduction of cytochrome c at 550 nm with reduced decylubiquinone in the assay buffer. Complex IV activity was measured by the addition of 2 mM KCN by monitoring the oxidation of reduced cytochrome c as a decrease of absorbance at 550 nm. All assays were performed by using Synergy H1 (Biotek, Winooski, VT, USA). Complex I to IV activities were normalized by citrate synthase activity. 
Haplogroup Analyses
The mtDNA sequences of the 10 subjects carrying the putative mtDNA mutations(s), 120 Chinese probands carrying the m.11778G>A mutation, and 41 patients carrying the m.11696G>A variant were assigned to the Asian mitochondrial haplogroups by using the nomenclature of mitochondrial haplogroups.28,29 
Computer Analysis
Statistical analysis was carried out by using the unpaired, two-tailed Student's t-test contained in the Microsoft-Excel program for a Macintosh (Microsoft Corp., Redmond, WA, USA). Differences were considered significant at P < 0.05. 
Results
Study Samples
The study samples with LHON consisted of 963 males and 318 females. All participants were recruited from eye clinics across 29 provinces in China. Ophthalmologic evaluation showed that all affected subjects exhibited the variable severity and age at onset of optic neuropathy. Of these, 240 subjects exhibited profound visual impairment, 389 subjects had severe visual impairment, 340 individuals had moderate visual impairment, and 312 subjects had mild visual impairment. A shown in Table 1, the age at onset of optic neuropathy ranged from 5 to 53 years, with an average of 19.5 years. The distribution of age at onset of LHON in 1281 probands was 5 to 10 years (146 subjects), 11 to 20 years (564 individuals), 21 to 30 years (406 patients), 31 to 40 years (106 probands), 41 to 50 years (50 subjects), and 50 to 60 years (9 patients). The age of the 478 Han Chinese control subjects, who were recruited from the same region, ranged from 8 to 58 years with an average of 21 years. The age and sex of these controls were comparable to those of affected subjects. Comprehensive family medical histories of those probands showed no other clinical abnormalities, including diabetes, muscular diseases, hearing impairment, and neurologic disorders. Pedigree analysis showed that 503 probands had a family history of optic neuropathy and 778 subjects were sporadic cases. 
Table 1
 
Sex and Age Distributions for the 1281 Chinese Probands With LHON and 478 Control Subjects
Table 1
 
Sex and Age Distributions for the 1281 Chinese Probands With LHON and 478 Control Subjects
Mutational Analysis of MitochondrialND4Gene
DNA fragments spanning the ND4 gene were PCR amplified from genomic DNA of 1281 Chinese subjects with LHON and 478 Han Chinese control individuals. Each fragment was purified and subsequently analyzed by DNA sequencing. Comparison of the resultant sequences in 1281 affected subjects with the Cambridge consensus sequence identified 149 (102 known and 47 novel) nucleotide changes in the ND4 gene,21 as shown in Table 2 and Supplementary Table S1. These variants included 32 (14 novel and 18 known) missense mutations and 117 synonymous mutations. The known missense mutations included the m.11778G>A mutation as well as putative LHON-associated m.11253T>C and m.11696G>A variants.30,31 All the nucleotide changes were verified by sequence analysis of both strands and appeared to be homoplasmic. In the mutational screening, 481 individuals carried one of the known LHON-associated ND4 mutations: 2 subjects with the m.11253T>C mutation, 438 individuals carrying the m.11778G>A mutation, 25 patients harboring the m.11696G>A mutation, 15 subjects carrying both m.11696G>A and m.11778G>A mutations, and 1 subject carrying both m.11696G>A and m.14484T>C mutations. This translated to a frequency of 37.55% for these known LHON-associated mtDNA mutations (34.19% in only m.11778G>A mutation, 1.17% in both m.11696G>A and m.11778G>A mutations, 0.08% in both m.11696G>A and m.14484T>C mutations, 1.95% in only m.11696G>A variant, and 0.16% in m.11253T>C) in this cohort. 
Table 2
 
Missense Mutations in the ND4 Gene in 1281 Chinese Subjects With LHON
Table 2
 
Missense Mutations in the ND4 Gene in 1281 Chinese Subjects With LHON
Evaluation of Missense Variants inND4Gene
These 32 missense variants in the ND4 gene were first evaluated by the phylogenetic analysis of these variants and amino acid sequences from other 16 vertebrates. The CI among these residues ranged from 5.88% to 100%, as shown in Table 2. Of these, the CI of 10 variants, including 5 novel variants (m.10934G>A, m.11096A>G, m.11213T>G, m.11861G>T, and m.11430C>G), was >75%, with potential functional significance.32 However, the CI of 22 other variants, including nine novel variants (10908T>C, 10913T>G, 10928T>C, 11223T>G, 11373T>G, 11501C>T, 11522A>G, 11792T>G, and 11906G>A), was <75%. These variants were then evaluated by examining the allelic frequency in a cohort of 478 Han Chinese controls. As shown in Table 2, twenty-four variants were absent in 478 controls, while the frequencies of nine variants ranged from 0.29% to 3.14% in this control population. In addition to the known LHON-associated m.11253T>C, m.11778G>A, and m.11696G>A mutations, seven missense mutations (two known [11204T>C, 11447G>A] and five novel (10934G>A, 11096A>G, 11213T>G, 11861G>T, and 11430C>G), which were absent in 478 controls and whose CIs were >75%, were the putative LHON-associated mutations. On the other hand, 22 other missense variants, which were present in the controls or had lower CIs, appeared to be polymorphisms. 
Furthermore, we analyzed the structural alteration of ND4 by three known and seven putative mutation variants on the basis of the predicated secondary structure. As shown in Figure 1, ND4 polypeptide consists of transmembrane, outer membrane, and intermembrane domains. Five variants (11096A>G [p.113T>A], 11253T>C [p.165I>T], 11447G>A [p.230V>M], 11696G>A [p.313V>I], and 11861G>T [p.368A>S]) localized at the transmembrane domain, and five variants (10934G>A [p.59D>N], 11204T>C [p.149F>L], 11213T>G [p.152Y>D], 11430C>G [p.224P>R], and 11778G>A [p.340R>H]) resided at the intermembrane domain. 
Figure 1
 
Locations in the secondary structure of ND4 polypeptide. The secondary structure was predicted by submitting the amino acid sequence to an online software (available in the public domain at http://bp.nuap.nagoya-u.ac.jp). Sites of 10 mutations in the domains were indicated by arrows.
Figure 1
 
Locations in the secondary structure of ND4 polypeptide. The secondary structure was predicted by submitting the amino acid sequence to an online software (available in the public domain at http://bp.nuap.nagoya-u.ac.jp). Sites of 10 mutations in the domains were indicated by arrows.
Characterization of 10 Chinese Probands Carrying the PutativeND4Mutations
Comprehensive medical histories of 10 probands carrying one of seven putative ND4 mutations and other members in these families showed no other clinical abnormalities, including diabetes, muscular diseases, hearing loss, and neurologic disorders. As shown in Table 3, 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 had optic neuropathy, while two pedigrees (WZ1233 and WZ1165) had a history of optic neuropathy. 
Table 3
 
Summary of the Clinical and Molecular Data for 10 Han Chinese Probands Carrying One of the Putative ND4 Mutations
Table 3
 
Summary of the Clinical and Molecular Data for 10 Han Chinese Probands Carrying One of the Putative ND4 Mutations
Figure 2
 
Ten Han Chinese pedigrees with LHON. Vision-impaired individuals were indicated by filled symbols. Arrowhead denotes probands.
Figure 2
 
Ten Han Chinese pedigrees with LHON. Vision-impaired individuals were indicated by filled symbols. Arrowhead denotes probands.
The putative mutation(s) was(were) first examined in all available members of these pedigrees. The mtDNA mutation(s) was(were) presented in matrilineal relatives in each family in the homoplasmic form, but not in other members of these families (data not shown). Then, we performed the mutational screening of three known LHON-associated mtDNA mutations in these probands. As shown in Table 3, the pedigree WZ410 carried both m.11778G>A and m.11096A>G mutations, the pedigrees WZ511 and WZ987 harbored both m.14484T>C and m.11204T>C or m.11861G>T mutations, while seven pedigrees (WZ1183, WZ317, WZ1138, WZ1016, WZ1233, WZ1165, and WZ1168) lacked three primary mtDNA mutations. 
To assess if mtDNA variants affect the phenotypic variability of optic neuropathy in these Chinese pedigrees, we analyzed entire mtDNA sequences in 10 probands. As shown in Supplementary Table S2, these probands exhibited distinct sets of mtDNA polymorphisms including 151 known and 26 novel variants, belonging to the Eastern Asian haplogroups H2, B4a2, B4, H2, R, M8, R, F1a'c, R, and N. These variants in RNAs and polypeptides were further evaluated by phylogenetic analysis of these variants and sequences from other 16 vertebrates. Of these, the ND2 4677C>G (p.70L>V) and 4683A>G (p.72M>V) variants at the pedigree WZ1183, and 4699A>G (p.77N>S), 4722A>G (p.85T>A), 4828A>T (p.120Q>L), 4870A>G (p.134Q>R), 4987C>T (p.173T>I), 4998A>C (p.177K>Q), 5007G>T (p.180A>S), 5200T>G (p.244I>S), 5304C>T (p.279P>A) variants in the ND2 gene and tRNAThr 15949G>A variant at the pedigree WZ1165 showed a evolutionary conservation in these species and absence in 478 Chinese controls. However, none of other variants showed evolutionary conservation or presence of controls. 
Enzymatic Assays for Cells Carrying the Putative mtDNA Mutations
To investigate the effect of the seven putative LHON-associated ND4 mutations on oxidative phosphorylation, we measured the activities of respiratory complexes by isolating mitochondria from nine mutant cell lines carrying m.11778G>A, m.14484T>C, one of seven putative mtDNA mutations, and three control cell lines lacking these mtDNA mutations. Complex I (NADH ubiquinone oxidoreductase) activity was determined by following the oxidation of NADH with ubiquinone as the electron acceptor.27 Complex III (ubiquinone–cytochrome c oxidoreductase) activity was measured as reduction of cytochrome c (III) by using D-ubiquinol-2 as the electron donor. The activity of complex IV (cytochrome c oxidase) was monitored by following the oxidation of cytochrome c (II). As shown in Figure 3, the activities of complex I in mutant cell lines derived from subjects WZ4-IV-2 carrying only m.11778G>A mutation, WZ6-III-1 harboring only m.14484T>C mutation, WZ1183-IV-1 harboring m.10934G>A mutation, WZ317-III-3 with 11204T>C mutation, WZ1165-IV-1 harboring 11430C>G mutation, WZ1168-III-3 carrying 11447G>A mutation, WZ1223-IV-1 containing m.11213T>G mutation, WZ410-III-1 carrying both 11778G>A and m.11096A>G mutations, and WZ987-III-2 carrying both 14484T>C and m.11861G>T mutations were 65%, 71%, 68%, 71%, 65%, 76%, 65%, 66%, and 70%, respectively, relative to the mean value measured in the control cell lines. The absence of significant differences between cell lines carrying only m.11778G>A and both m.11778G>A and m.11096A>G mutations or cells harboring only m.14484T>C mutation and m.14484T>C and m.11861G>T mutations suggested that m.11096A>G and m.11861G>T mutations may not be deleterious mutations. However, the activities of complex II, III, and IV in nine mutant cell lines were comparable with those of three control cell lines (Supplementary Fig. S1). 
Figure 3
 
Enzymatic activity of complex I. The activities of complex I were assayed as detailed in Subjects and Methods in isolated mitochondria derived from lymphoblastoid cell lines. These cell lines were derived from subjects WZ4-IV-2 carrying only m.11778G>A mutation, WZ6-III-1 harboring only m.14484T>C mutation, and seven probands (WZ1183-IV-1 harboring m.10934G>A mutation, WZ410-III-1 carrying both m.11096A>G and m.11778G>A mutations, WZ317-III-3 with m.11204T>C mutation, WZ1165-IV-1 harboring m.11430C>G mutation, WZ1168-III-3 carrying m.11447G>A mutation, WZ1233-IV-1 containing m.11213T>G mutation, and WZ987-III-2 carrying both m.11861G>T and m.14484T>C mutations) and three genetically unrelated control individuals (A40, A41, and A42). The values for the mutant cell lines are expressed as percentages of the average values for the control cell lines. The calculations were based on three to five independent determinations in each cell line. The error bars indicate two standard errors of the mean. P indicates the significance, according to the Student's t-test, of the differences between mean of mutant cell lines and mean of control cell lines.
Figure 3
 
Enzymatic activity of complex I. The activities of complex I were assayed as detailed in Subjects and Methods in isolated mitochondria derived from lymphoblastoid cell lines. These cell lines were derived from subjects WZ4-IV-2 carrying only m.11778G>A mutation, WZ6-III-1 harboring only m.14484T>C mutation, and seven probands (WZ1183-IV-1 harboring m.10934G>A mutation, WZ410-III-1 carrying both m.11096A>G and m.11778G>A mutations, WZ317-III-3 with m.11204T>C mutation, WZ1165-IV-1 harboring m.11430C>G mutation, WZ1168-III-3 carrying m.11447G>A mutation, WZ1233-IV-1 containing m.11213T>G mutation, and WZ987-III-2 carrying both m.11861G>T and m.14484T>C mutations) and three genetically unrelated control individuals (A40, A41, and A42). The values for the mutant cell lines are expressed as percentages of the average values for the control cell lines. The calculations were based on three to five independent determinations in each cell line. The error bars indicate two standard errors of the mean. P indicates the significance, according to the Student's t-test, of the differences between mean of mutant cell lines and mean of control cell lines.
Assessment of 41 Chinese Probands Carrying the m.11696G>A Mutation
A total of 41 Chinese probands carrying the m.11696G>A mutation consisted of 11 females and 30 males. Twenty-five subjects carried only the m.11696G>A mutation, while 16 patients harbored the m.11696G>A and one additional mutation(s) (15 subjects carrying the m.11778G>A mutation, 1 individual harboring the m.14484T>C mutation).3336 As shown in Table 4, four individuals had profound visual impairment, 6 subjects exhibited severe visual impairment, 15 patients had moderate visual impairment, and 16 subjects exhibited mild visual impairment. The age at onset of visual loss in these subjects ranged from 4 to 46 years, with an average of 19.4 years. Of these, 28 probands (23 carrying only the m.11696G>A mutation, 5 carrying both m.11696G>A and m.11778G>A mutations) did not have a history of optic neuropathy, while 13 subjects (2 carrying only the m.11696G>A mutation, 10 individuals carrying both m.11696G>A and m.11778G>A mutations, and 1 patient carrying both m.11696G>A and m.14484T>C mutations) had a family history of visual loss. In previous investigations,3336 we have performed the sequence analysis of entire mtDNA in seven probands carrying the m.11696G>A mutation. For defining the mtDNA haplogroups, we performed the sequence analysis of the PCR-amplified fragments spanning the D-loop region from 34 probands carrying the m.11696G>A mutation. As shown in Supplementary Table S3, 35 variants were identified in the D-loop region. These included the haplogroup D–specific variants 489T>C, 16223C>T, 16362T>C, and 16169G>A; haplogroup M–specific variants 489T>C and 16362T>C; and haplogroup B–specific variants 16183A>C, 16189T>C, and 16217T>C.28,29 As shown in Table 5, the frequencies of mtDNA haplogroups B, D, M, and M8 in 41 LHON families carrying the m.11696G>A mutation were 2.44%, 90.24%, 2.44%, and 4.88%, respectively. 
Table 4
 
Summary of the Clinical and Molecular Data for 41 Han Chinese Probands Carrying the m.11696G>A Variant
Table 4
 
Summary of the Clinical and Molecular Data for 41 Han Chinese Probands Carrying the m.11696G>A Variant
Table 5
 
Mitochondrial DNA Haplogroups From 169 Han Chinese Probands Carrying ND4 Mutation(s) and 478 Han Chinese Control Subjects
Table 5
 
Mitochondrial DNA Haplogroups From 169 Han Chinese Probands Carrying ND4 Mutation(s) and 478 Han Chinese Control Subjects
Analysis of mtDNA in 120 Probands Carrying the m.11778G>A Mutation
As shown in Supplementary Table S4, 120 Chinese probands carrying the m.11778G>A mutation consisted of 12 females and 108 males. The age at onset of visual loss in these subjects ranged from 6 to 48 years, with an average of 19.5 years. Previous investigations23,3644 have examined the entire mtDNA sequences of 44 subjects carrying the m.11778G>A mutation. In the present investigation, we performed the sequence analysis of the PCR-amplified fragments spanning the D-loop region from an additional 76 probands carrying the m.11778G>A mutation. As shown in Supplementary Table S5, 135 (126 known and 9 novel) variants were identified in the D-loop region. From these data, mtDNAs carrying the m.11778G>A mutation were assigned to haplogroups.24,25 As shown in Table 5, the mtDNAs from 120 Chinese probands carrying the m.11778G>A mutation were distributed among 12 different haplogroups. The frequencies of mtDNA haplogroups A, B, C, N, H, D, M, M7, M8, M9, M10, and M11 in 120 probands carrying the m.11778G>A mutation were 6.67%, 11.67%, 4.17%, 2.50%, 8.33%, 28.33%, 5.0%, 6.67%, 8.33%, 3.33%, 14.17%, and 0.83%, respectively, while those of 478 Chinese controls were 7.11%, 16.53%, 5.86%, 2.51%, 2.72%, 26.78%, 5.23%, 8.16%, 1.88%, 4.39, 0.84%, and 0.42%, respectively. 
Discussion
Incidence and Genetic Feature of the m.11778G>A Mutation
In the present study, mutational analysis of the ND4 gene identified 453 probands carrying the m.11778G>A mutation in a cohort of 1281 Chinese subjects with LHON. Of these, 292 probands had a family history of LHON, while 161 subjects were sporadic cases. This was in contrast with the rare occurrence of sporadic cases carrying the primary LHON mutations in the Caucasian population.1,6 The 35.36% prevalence of m.11778G>A mutation in this cohort was comparable to 34.55% in another cohort of 903 Han Chinese patients with LHON.13 In other Asian populations, the incidence of the m.11778G>A mutation was 73.35% in 80 Japanese probands, 56% in 82 Korean patients, and 8.9% in 90 Indian subjects.11,4548 However, the incidence of the m.11778G>A mutation varied from 36% to 77% in several European cohorts.1,9,4850 The discrepancy in the prevalence of m.11778G>A mutation between Asian and Caucasian origins may be attributed to evolution.51 Alternatively, this difference may be because a proportion of the 1281 cases recruited from 29 provinces across China may have other inherited or acquired optic neuropathies. 
The Prevalence and Pathogenicity of m.11696G>A Mutation
The incidence of the m.11696G>A mutation was 3.2% in this Chinese cohort. Of these, 23 patients carrying only the m.11696G>A mutation and 5 subjects harboring both m.11696G>A and one of the primary mtDNA mutations did not have a family history of optic neuropathy, while 2 probands carrying only the m.11696G>A mutation and 11 subjects carrying both m.11696G>A and one of the primary mtDNA mutations had a family history of visual loss. To assess if the differences in the penetrance of visual loss differ by the presence and absence of m.11696G>A mutation, a statistical analysis was performed with the unpaired, two-tailed Student's t-test contained in Microsoft Excel. The penetrance of visual loss among Chinese families carrying both the m.11696G>A and m.11778G>A mutations was significantly higher than that of pedigrees carrying the single mtDNA mutation(s) (P < 0.001 and P < 0.0001). This provides additional evidence that the m.11696G>A mutation is itself not sufficient to produce the clinical phenotype but may play a synergistic role in modulating the phenotypic manifestation of the primary LHON-associated mtDNA mutations.31,35,36 Furthermore, additional mtDNA variant(s) or a nuclear modifier gene may contribute to the phenotypic manifestation of 25 patients carrying only the m.11696G>A mutation, as in the case of m.11778G>A mutation.23,52,53 
Prevalence and Pathogenicity of Other Putative LHON-AssociatedND4Mutations
In the present study, we identified five putative LHON-associated mtDNA mutations. In contrast with m.11778G>A mutation, these mutations occurred at very low frequency in the Chinese cohort. In particular, the incidence of m.10934G>A, m.11204T>C, m.11213T>G, m.11447G>A, and m.11430C>G mutations was 0.078%, 0.312%, 0.078%, 0.078%, and 0.078%, respectively, in this cohort. These mutations were present alone among seven probands. In fact, pedigree analysis showed that two probands carrying either m.11213T>G or m.11430C>G mutation had a family history of visual loss, and other five probands carrying one of the putative mutations did not have a family history of optic neuropathy. 
Furthermore, these mutations may alter the structure and function of ND4 polypeptide. In fact, the 11447G>A (p.230V>M) mutation localized at the transmembrane domain, while 10934G>A (p.59D>N), 11204T>C (p.149F>L), 11213T>G (p.152Y>D), and 11430C>G (p.224P>R) resided at the intermembrane domain, which interacts with other subunits of complex I.54,55 These putative ND4 mutations, similar to the m.11778G>A mutation, may affect the stability of this polypeptide. In the present investigation, five mutant cell lines carrying only one of five putative ND4 mutations (m.10934G>A, 11204T>C, 11430C>G, 11447G>A, and 11213T>G) showed mild decrease in the activity of complex I, ranging from 24% to 35%. These data are in good agreement with the observation that there was 27% to 40% reduction in NADH dehydrogenase–dependent respiration in cell lines carrying the LHON-associated m.11778G>A, m.T14484T>C, m.3866T>C, or m.3635G>A mutations.27,5658 However, the reduced levels of complex I activity in mutant cell lines carrying both m.11778G>A and m.11096A>G mutations or both m.14484T>A and m.11861G>T mutations were comparable to those in cell lines carrying the m.11778G>A or m.14484T>C mutation alone. These data suggest that the m.10934G>A, 11204T>C, 11430C>G, 11447G>A, and 11213T>G mutations may be the putative LHON-associated mutations, and m.11096A>G and 11861G>T mtDNA mutations may not be deleterious mutations. These observations indicate that the primary defect in these five ND4 mutations was reduced activity of NADH dehydrogenase. This may lead to deficient function of oxidative phosphorylation, decrease in ATP synthesis, and increase in reactive oxygen species, as in the case of cell lines carrying the m.11778G>A, m.3635G>A, or 3866T>C mutation.27,58,59 The low penetrance of optic neuropathy and mild biochemical defects in these Chinese pedigrees carrying only one putative LHON mutation suggest that the mutation(s) is (are) necessary but is (are) itself (themselves) insufficient to produce a clinical phenotype. Therefore, the nuclear modifiers, or environmental factors, should play a role in the phenotypic manifestation of these mtDNA mutations. 
The Phylogenetic and Haplogroup Analysis of Probands Carrying theND4Mutations
In the present investigation, mtDNAs in 120 LHON probands carrying the m.11778G>A mutation were widely dispersed among 12 Eastern Asian subhaplogroups, while mtDNAs of European pedigrees carrying this mutation belonged to the European haplogroups H, I, J, K, T, U, V, and L.48,49,60 Notably, the frequencies of mtDNA haplogroups D, M8, M10, M11, and H in these Chinese probands harboring the m.11778G>A mutation were higher than those in the Chinese control population.28,29 Moreover, the occurrence of mtDNAs in haplogroups D and M8 in 41 Chinese families harboring the m.11696G>A mutation and haplogroups M8, H, and R in eight pedigrees carrying one of five putative mutations was markedly higher than that in this cohort of Chinese controls and other cohorts.28,29 Thus, the frequencies of haplogroups D, M8, M10, M11, and H in the LHON probands carrying the ND4 mutations were significantly higher than those in 478 Chinese controls and other Asian populations.28,29,61 This discrepancy implicates a role of mtDNA haplotypes in the phenotypic manifestation of LHON-associated mtDNA mutations.34,52,60,61,62 
In summary, this is the first comprehensive study to investigate the mutational spectrum and incidence in ND4 gene in a larger cohort of Chinese subjects with LHON. In addition to the known m.11778G>A, m.11696G>A, and m.11253T>C mutations, we identified five putative LHON-associated mtDNA mutations. A total of 488 subjects carrying one of the ND4 pathogenic mutations accounted for 38.10% of cases among 1281 Han Chinese subjects with LHON. These results suggest that the ND4 gene is the hot spot for mutations associated with LHON. Thus, our findings may provide valuable information for the further understanding of pathogenic mechanism of LHON. 
Acknowledgments
Supported by the National Key Technologies R&D Program Grant 2012BAI09B03 from the Ministry of Science and Technology of China (MXG and PJ) and Grants 81200724, 31471191, and 81400434 from the National Nature Science Foundation of China (MXG, JZ, YJ), and the Ministry of Education of Zhejiang Provincial Program Y201017001 and the Ministry of Science and Technology of Wenzhou City Y20100272 (ML). 
Disclosure: P. Jiang, None; M. Liang, None; J. Zhang, None; Y. Gao, None; Z. He, None; H. Yu, None; F. Zhao, None; Y. Ji, None; X. Liu, None; M. Zhang, None; Q. Fu, None; Y. Tong, None; Y. Sun, None; X. Zhou, None; T. Huang, None; J. Qu, None; M.-X. Guan, None 
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Figure 1
 
Locations in the secondary structure of ND4 polypeptide. The secondary structure was predicted by submitting the amino acid sequence to an online software (available in the public domain at http://bp.nuap.nagoya-u.ac.jp). Sites of 10 mutations in the domains were indicated by arrows.
Figure 1
 
Locations in the secondary structure of ND4 polypeptide. The secondary structure was predicted by submitting the amino acid sequence to an online software (available in the public domain at http://bp.nuap.nagoya-u.ac.jp). Sites of 10 mutations in the domains were indicated by arrows.
Figure 2
 
Ten Han Chinese pedigrees with LHON. Vision-impaired individuals were indicated by filled symbols. Arrowhead denotes probands.
Figure 2
 
Ten Han Chinese pedigrees with LHON. Vision-impaired individuals were indicated by filled symbols. Arrowhead denotes probands.
Figure 3
 
Enzymatic activity of complex I. The activities of complex I were assayed as detailed in Subjects and Methods in isolated mitochondria derived from lymphoblastoid cell lines. These cell lines were derived from subjects WZ4-IV-2 carrying only m.11778G>A mutation, WZ6-III-1 harboring only m.14484T>C mutation, and seven probands (WZ1183-IV-1 harboring m.10934G>A mutation, WZ410-III-1 carrying both m.11096A>G and m.11778G>A mutations, WZ317-III-3 with m.11204T>C mutation, WZ1165-IV-1 harboring m.11430C>G mutation, WZ1168-III-3 carrying m.11447G>A mutation, WZ1233-IV-1 containing m.11213T>G mutation, and WZ987-III-2 carrying both m.11861G>T and m.14484T>C mutations) and three genetically unrelated control individuals (A40, A41, and A42). The values for the mutant cell lines are expressed as percentages of the average values for the control cell lines. The calculations were based on three to five independent determinations in each cell line. The error bars indicate two standard errors of the mean. P indicates the significance, according to the Student's t-test, of the differences between mean of mutant cell lines and mean of control cell lines.
Figure 3
 
Enzymatic activity of complex I. The activities of complex I were assayed as detailed in Subjects and Methods in isolated mitochondria derived from lymphoblastoid cell lines. These cell lines were derived from subjects WZ4-IV-2 carrying only m.11778G>A mutation, WZ6-III-1 harboring only m.14484T>C mutation, and seven probands (WZ1183-IV-1 harboring m.10934G>A mutation, WZ410-III-1 carrying both m.11096A>G and m.11778G>A mutations, WZ317-III-3 with m.11204T>C mutation, WZ1165-IV-1 harboring m.11430C>G mutation, WZ1168-III-3 carrying m.11447G>A mutation, WZ1233-IV-1 containing m.11213T>G mutation, and WZ987-III-2 carrying both m.11861G>T and m.14484T>C mutations) and three genetically unrelated control individuals (A40, A41, and A42). The values for the mutant cell lines are expressed as percentages of the average values for the control cell lines. The calculations were based on three to five independent determinations in each cell line. The error bars indicate two standard errors of the mean. P indicates the significance, according to the Student's t-test, of the differences between mean of mutant cell lines and mean of control cell lines.
Table 1
 
Sex and Age Distributions for the 1281 Chinese Probands With LHON and 478 Control Subjects
Table 1
 
Sex and Age Distributions for the 1281 Chinese Probands With LHON and 478 Control Subjects
Table 2
 
Missense Mutations in the ND4 Gene in 1281 Chinese Subjects With LHON
Table 2
 
Missense Mutations in the ND4 Gene in 1281 Chinese Subjects With LHON
Table 3
 
Summary of the Clinical and Molecular Data for 10 Han Chinese Probands Carrying One of the Putative ND4 Mutations
Table 3
 
Summary of the Clinical and Molecular Data for 10 Han Chinese Probands Carrying One of the Putative ND4 Mutations
Table 4
 
Summary of the Clinical and Molecular Data for 41 Han Chinese Probands Carrying the m.11696G>A Variant
Table 4
 
Summary of the Clinical and Molecular Data for 41 Han Chinese Probands Carrying the m.11696G>A Variant
Table 5
 
Mitochondrial DNA Haplogroups From 169 Han Chinese Probands Carrying ND4 Mutation(s) and 478 Han Chinese Control Subjects
Table 5
 
Mitochondrial DNA Haplogroups From 169 Han Chinese Probands Carrying ND4 Mutation(s) and 478 Han Chinese Control Subjects
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