Mitochondrial ND1 Variants in 1281 Chinese Subjects With Leber's Hereditary Optic Neuropathy

Abstract

Purpose: The purpose of this study was to investigate the mutational incidence and spectrum of mitochondrial ND1 gene in subjects with Leber's hereditary optic neuropathy (LHON).

Methods: A cohort of 1281 Han Chinese probands and 478 control subjects underwent sequence analysis of mitochondrial (mt)DNA. Resultant variants were evaluated for evolutionary conservation, allelic frequencies, and structural and functional consequences. Respiratory complex activities were measured using lymphoblastoid cell lines derived from 25 probands carrying the mtDNA mutation and 3 controls.

Results: Mutational analysis identified 178 (70 missense and 108 silent) variants in the MT-ND1 gene. The incidences of known m.3460G>A, m.3635G>A, m.3733G>A, m.3866T>C, and m.3394T>C mutations were 1.33%, 0.86%, 0.08%, 0.55%, and 2.97%, respectively. Fifteen novel putative mutations were identified in 27 probands, translated into 2.1% cases of this cohort. The activity of complex I in mutant cell lines carrying one of putative mutations ranged from 66% to 76% of the average values in control cell lines, whereas activities of complexes II, III, and IV in mutant cells were comparable with those in controls. The low penetrances of optic neuropathy were observed in pedigrees carrying novel putative mutation(s). Moreover, mtDNAs in 101 probands carrying the MT-ND1 mutation(s) were widely dispersed among 15 Eastern Asian haplogroups. In particular, the occurrences of haplogroups M, M9, and M10 in patients carrying the ND1 mutations were higher than those in controls.

Conclusions: These data demonstrated that the MT-ND1 gene is a hot spot for mutations associated with LHON. Our findings may provide valuable information for pathophysiology, management, and genetic counseling of LHON.

Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disorder leading to severe visual impairment or even blindness by death of retinal ganglion cells, affecting children through adults.^1–5 In the majority of cases worldwide, LHON is due to one of three point mutations in the mitochondrial DNA (mtDNA) encoding three subunits of respiratory chain complex I (Nicotinamide adenine dinucleotide [NADH] dehydrogenase): m.3460G>A(MT-ND1), m.11778G>A(MT-ND4), and m.14484T>C(MT-ND6).^5–10 These LHON-associated mtDNA mutations occurred in heteroplasmy (mixture of wild-type and mutated molecules) or homoplasmy (only mutated molecules). These three mutations account for approximately 90% of LHON pedigrees in some countries, whereas these mutations are only responsible for 38.3% and 46.5% cases in two large cohorts of Chinese Han subjects with LHON.^11,12 In particular, the incidences of the m.3460G>A mutation in two large cohorts of Chinese Han subjects with LHON and 159 Caucasian pedigrees with LHON were 0.44%, 2.11%, and 13%, respectively,^10–12 suggesting the variable incidence and spectrum of mtDNA mutations among the different ethnic origins.^12–14 The other LHON-associated mutations in the MT-ND1 gene included the m.3866T>C, m.3635G>A, and m.3733G>A mutations.^15–17 Thus, the MT-ND1 gene is proposed to be another hot spot for mutations associated with LHON.¹⁸ However, the mutational spectrum and incidence in the MT-ND1 gene are poorly understood in the LHON patients. Furthermore, it is anticipated that additional mutations can be found in the MT-ND1 gene. 

To further understand the pathophysiology of LHON, we performed the clinical and genetic analysis of 1281 Han Chinese probands (patient or member of the family that brings a family under study) with LHON.^7,19,20 In this cohort, 503 probands had a family history of optic neuropathy, and 778 subjects were sporadic cases.⁷ Mutational analysis showed that mutations in MT-ND4 and MT-ND6 genes are responsible for 39.5% and 9.3% cases of LHON subjects in this large cohort, respectively.^7,19 In the present study, we carried out a systematic and extended mutational screening of the MT-ND1 gene in a cohort of 1281 genetically unrelated Han Chinese subjects with LHON. This analysis identified 178 nucleotide changes, including the known m.3460G>A, 3866T>C, m.3733G>A, and m.3635G>A mutations. These MT-ND1 variants have been evaluated by phylogenetic analysis, structure–function relation, and allelic frequency of these variants in 478 Han Chinese controls from the same region. Furthermore, we performed the clinical and genetic evaluation and mtDNA analysis of 36 probands carrying the known primary m.3460G>A, m.3635G>A, m.3866T>C and, m.3733G>A mutations, 38 subjects harboring the secondary m.3394T>C mutation, and 39 subjects carrying 1 of 23 putative LHON-associated MT-ND1 mutations. Functional significance of 23 putative LHON-associated mutations was further assessed for the enzymatic activities of electron transport chain complexes in cell lines derived from probands carrying one of these mtDNA putative mutations and three control subjects lacking these mutations. Moreover, these mtDNAs from subjects carrying the known and putative mutations were assigned to the Asian mtDNA haplogroups using the nomenclature of mtDNA haplogroups.²¹ 

Subjects and Methods

Subjects and Ophthalmologic Examinations

A total of 1281 genetically unrelated Han Chinese subjects with LHON were recruited from the eye clinics across the China, as described previously.^7,19 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 Ethic Committees of Zhejiang University and the Wenzhou Medical University. 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 ophthalmic examinations of probands and other members of these families were conducted as detailed previously.^7,19 A total of 478 Han Chinese control subjects used for screening for the presence of variants in the MT-ND1 gene were obtained from same regions. 

Mutational Analysis of Mitochondrial Genomes

Genomic DNA was isolated from whole blood of 1281 probands and 478 Chinese control subjects using QIAamp DNA Blood Mini Kit (No.51104; Qiagen, Suzhou, China). Subject's DNA fragments spanning the MT-ND1 gene were amplified by PCR using oligodeoxynucleotides corresponding to mtDNA at positions 3307–4262.²² Each fragment was purified and subsequently analyzed by direct sequencing as described elsewhere. These sequence results were compared with the updated consensus Cambridge sequence (GenBank accession number: NC_012920).²³ The homoplasmy of the m.3460G>A, m.3635G>A, m.3733G>A, and m.3866T>C mutations in these subjects was determined as detailed previously.^{15–17,19,24,25} The allelic frequencies in the MT-ND1 gene in 478 Chinese control subjects were 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 15,811 to 775, from 28 probands carrying the m.3394T>C mutation. The entire mitochondrial genomes of 8 probands carrying the known m.3460G>A, m.3635G>A, m.3733G>A, and m.3866T>C mutations and 37 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.²² These sequence results were compared with the updated consensus Cambridge sequence, as described above. 

Cell Cultures

Lymphoblastoid cell lines were immortalized by transformation with the Epstein-Barr virus, as described elsewhere.²⁶ Cell lines derived from 25 probands (1 carrying only m.11778G>A mutation,²⁷ 1 harboring only m.14484T>C,²⁸ and 23 subjects carrying one putative mutation and from three genetically unrelated control individuals (LC1, LC2, and LC3) were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum. 

Enzymatic Assays

The enzymatic activities were assayed following the modified protocol by Birch-Machin and Turnbull.^16,29,30 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 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 in the addition of 2 mM of 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–IV activities were normalized by citrate synthase activity. 

Phylogenetic Analysis

For interspecific analysis of these mitochondrial DNA variants identified, a total of 17 mitochondrial DNA sequences were used as detailed elsewhere.^7,19 The conservation index (CI) was calculated by comparison of the human mitochondrial DNA variants with the other 16 species. 

Haplogroup Analyses

The mtDNA sequences of the 37 subjects carrying the putative mtDNA mutations(s) and 74 Chinese probands carrying one of the known mutations (m.3460G>A, m.3394T>C, m.3635G>A, m.3733G>A, m.3866T>C) were assigned to the Asian mitochondrial haplogroups using the nomenclature of mitochondrial haplogroups or using online software (provided the whole mtDNA mutation at http://www.mitotool.org/genomeRSRS.html).^21,31 

Computer Analysis

Statistical analysis was carried out by using the unpaired, 2-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

Mutational Analysis of the Mitochondrial ND1 Gene

The study samples with LHON consisted of 963 males and 318 females, who were Han Chinese recruited from Eye Clinics across 29 Provinces in China.^7,19 DNA fragments spanning the whole ND1 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 these affected subjects with the Cambridge consensus sequence identified 178 (61 novel and 117 known) nucleotide changes in the MT-ND1 gene, as shown in Table 1 and Supplementary Table S1. These variants included 70 (31 novel and 39 known) missense mutations and 108 silent variants. The known primary mutations were the m.3460G>A (17 subjects), m.3635G>A (11 patients), m.3866T>C (7 individuals), and m.3733G>A (1 proband).^15–18,32 Furthermore, 38 probands harbored the putative known m.3394T>C mutation.^33,34 All the nucleotide changes were verified by sequence analysis of both strands and appeared to be homoplasmy. This translated to a frequency of 5.78% for these known LHON-associated mtDNA mutations (1.33% in m.3460G>A, 2.97% in m.3394T>C, 0.86% in m.3635G>A, 0.55% in m.3866T>C, and 0.08% in m.3733G>A) in this cohort. 

Table 1

 

Missense Mutations in the MT-ND1 Gene in 1281 Chinese Subjects With LHON

 

Evaluation of the MT-ND1 Missence Variants

These 70 missense variants in the MT-ND1 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 1. Of these, CIs of 37 variants including 17 novel variants were >75%, with potential functional significance.³⁵ However, CIs of other 33 variants including 11 novel variants were <75%. The allelic frequencies of these variants were then examined in 478 Han Chinese controls. As shown in Table 1, 46 variants were absent in 478 Chinese controls, whereas the frequencies of 24 variants ranged from 0.21% to 2.72% in this control population. In addition to the known LHON-associated m.3394T>C, m.3460G>A, m.3635G>A, m.3733G>A, and m.3866T>C mutations, 23 missense (7 known and 16 novel) mutations, which were absent in 478 Chinese controls and whose CIs were >75%, were the putative LHON-associated mutations. On the other hand, 43 other missense variants, which were present in the controls or lower CIs, appeared to be the polymorphisms. 

Furthermore, we analyzed the structural alteration of mitochondrial ND1 by m.3394T>C, m.3460G>A, m.3635G>A, m.3733G>A, and m.3866T>C mutations, which are 23 putative missense mutations on the basis of the predicated secondary structure. As shown in Figure 1, the ND1 polypeptide consists of transmembrane, outer membrane, and intermembrane domains. Sixteen variants localized at the transmembrane domain, 11 variants localized at the outer membrane, and 1 variant resided at the intermembrane domain. 

Figure 1

 

Locations of the putative LHON-associated mutations in the secondary structure of ND1 polypeptide. The secondary structure was predicted by submitting the amino acid sequence to online software (available in the public domain at http://bp.nuap.nagoya-u.ac.jp). Arrows indicated the sites of 28 mutations in the domains.

Characterization of 37 Chinese Probands Carrying the Putative MT-ND1 Mutations

Comprehensive medical histories of 37 probands carrying 1 of 23 putative MT-ND1 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 2 and Figure 2, these families exhibited a wide range of severity, age at onset, and penetrance of optic neuropathy. Twenty-two probands did not have a history of optic neuropathy, whereas at least matrilineal relatives of each family in 22 pedigrees suffered from optic neuropathy. 

Table 2

 

Summary of the Clinical and Molecular Data for 37 Han Chinese Probands Carrying One of the Putative ND1 Mutations

 

Figure 2

 

Thirty-seven Han Chinese pedigrees carrying one of putative MT-ND1 mutations. Vision-impaired individuals are indicated by filled symbols. Arrowhead denotes probands.

These putative mutations were first examined in all available members of these pedigrees. These mtDNA mutations were presented in matrilineal relatives in each family in the homoplasmy, but not in other members of every family (data not shown). Then, we performed the mutational screening of the known LHON associated mtDNA mutations in these probands. As showed in Table 2, 13 pedigrees carried both m.11778G>A and one putative mutation, 2 pedigrees harbored both m.14484T>C and one putative mutation, and the other 23 pedigrees lacked three primary mtDNA mutations. 

To assess the contribution that mtDNA variants make toward the phenotypic variability of optic neuropathy in these Chinese pedigrees, we analyzed entire mtDNA sequences of 37 probands. As shown in Supplementary Table S2, these probands exhibited distinct sets of mtDNA polymorphisms including 368 known and 30 novel variants, belonging to the Eastern Asian haplogroups A, B4, B5, C, D, F, G, M, M7, M10, N9, and R.^21,34 These variants in RNAs and polypeptides were further evaluated by phylogenetic analysis of these variants and sequences from other 16 vertebrates. In particular, the m.1452T>C (MT-RNR1) variant at the pedigree WZ437, the m.2690G>A (MT-RNR2) and m.8962A>G (MT-ATP6) variants at the pedigree WZ278, m.12338T>C (MT-ND5) variant at the pedigrees WZ1405, m.14502T>C (MT-ND6) variant at the pedigrees WZ422, WZ734, WZ939, and WZ1063, and m.15910G>A (MT-TT) variants at the pedigree WZ1364 showed evolutionary conservation and absence of 478 Chinese controls in these species. However, none of the other variants showed evolutionary conservation or presence of controls. 

Enzymatic Assays for Cells Carrying the Putative mtDNA Mutations

To investigate the effect of putative LHON-associated MT-ND1 mutations on the oxidative phosphorylation, we measured the activities of respiratory complexes by isolating mitochondria from 25 mutant cell lines (1 carrying only the m.11778G>A mutation, 1 harboring the m.14484T>C mutation, 15 bearing only one putative mtDNA mutation, and 8 carrying both m.11778G>A and one putative mutation) and 3 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.^7,30 Complex III (ubiquinone-cytochrome c) activity was measured as reduction of cytochrome c (III) 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 WZ4-IV-2 carrying only the m.11778G>A mutation, WZ6-III-1 harboring only the m.14484T>C mutation, and 15 probands carrying only one putative mtDNA mutation (m.3308T>C, m.3335T>C, m.3391G>A, m.3472T>C, m.3488T>C, m.3551C>T, m.3632C>T, m.3713T>C, m.3769C>G, m.3781T>C, m.3958G>A, m.4081T>C, m.4213A>T, and m.4163T>C mutations) were 63%, 69%, 73%, 70%, 76%, 69%, 73%, 76%, 66%, 76%, 71%, 68%, 69%, 70%, 73%, 68%, and 70%, relative to the mean value measured in three control cell lines (P = 0.0001–0.031), respectively. Furthermore, mutant cell lines derived from eight probands carrying both m.11778G>A and one putative mutation exhibited significant reductions in complex I compared with those in controls. However, the absence of significant differences between cell lines carrying only the m.11778G>A or the m.14484T>C mutation and mutant cell lines bearing both m.11778G>A and one of eight putative mutations suggested that these eight putative mutations may not be deleterious mutations. In addition, the activities of complex II, III, and IV in 25 mutant cell lines were comparable with those of three control cell lines. 

Figure 3

 

Respiratory complex activities. The activities of respiratory complexes were investigated by enzymatic assay on complexes I, II, III, and IV in isolated mitochondria from lymphoblastoid cell lines derived from 25 probands (1 carrying only m. 11778G>A mutation, 1 bearing only m. 14484T>C mutation, 15 harboring only one putative mutation [single mutation], 8 carrying both one putative and m.11778G>A mutations [double mutation]) and 3 control individuals (LC1, LC2, and LC3). Activities of complexes I, II, III, and IV were normalized by citrate synthase activity. The values for the mutant cell lines are expressed as percentages of the average values for three control cell lines. The calculations were based on three to five independent determinations in each cell line. Error bars denote 2 SEM. P indicates 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 38 Chinese Probands Carrying the m.3394T>C Mutation

A total of 38 Chinese probands carrying the m.3394T>C mutation consisted of 11 females and 27 males. Twenty-three subjects carried only the m.3394T>C mutation, whereas 15 patients harbored the m.3394T>C and one additional mutation(s) (11 subjects carrying the m.11778G>A mutation and 4 individuals harboring the m.14484T>C mutation).^28,33,34 As shown in Table 3, 5 individuals suffered from profound visual impairment, 11 subjects exhibited severe visual impairment, 9 patients had moderate visual impairment, and 13 subjects exhibited mild visual impairment. The age of onset of visual loss in these subjects ranged from 1 to 41 years, with an average of 19 years. Of these, 22 probands (18 carrying only the m.3394T>C mutation, 3 carrying both m.3394T>C and m.11778G>A mutations, 1 carrying both m.3394T>C and m.11778G>A mutations) did not have a history of optic neuropathy, whereas 16 subjects (4 carrying only the m.3394T>C mutations, 8 individuals carrying both m.3394T>C and m.11778G>A mutations, and 4 patients carrying both m.3394T>C and m.14484T>C mutations) had a family history of visual loss. In the previous investigation, we performed the sequence analysis of entire mtDNA in 10 probands carrying the m.3394T>C mutation.^28,33,34 For defining the mtDNA haplogroups, we performed the sequence analysis of the PCR-amplified fragments spanning the D-loop region from 28 probands carrying the m.3394T>C mutation. As shown in Supplementary Table S3, 62 variants were identified in the D-loop region. These included the haplogroup D–specific variants 489T>C, m.16223C>T, and m.16362T>C, haplogroup M9–specific variants m.153A>G, m.489T>C, and 16362T>C, and haplogroup B–specific variants m.16183A>C, m.16189T>C, and m.16217T>C.^21,31 As shown in the Table 4, the frequencies of mtDNA haplogroups B4, C, D, M, M7, M9, and R in 38 LHON families carrying the m.3394T>C mutation were 7.9%, 2.63%, 7.9%, 7.9%, 2.63%, 68.41%, and 2.63%, respectively. 

Table 3

 

Summary of the Clinical and Molecular Data for 38 Han Chinese Probands Carrying the m.3394T>C Mutation

 

Table 4

 

Summary of Clinical and Molecular Data for 36 Chinese Probands Carrying One of the Four Known MT-ND1 Mutations

 

Table 5

 

mtDNA Haplogroup From 101 Han Chinese LHON Probands Carrying MT-ND1 Mutations and 478 Control Subjects

 

Analysis of mtDNA in 36 Chinese Probands Carrying Four Known LHON-Associated MT-ND1 Mutations

As shown in Table 4, 36 Chinese probands carryied one of the known mutations: m.3460G>A (17 probands), m.3635G>A (11 patients), m.3866T>C (7 probands), and m.3733G>A (1 individual). The previous investigations examined the entire mtDNA sequences of 16 subjects carrying the m.3460G>A mutation, 9 subjects carrying the m.3635G>A mutation, and 3 subjects carrying the m.3866T>C mutation.^15,16,28 In the present investigation, we performed the sequence analysis of entire mtDNA genomes from additional eight probands carrying one of these known mutations. As shown in Supplementary Table S4, these probands exhibited distinct sets of mtDNA polymorphisms A, D4, F1a1, G2a1a, M7, and M10. In particular, three pedigrees exhibited the coexistence of both m.3866T>C and 11778G>A mutations. 

Discussion

Prevalence and Genetic Features of the Known LHON-Associated MT-ND1 Mutations

In the present study, mutational analysis of the MT-ND1 gene identified 36 probands carrying the known m.3460G>A, m.3635G>A, m.3733G>A, and m.3866T>C mutations in the cohort of 1281 Chinese subjects with LHON. Of 17 probands carrying the m.3460G>A mutation, 9 probands had a history of LHON, whereas 8 subjects were sporadic cases. Among 11 subjects bearing m.3635G>A mutations, 3 subjects did not have a family history of LHON, whereas 8 probands had a history of LHON. Of seven probands harboring the m.3866T>C mutation, three individuals did not have a history of LHON, whereas four patients exhibited a maternal transmission of LHON. In addition, the proband carrying the m.3733G>A mutation did not have a history of LHON. 

In fact, the incidence of the m.3460G>A mutation in this cohort was 1.33%, whereas those of other Asian cohorts were 0%, 0.44%, 2.11%, and 4.41%, respectively.^{9,11,12,36,37} However, the prevalence of the m.3460G>A mutation in the European origins appeared to be higher than that in Asians. Indeed, the incidences of the m.3460G>A mutation varied from 0% to 13% in several European cohorts.^1,10,38–41 In addition, the m.3733G>A mutation was a rare mutation, as in the case of European origin.^17,18 In this cohort, the m.3635G>A mutation in the Chinese population appeared to be more common than European patients.^16,42 Furthermore, the m.3866T>C mutation was presented more in Asian than in European patients with LHON.^6,15,43 Therefore, these known LHON-associated MT-ND1 mutations accounted for 2.81% cases of this cohort (1.33% in only m.3460G>A mutation, 0.86% in only m.3635G>A mutations, 0.55% in m.3866T>C mutation, and 0.08% in m.3733G>A mutation). 

Prevalence and Pathogenecity of the m.3394T>C Mutation

The incidence of the m.3394T>C mutation was 2.97% in this Chinese cohort. Of these, 22 patients (18 carrying only m.3394T>C mutation, 4 carrying both m.3394T>C, and 1 of primary mtDNA mutations) did not have a family history of optic neuropathy, whereas 16 probands (4 carrying only the m.3394T>C mutation, 12 carrying both m.3394T>C and one primary mtDNA mutation) had a family history of visual loss. To assess whether the differences in the penetrance of visual loss differ based on the presence and absence of the m.3394T>C mutation, a statistical analysis was performed by the unpaired, 2-tailed Student's t-test contained in Microsoft Excel. The penetrances of visual loss among Chinese families carrying both m.3394T>C and m.11778G>A mutations were significantly higher than those pedigrees carrying the single mtDNA mutation(s) (P < 0.001). This provides additional evidence that the m.3394T>C mutation is itself not sufficient to produce the clinical phenotype, but triggers, acting as a mitochondrial modifier, the optic neuropathy in subjects harboring m.11778G>A or m.14484T>C mutations, as in the cases of m.14502T>C or m.11696G>A mutations.^7,19,44 

Prevalence and Pathogenicity of Other Putative LHON-Associated MT-ND1 Mutations

In the present study, we identified 15 putative LHON-associated mtDNA mutations occurring in 27 probands. In contrast with the m.11778G>A mutation, these mutations appeared at very low frequency in the Chinese cohort. In particular, the incidence of these 15 putative mutations ranged from 0.08% to 0.47%. Thus, this translated into 2.1% cases of this cohort. Among these subjects, 10 probands (WZ705IV-3 with the m.3472T>C mutation, WZ546-II-3 with m.3713T>C mutation, WZ437-III-2 with m.4123A>T mutation, WZ787-III-3 with m.4163T>C mutation, and 6 subjects carrying both one putative mutation and m.11778G>A or 14484T>C mutation) had a family history of visual loss, whereas the other 17 probands carrying only one putative mutation were sporadic cases. 

Furthermore, these mutations may alter the structure and function of the ND1 polypeptide. In fact, these 15 variants localized at the transmembrane domain, outer membrane and intermembrane domain, which interacts with other subunits of complex I.^45,46 These putative mutations, similar to the m.3460G>A mutation, may affect the stability of this polypeptide. In the present investigation, 15 mutant cell lines carrying only one putative mutation(s) exhibited mild decreases in the activity of complex I, ranging from 23.7% to 37%. These data were in good agreement with the observation that there were 27%–40% reductions in NADH dehydrogenase-dependent respiration in cell lines carrying the LHON-associated m.11778G>A, m.14484T>C, m.3866T>C, or m.3635G>A mutations.^{15,16,47–50} However, 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, may play a role in the phenotypic manifestation of the mtDNA mutation(s).^50–52 

Phylogenetic and Haplogroup Analysis of Probands Carrying the MT-ND1 Mutations

Here, mtDNAs in 17 LHON probands carrying the m.3460G>A mutation were widely dispersed among 10 Eastern Asian subhaplogroups, whereas mtDNAs of European pedigrees carrying this mutation belonged to the European haplogroups H, I, J, K, T, U, V, and L.^53,54 Indeed, the occurrences of mtDNA haplogroups B5, C, D, M, M7, M8, H, and R in Chinese families carrying the m.3460G>A mutation were higher than those in the Chinese control population.^21,33 Moreover, the frequencies of mtDNAs in haplogroups M, M9, and R in 38 Chinese families carrying the m.3394T>C mutation, haplogroups G, M7, and F in 11 Chinese families carrying the m.3635G>A mutation, haplogroups G and M10 in 7 Chinese families carrying the m.3866T>C mutation, and haplogroups B and M in 15 pedigrees carrying one of 15 putative mutations were markedly higher than those in Chinese controls.^21,33 Thus, the frequencies of haplogroups M, M9, M10, and R in the Chinese pedigrees carrying the ND1 mutations were significantly higher than those in 478 Chinese controls and other Asian populations.^21,33 This discrepancy between the different ethnic origins may be attributed to evolution.⁵⁵ 

In summary, this is the first comprehensive study to investigate the spectrum and incidence of mutations in the MT-ND1 gene in the Chinese population. In addition to five known MT-ND1 m.3460G>A, m.3394T>C, m.3635G>A, m.3733G>A, and m.3866T>C LHON-associated mutations, we identified 15 putative LHON-associated mtDNA mutations in a large cohort of 1281 Chinese subjects with LHON. A total of 111 subjects carrying one of the LHON-associated MT-ND1 mutations accounted for 7.6% cases of 1281 Chinese probands with LHON. These results support that the MT-ND1 gene is another hot spot for mutations associated with LHON. These data may provide valuable information for pathophysiology, management, and genetic counseling of LHON. 

Acknowledgments

Supported by National Key Technologies R&D Program Grant 2012BAI09B03 from the Ministry of Science and Technology of China (MXG and PJ) and Grants 31471191, 81400434, and 81200724 from the National Nature Science Foundation of China (MXG, YJ, and JZ). 

Disclosure: Y. Ji, None; M. Liang, None; J. Zhang, None; L. Zhu, None; Z. Zhang, None; R. Fu, None; X. Liu, None; M. Zhang, None; Q. Fu, None; F. Zhao, None; Y. Tong, None; Y. Sun, None; P. Jiang, None; M.-X. Guan, None