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Mol Vis. 2010; 16: 782–792.
Published online 2010 April 30.
PMCID: PMC2862244

Identification of novel mitochondrial mutations in Leber’s hereditary optic neuropathy

Abstract

Purpose

To screen mitochondrial DNA (mtDNA) variations in Leber hereditary optic neuropathy (LHON).

Methods

Ten LHON patients were selected from neuro-ophthalmology clinics of All India Institute of Medical Sciences (AIIMS), New Delhi, India. Clinical evaluation included slit-lamp biomicroscopy, fundus examination, and neuroimaging. DNA was isolated from whole blood samples. The entire coding region of the mitochondrial genome was amplified by PCR in ten patients and 20 controls. The full mtDNA genome except D-loop was sequenced. All sequences were analyzed against mitochondrial reference sequence NC_012920.

Results

MtDNA sequencing revealed a total of 30 nucleotide variations in the ten LHON patients and 29 in the 20 controls. Of 30 changes, 30.00% (9/30) were nonsynonymous, and the remaining 70.00% (21/30) were synonymous. In controls, a total of five changes were nonsynonymous. Out of the total 14 nonsynonymous changes observed in cases and controls, four (p.A52T in nicotinamide adenine dinucleotide [NADH] dehydrogenase [ND1] protein; p.L128Q in ND2; p.W48R in ATPase6; p.R340H in ND4 protein) were pathogenic. Four patients were positive for either of pathogenic changes. In total, 16.66% (5/30) variations were novel out of which 40.00% (2/5) were nonsynonymous. All novel variations were submitted to the GenBank database, and accession numbers were obtained. Primary LHON mutations in complex I genes have been considered a hallmark feature of LHON patients, and primary LHON mutations were present in two cases in this study. Mutations in complex I genes (ND genes) account for 50%–90% of LHON pedigrees in different ethnic pedigrees. In this study the highest numbers of changes were also present in complex I genes (46.66%; 14/30) followed by complex IV (30.00%; 9/30), complex III (16.66%; 5/30), and then complex V (6.66%; 2/30). Complex I had 5/30 (16.66%) nonsynonymous changes, complex III had 1/30 (3.33%), complex IV had 1/30 (3.33%), and complex V had 2/30 (6.66%) nonsynonymous changes. Nonsynonymous mutations in cytochrome c oxidase (COX) genes have been reported previously in LHON patients. Nonsynonymous mtDNA variations may adversely affect the respiratory chain and impair the oxidative phosphorylation (OXPHOS) pathway, resulting in low ATP production and elevated reactive oxygen species (ROS) levels, which cause oxidative stress. It has previously been reported that oxidative stress (OS) leads to oxidative damage of cellular macromolecules, such as mitochondrial and nuclear DNA, proteins, and lipids along with energy depletion and a local imbalance of calcium homeostasis, resulting in neuronal degeneration. OS is the underlying etiology in several ocular diseases and also plays an essential role in LHON.

Conclusions

A total of five novel mtDNA variations were identified in this study. Nonsynonymous mtDNA variations may adversely affect the respiratory chain and impair the OXPHOS pathway, resulting in low ATP production and elevated ROS levels. OS further damages both nuclear and mtDNA. This preliminary study describes mtDNA sequence variations in a relatively small number of LHON patients of north Indian ethnic origin. However, these results should be confirmed in other populations. Early diagnosis of mtDNA variations and prompt anti-oxidant administration in these cases may delay OS-induced injury to retinal ganglion cells (RGCs) and hence improve visual prognosis.

Introduction

Leber hereditary optic neuropathy (LHON; OMIM 535000) was first described as a distinctive clinical entity in 1871 by the German ophthalmologist Theodore Leber (1840–1917) [1]. LHON is a maternally inherited disease leading to acute bilateral blindness due to loss of the optic nerve and papillomacular bundle nerve fibers, predominantly in young men [2]. The prevalence is estimated to be 1:50,000. LHON begins generally in young adults, with a mean age of onset between 18 and 35 years. Nearly all patients worldwide carry one of three mitochondrial (mt) DNA pathogenic point mutations at positions 11778/nicotinamide adenine dinucleotide (NADH) dehydrogenase (ND)4, 3460/ND1, and 14484/ND6, which affect complex I subunit genes [3]. Other pathogenic point mutations have also been described in a minority of patients [4,5]. Although visual failure is the defining feature in this mitochondrial disorder, cardiac arrhythmias, and neurologic abnormalities, such as postural tremor, peripheral neuropathy, nonspecific myopathy [6], and movement disorders [7], have also been reported to be more common in LHON patients compared to controls. Pathogenic mutations in different mitochondrial genes in LHON patients have been reported in previous studies [8-13]. In this preliminary study we investigated a series of patients presenting at ophthalmology clinics with subacute visual failure and who were suspected of having LHON. This study was planned with the aim to screen LHON cases for mtDNA sequence variations by PCR–DNA sequencing.

Methods

Clinical examination and selection of cases

After receiving ethical approval from the institutional review board (IRB#00006862; All India Institute of Medical Sciences, Delhi, India), ten clinically diagnosed LHON cases from northern India, presenting at the Dr. R. P. Centre for Ophthalmic Sciences (AIIMS, New Delhi, India), were enrolled for this study only after informed consent. LHON was diagnosed on the basis of a high index of suspicion. Since it is mainly a diagnosis of exclusion, all other causes were considered and ruled out. Detailed family history was taken, including associated peri-ocular pain to differentiate from papillitis, and the history of chronic tobacco or alcohol or chronic systemic medication was noted to rule out toxic optic neuropathy. Family history of any similar episode was noted, and the family tree was charted. Detailed systemic and neurologic examinations were done to check the involvement of other cranial and peripheral nerves. Visual field evaluation, electrophysiological electro-retinogram, and visual-evoked potential were done in all patients. All patients underwent a complete ophthalmic examination, including visual acuity measurement, slit-lamp observation of the anterior segment, indirect ophthalmoscopy, and applanation tonometry. Visual field testing was performed in all patients with good fixation. All patients underwent magnetic resonance imaging (MRI) of the brain and orbit and flourescein angiography.

Hospital records were reviewed, and full neuro-ophthalmologic examinations were performed on all patients. The mean age of onset of symptoms was 24.9 years (all patients were male). No patient reported any drastic changes in their diet or intake of any drug or exposure to any toxic agent/pollutant around the time of visual loss. All patients had normal erythrocyte sedimentation rate, antinuclear antibodies, and syphilis serology. None of the patients reported myotonia, exercise intolerance, palpitations, cardiac conduction abnormalities, oral or genital ulcers, erythema nodosum, or somatic anomalies. Patients were followed in a neuro-ophthalmology clinic. The clinical manifestation of all LHON patients has been tabulated (Table 1).

Table 1
Clinical phenotypes of Leber hereditary optic neuropathy patients

A total of 20 ethnically and age-matched normal individuals without any history of ocular disorders was enrolled as controls. All control subjects were AIIMS blood donors who reported no symptomatic metabolic, genetic, or ocular disorders on an extensive questionnaire regarding family history, past medical problems, and current health. The control group for mtDNA sequencing consisted of 20 male individuals (mean age, 23.85±3.34 years). Family information was obtained by history.

Sample collection and DNA isolation

Peripheral blood samples (5 ml, blood drawn by venipuncture) were collected in EDTA vacutainers (Greiner Bio-one Catalogue no. 455036; Frickenhausen, Germany) after obtaining written consent and stored at −80 °C until further use. DNA was extracted from whole blood samples of all LHON patients and controls, using the phenol chloroform method [14].

PCR amplification and sequence analysis of the mitochondrial DNA coding region

The entire coding region of the mtDNA was amplified in LHON patients and controls using 24 pairs of primers (Table 2) [15]. PCR amplifications for all primer sets were performed in a 40-μl volume containing 1.0 μl of 20 μM stock solution for each primer (Eurofins Genomics India pvt Ltd, Bangalore, India), 100 ng of genomic DNA, 1 unit of Taq polymerase (Banglore Genei, Bengaluru, Karnataka, India), 0.1 mM of each deoxynucleotide triphosphate (dNTP), and 4 μl of 10X PCR buffer (with 15 mM MgCl2) by means of 30 cycles of amplification, each consisting of 30 s denaturation at 94 °C, 30 s annealing at 56 °C, and 1 min extension at 72 °C. Finally, an extension for 5 min at 72 °C was performed.

Table 2
Primers used for polymerase chain reaction used for amplification of mitochondrial genome.

Amplified PCR products were purified using a gel/PCR DNA fragments extraction kit (Geneaid Biotech Ltd., Sijhih City, Taiwan). Purified PCR products were sent for sequencing to Molecular Cloning Laboratories (South San Francisco, CA). The full mtDNA genome was sequenced except D-loop, as this part of the mitochondrial genome is a hyper-variable region. All fragments were sequenced in both forward and reverse directions for confirmation of any nucleotide variation. All sequence variants from both LHON patients and controls were compared to the Human Mitochondrial Reference Sequence NC_012920 provided by the National Center for Biotechnology Information (NCBI), using ClustalW2 (multiple sequence alignment program for DNA; European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK).

Computational assessment of missense mutations

Two homology-based programs, Polymorphism Phenotyping (PolyPhen) and Sorting Intolerant From Tolerant (SIFT) analysis tool, were used to predict the functional impact of missense changes identified in this study. PolyPhen structurally analyzes an amino acid polymorphism and predicts whether that amino acid change is likely to be deleterious to protein function [16,17]. PolyPhen scores of >2.0 indicate the polymorphism is probably damaging to protein function. Scores of 1.5–2.0 are possibly damaging, and scores of <1.5 are likely benign. SIFT is a sequence homology-based tool that sorts intolerant from tolerant amino acid substitutions and predicts whether an amino acid substitution in a protein will have a phenotypic effect [18]. SIFT is based on the premise that protein evolution is correlated with protein function. Positions with normalized probabilities less than 0.05 are predicted to be deleterious and those greater than or equal to 0.05 are predicted to be tolerated in the case of SIFT.

The substitution may occur at a specific site, e.g., active or binding, or in a nonglobular, e.g., transmembrane, region. PolyPhen uses the predicted hydrophobic and transmembrane (PHAT) matrix score to evaluate the possible functional effect of a substitution in the transmembrane region. At this step Polyphen memorizes all positions that are annotated in the query protein as BINDING, ACT_SITE, LIPID, or METAL. At a later stage, if the search for a homologous protein with known three-dimensional structure is successful, PolyPhen is checked whether the substitution site is in spatial contact with these critical residues. Positions important for function should be conserved in an alignment of the protein family, whereas unimportant positions should appear diverse in an alignment.

Results

MtDNA sequencing revealed a total of 30 nucleotide variations in the ten LHON patients, of which 30.00% (9/30) were nonsynonymous (Table 3), and 29 nucleotide changes in 20 controls, of which 17.24% (5/29) were nonsynonymous (Table 4). Of the 30 changes 30.00% (9/30) were nonsynonymous and the remaining 70% (21/30) were synonymous. Of the total changes 96.66% were transitions and the remaining were transversions. In total, 16.66% (5/30) variations were novel, out of which 40.00% (2/5) were nonsynonymous. Twenty-five other mtDNA sequence variations (see the MITOMAP and DB) were also identified in patients. The highest number of changes were present in complex I genes (46.66%; 14/30), followed by complex IV (30%; 9/30), then complex III (16.66%; 5/30), then complex V (6.66%; 2/30) genes. Complex I had 5/30 (16.66%) nonsynonymous changes, complex III had 1/30 (3.33%), complex IV had 1/30 (3.33%), and complex V had 2/30 (6.66%) nonsynonymous changes. All nucleotide variations identified in the current study were homoplasmic. All novel variations were submitted to the GenBank database, and accession numbers were obtained (Table 3).

Table 3
Mitochondrial DNA sequence changes in Leber hereditary optic neuropathy patients.
Table 4
Mitochondrial DNA sequence changes in controls

Sorting Intolerant From Tolerant and Polymorphism Phenotyping analysis

SIFT and PolyPhen analysis of all 14 nonsynonymous changes (9+5) from cases and controls revealed four pathogenic changes (p.A52T in ND1 protein; p.L128Q in ND2; p.W48R in ATPase6; p.R340H in ND4 protein; Table 3). Four patients (LHON 5–8, Table 5) were positive for at least one of these pathologic mtDNA nucleotide changes, but none of the controls harbored any pathogenic nucleotide change (Table 4). Patient and gene-wise distribution of mtDNA sequence changes have been tabulated (Table 5).

Table 5
Patient and gene wise distribution of mitochondrial DNA variations

Discussion

In this preliminary pilot study, we enrolled ten LHON patients who experienced acute or subacute, bilateral, persistent, optic neuropathies characterized by central visual loss that occurred simultaneously or sequentially within a period of 1 year. Leber described a maternal inheritance pattern; however, some patients with primary LHON mutations deny having a family history.

LHON-associated mtDNA mutations have been identified in various ethnic populations [19,20]. Mutations in complex I genes (ND genes) account for 50%–90% of LHON pedigrees in different ethnic pedigrees [11,13,21,22]. Primary LHON mutations have been considered a hallmark feature of LHON patients, and patients without primary LHON mutations have also been reported [23,24]. In this study 46.66% variations were reported in complex I (ND group of genes) genes, of which three were novel changes. Three pathogenic mutations (p.A52T in ND1; p.L128Q in ND2; and p.R340H in ND4) were present in complex I genes. Primary LHON mutation 3460G>A (p.A52T) was present in one patient (LHON 8), 11778G>A (p.R340H) was present in one patient (LHON 5), and 14484T>C (p.M64V in ND6) was absent in our patients.

Altered ubiquinone binding to complex I has been previously shown in the presence of several LHON-causing mutations. Decreased affinities for ubiquinone or the complex I inhibitor rotenone have been shown in the 3460G>A (p.A52T) and 11778G>A (p.R340H) mutations [25-28]. Substrate inhibition at higher concentrations of ubiquinone has previously been observed in the mitochondrial membrane preparations from Ebstein-Barr virus-transformed cell lines carrying the 3460G>A (p.A52T) or 14459G>A (p.A72V in ND6) mutations [25,29]. No primary LHON mutation or pathogenic mutation was present in control subjects. The age of onset symptoms did not differ significantly in patients with and without pathogenic mutations in the current study.

In the current study two patients (LHON 8 and LHON 5) had primary LHON mutations (3460G>A [p.A52T]; 11778G>A [p.R340H]). But none of the patients with primary LHON mutations reported a multigenerational history compatible with maternal inheritance. Two patients (LHON 6 and LHON 7) had pathogenic mtDNA sequence changes other than primary LHON mutations, while others had no suspicious mtDNA changes. Patients with no obvious mtDNA abnormalities might have no mitochondrial disease or they might have mtDNA abnormalities isolated to the optic nerve, conceptually similar to mitochondrial myopathies [30]. However, these patients may have elevated oxidative stress comparable to patients with primary LHON mutations [31]. Previous studies have suggested similar patients with LHON-like optic neuropathies but no identified pathogenic mtDNA mutations after sequencing all [24] or part [32] of the mitochondrial coding region. The phenotypic expression of the primary LHON mutations is very complex. Only about one-third of individuals harboring one of these three mutations eventually develops LHON, and the penetrance varies among different families [33-35]. Therefore, identification of other factors affecting LHON penetrance would be of value in elucidating the pathophysiology of retinal neuron loss as well as in searching for clues that might relieve visual loss or prevent the onset of LHON. Many factors, such as mtDNA background, heteroplasmy of the mtDNA mutation, nuclear gene(s), and environmental factors, have been shown to play active roles in the phenotypic expression of LHON [36-38].

In this study one nonsynonymous sequence change and four synonymous sequences in the cytochrome c oxidase III (COIII) gene were also identified of which two were novel. Nonsynonymous mutations in the COIII gene have been reported previously in LHON patients [12,39]. Other human diseases associated with cytochrome c oxidase (COX) mutations include primary congenital glaucoma (PCG), primary open angle glaucoma (POAG), and Leigh syndrome [15,40,41].

In this study one patient (LHON 10) had a nonsynonymous sequence change in cytochrome b (complex III). Nonsynonymous mutations in the cytochrome b gene has previously been described in LHON [42]. Two nonsynonymous changes in ATPase6 (p.T53I and p.W48R) were also identified, one of which (p.W48R) was pathogenic and present in one patient (LHON 5). ATPase6 mutations in LHON patients have been described previously [43]. Mutations in ATPase6 have been reported in PCG, POAG, neuropathy, ataxia, retinitis pigmentosa, and mitochondrial DNA-associated Leigh Syndrome patients [15,40,44,45]. In this study eight patients had no primary LHON mutations. However, two other pathogenic mutations were present in three patients (Table 5).

An intriguing feature of LHON is that only 50% of males and 10% of females who harbor one of the three primary mutations actually develop the optic neuropathy. This incomplete penetrance and predilection for males to lose vision imply that additional genetic and/or environmental factors must modulate the phenotypic expression of LHON. No generally accepted measures have been shown to either prevent or delay the onset of blindness in LHON. The long-term management of visually impaired patients remains supportive, with provision of visual aids and registration with the relevant social services.

Many mechanisms have been studied and proposed as the bases for the pathogenesis of mitochondrial optic neuropathies, including bioenergetic failure, oxidative stress, glutamate toxicity, abnormal mitochondrial dynamics and axonal transport, and susceptibility to apoptosis [3]. It has been well established that optic atrophy is a very common and sometimes the singular pathological feature in mitochondrial disorders [41]. Several human diseases have been associated with mtDNA mutations, indicating that dysfunction of the components of oxidative phosphorylation encoded by the mitochondrial genome can be deleterious [46]. Abnormalities in mtDNA have proven to be associated with LHON [40], POAG, pseudoexfoliation glaucoma (PEG), primary angle-closure glaucoma (PACG), PCG, and other spontaneous optic neuropathies [15,47-50]. It is generally agreed that there are two main sites in the respiratory chain where superoxide anions are generated, viz., complex I and complex III [51,52]. In the current study complex I genes had 16.66% nonsynonymous sequence changes, and of these three were pathogenic mutations.

The distribution of high numbers of mitochondria in the optic nerve head reflects the high energy requirement of the human optic nerve head. Neurons, because of their high energy requirement, are heavily dependent on mitochondria for survival [53]. Any malfunction of the mitochondrial electron transport chain results in an excessive generation of free radicals and low ATP production. It has previously been reported that oxidative stress (OS) leads to oxidative damage of cellular macromolecules, such as mitochondrial and nuclear DNA, proteins, and lipids, along with energy depletion and a local dysregulation of calcium homeostasis, resulting in neuronal degeneration [54]. OS is the underlying etiology in several ocular diseases, such as glaucoma, LHON, proliferative vitreoretinopathies, and cataract [40,55-57]. It has been established that pathogenic mitochondrial mutations can cause mitochondrial dysfunction and enhance OS, which in turn lead to apoptosis in affected tissue and primary culture of human cells that harbor mtDNA mutations [58]. Nonsynonymous mtDNA alterations may lead to mitochondrial dysfunction, which leads to reduced mitochondrial respiration, OS, damage to mtDNA, altered mitochondrial morphology, alterations in mitochondrial fission and fusion, and ultimately the cell’s demise. Oxidative stress-induced mtDNA damage has also been reported in other diseases, such as premature ovarian insufficiency [59]. LHON and LHON-like optic neuropathies might approach 20 per 100,000 individuals [60], and this diagnosis may be responsible for as much as 10% of blindness in individuals under age 65 [61].

This study describes mtDNA sequence variations in a relatively small number of LHON patients of north Indian ethnic origin. Nonsynonymous mitochondrial variations adversely affect oxidative phosphorylation, resulting in decreased mitochondrial respiration and increased free radical production [62]. However, these results should be confirmed by larger studies in other populations. Knowledge of mtDNA mutations and/or mitochondrial dysfunction in LHON may lead to a better understanding of optic atrophy in LHON. Novel approaches are now available for studying mitochondrial disease in the eye, and a novel in vitro treatment has already been devised for the metabolic defect of at least one mtDNA mutation in LHON [63]. Further work and ideas must be forthcoming to realistically treat mtDNA disease or prevent the transmission of mtDNA disease to future generations. Early diagnosis and prompt management by anti-oxidants may delay the progression of disease.

Conclusion

A total of five novel mtDNA variations were identified in this study, of which one was pathogenic. Nonsynonymous mtDNA variations may adversely affect the respiratory chain and impair the OXPHOS pathway, resulting in low ATP production and elevated ROS levels. OS further damages both nuclear and mtDNA. This preliminary study describes mtDNA sequence variations in a relatively small number of LHON patients of north Indian ethnic origin. However, these results should be confirmed in other populations.

Acknowledgments

The authors would like to thank the patients for their cooperation without whom the study would not be possible.

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