DEFINITION
A maternally linked genetic disorder that presents in mid-life as acute or subacute central vision loss leading to central scotoma and blindness. The disease has been associated with missense mutations in the mtDNA, in genes for Complex I, III, and IV polypeptides, that can act autonomously or in association with each other to cause the disease. (from Online Mendelian Inheritance in Man)
EPIDEMIOLOGY
In Northern European populations about one in 9000 people carry one of the three primary LHON mutations. There is a prevalence of between 1:30,000 to 1:50,000 in Europe.
The LHON ND4 G11778A mutation dominates as the primary mutation in most of the world with 70% of European cases and 90% of Asian cases. Due to a Founder effect, the LHON ND6 T14484C mutation accounts for 86% of LHON cases in Quebec, Canada.
More than 50 percent of males with a mutation and more than 85 percent of females with a mutation never experience vision loss or related medical problems. The particular mutation type may predict likelihood of penetrance, severity of illness and probability of vision recovery in the affected. As a rule of thumb, a woman who harbors a homoplasmic primary LHON mutation has a ~40% risk of having an affected son and a ~10% risk of having an affected daughter.
The penetrance of LHON is age specific and in some studies, the median age of onset was a few years later in females. The 95th centile for age at onset is 50 years for all three primary mutations.
Incomplete penetrance and gender bias are two key features of LHON still remain unexplained..
SYMPTOMS
Leber hereditary optic neuropathy (LHON) typically presents in young adults as bilateral, painless, subacute visual failure. The peak age of onset in LHON varies between the second and third decades of life depending on the published case series, with 95% of those who lose their vision doing so before the age of 50 years. Very rarely, individuals first manifest LHON in the seventh and eighth decades of life Males are four to five times more likely to be affected than females, but neither gender nor mutational status significantly influences the timing and severity of the initial visual loss.
In the presymptomatic phase, fundal abnormalities such as peripapillary telangiectatic vessels and variable degrees of retinal nerve fiber layer edema have been previously documented and these can vary with time Using optical coherence tomography imaging, thickening of the temporal retinal nerve fiber layer was confirmed in clinically unaffected individuals with an LHON-causing mtDNA mutation, further evidence that the papillomacular bundle is selectively vulnerable in LHON On more detailed investigation, some individuals with an LHON-causing mtDNA mutation can also exhibit subtle impairment of optic nerve function including: (a) loss of color vision affecting mostly the red-green system, (b) reduced contrast sensitivity, and © subnormal electroretinogram and visual evoked potential
Following onset of the acute phase, affected individuals report worsening, blurring, or clouding of central vision. Both eyes are affected within six months. The most characteristic feature is an enlarging central or centrocecal scotoma and as the field defect increases in size, visual acuity deteriorates in approximately 80% of persons to the level of counting fingers or worse. Following the nadir, visual acuity may improve; such improvement is more likely in individuals with the m.14484T>C mutation than in those with the m.11778G>A mutation.
The atrophic phase is characterized by bilateral optic atrophy and dense central scotomata. Most persons remain severely visually impaired and are within the legal requirements for blind registration.
Other neurologic features associated with LHON. Minor neurologic abnormalities (e.g., postural tremor, peripheral neuropathy, nonspecific myopathy, movement disorders) are said to be common in individuals with LHON but are rarely clinically significant.
Some individuals with LHON, usually women, may develop a progressive multiple sclerosis (MS)-like illness. In addition to a severe bilateral optic neuropathy, these individuals manifest disseminated central nervous system demyelination, with characteristic periventricular white matter lesions and unmatched cerebrospinal fluid oligoclonal bands In a few families, mtDNA complex I mutations cause optic atrophy in association with severe neurologic deficits including ataxia, dystonia, and encephalopathy
Cardiac conduction defects and LHON. A number of studies have shown an increased incidence of cardiac accessory pathways in association with LHON.
Gene Review
DIAGNOSIS
Although visual failure is the defining clinical feature in this mitochondrial genetic disorder, analyzed by Electrophysiological studies and cranial neuroimaging (see above), cardiac arrhythmias and neurologic abnormalities such as postural tremor, peripheral neuropathy, nonspecific myopathy, and movement disorders have been reported to be more common in individuals with LHON than in controls. Because LHON is a mtDNA disease, molecular testing is necessary to confirm the clinical diagnosis.
Electrophysiologic studies (pattern electroretinogram and visual evoked potentials) confirm optic nerve dysfunction and the absence of retinal disease. Note: These ancillary investigations are not usually necessary unless the diagnosis is uncertain.
Cranial neuroimaging is necessary to exclude other compressive, infiltrative, and inflammatory causes of a bilateral optic neuropathy. In individuals presenting with LHON, magnetic resonance imaging (MRI) is often normal but may reveal a high signal within the optic nerves, the latter probably representing slight edema or gliosis in the acute and atrophic phase, respectively.
Biochemical studies. Although the three primary LHON-causing mtDNA mutations all affect different respiratory chain complex I subunit genes, the mutations are not always associated with a respiratory chain abnormality that can be measured in vitro. The absence of a respiratory chain complex defect therefore does not rule out the possibility of LHON.
A striking feature of all the biochemical studies is that none found a significant difference between affected and unaffected individuals with a disease-causing mtDNA LHON-causing mutation. Balancing the current weight of evidence, LHON is associated with a respiratory chain defect that is more subtle than that seen in other mitochondrial genetic disorders.
Biochemical studies have been superseded by molecular genetic testing and these are only indicated when establishing pathogenicity for novel putative LHON-causing mtDNA variants.
Table Respiratory Chain Dysfunction in LHON
| Mitochondrial DNA Mutation | Complex I Activity | Respiratory Rate |
| m.3460G>A | 60%-80% | 30%-35% |
| m.11778G>A | 0%-50% | 30%-50% |
| m.14484T>C | 0%-65% | 10%-20% |
Molecular Genetic Testing:
Mutations in the mitochondrial genes that encode subunits of NADH dehydrogenase, MT-ND1, MT-ND2, MT-ND4, MT-ND4L,MT-ND5, and MT-ND6, are known to be associated with LHON (Table above). Mutations in three additional mitochondrial genes, MT-CYB,MT-CO3, and MT-ATP6 are also thought to cause LHON but require further confirmation as they have only been found in single affected individuals or a single family.
Table. Leber Hereditary Optic Neuropathy: Genes and Databases
| Gene Symbol | Chromosomal Locus | Protein Name |
| MT-ND6 | Mitochondria | NADH-ubiquinone oxidoreductase chain 6 |
| MT-ND4 | Mitochondria | NADH-ubiquinone oxidoreductase chain 4 |
| MT-ND1 | Mitochondria | NADH-ubiquinone oxidoreductase chain 1 |
| MT-ATP6 | Mitochondria | ATP synthase subunit a |
| MT-ND5 | Mitochondria | NADH-ubiquinone oxidoreductase chain 5 |
| MT-CYB | Mitochondria | Cytochrome b |
| MT-CO3 | Mitochondria | Cytochrome c oxidase subunit 3 |
| MT-ND2 | Mitochondria | NADH-ubiquinone oxidoreductase chain 2 |
| MT-ND4L | Mitochondria | NADH-ubiquinone oxidoreductase chain 4L |
The primary pathogenic mtDNA mutations described below have been seen only in families with LHON. 90% of individuals with LHON were found to have one of three point mutations of mtDNA: m.11778G>A (MT-ND4) m.14484T>C (MT-ND6) or m.3460G>A (MT-ND1)
Approximately 10% of individuals with LHON do not harbor one of the three common mtDNA point mutations. A number of putative mtDNA LHON-causing mutations have been described in a single family or singleton cases; however, a novel mtDNA base change cannot be considered pathogenic until it has been observed independently on two or more occasions and only in association with LHON, showing clear segregation with affected disease status.
Sequence analysis and mutation scanning detect additional mtDNA nucleotide variants in the remaining 10% of individuals with LHON who do not harbor one of the three most common mtDNA mutations (m.3460G>A, m.11778G>A, and m.14484T>C).
Summary of Molecular Genetic Testing Used in LHON
Prenatal diagnosis and preimplantation genetic diagnosis (PGD) for at-risk pregnancies require prior identification of the disease-causing mutations in the family.
PATHOGENESIS
Analyzing mutations described above, the most severe known is 14459GNA, which shows a wide clinical variability mostly causing a LHON plusphenotype (additional neurological features accompanying typical atrophy of the optic nerve) however patients presenting only with classical LHON are also observed. 3460GNA mutation is considered to be severe, 11778GNA—intermediate, whereas 14484TNC is rather mild.
Although LHON is one of the best studied mitochondrial disorders, the exact mechanism of pathology is still not fully known: This is due the complications that can be find in studying biochemistry and physiology of LHON. In fact, the only affected tissues are retinal ganglion cells (RGC) and the optic nerve, which are not easily accessible for in vitro studies, while the mutations are present in the whole body at a very high, even homoplasmic level, giving no clinical phenotype.
Since the mitochondrion is the power factory of the cell, the most obvious explanation was that the mtDNA defect leads to a significant decrease in energy production and failure in optic nerve function. As the experimental data were confusing and the rather small decrease in energy production for some mutations could not explain all the observed phenotypic effects, alternative explanations such as increased oxidative stress and apoptosis have been proposed :
Energetic failure?
Previously studies show a connection between the severity of the mutation and the severity of the OXPHOS defect but they cannot explain the pathomechanism of the disease. A possible explanation is that mitochondria function differently in neuronal cells. No reduction in membrane potential was observed in both differentiated and undifferentiated states, but the differentiation process appeared to be altered for LHON mutation bearing cells, since the yield of obtained differentiated cells was decreased
Functional Analysis of Lymphoblast and Cybrid Mitochondria Containing the 3460, 11778, or 14484 Leber's Hereditary Optic Neuropathy Mitochondrial DNA Mutation,2000
Reactive oxygen species production?
Mitochondria are the biggest reactive oxygen species (ROS) factory in the cell, and ROS may damage the cell via numerous mechanisms. As dysfunction in the respiratory chain can lead to elevated ROS production, the mechanism of LHON mutation influence on cell physiology became an attractive hypothesis. Indeed, the above-mentioned neuronal cybrids revealed that ROS production was increased about 2.5-fold in cells bearing all three common LHON mutations, but the effect was observed only in differentiated cells, and was absent in undifferentiated ones with the same mutations. As LHON is mostly a defect in complex I genes, it is worth mentioning that inhibition of complex I by rotenone leads tooxygen radical overproduction in human skin fibroblasts, to increased complexity of mitochondrial reticulum and to general outgrowth of mitochondria, which may be a compensatory effect. At the same time, higher concentrations of rotenone cause apoptotic death of the cells with all the typical hallmarks like DNA laddering, cytochrome c release, or activation of caspase 3 by elevated ROS production, although the extent of the phenomenon was dependent on the cell type
Differentiation-specific effects of LHON mutations introduced into neuronal NT2 cells,2002
Oxidative stress and apoptosis,2000
Inhibition of complex I of the electron transport chain causes O2-. -mediated mitochondrial outgrowth,2005
Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production,2002
Apoptosis?
The next question is whether apoptosis is involved in the atrophy of the optic nerve in LHON. Indirect evidence comes from the fact that there are no signs of inflammation in the optic nerve. Are the cells harboring mtDNA Leber mutations more prone to apoptotic cell death? In the very well characterized apoptotic pathway, mitochondria play a central role in amplifying the apoptotic signal, thus apoptosis of cells containing mitochondria with these mutations would be a very reasonable explanation of LHON pathology. There is increasing support for this hypothesis. Cell lines with each of the three typical LHON mutations died within a few days, showing nuclear morphology typical for apoptosis as well as DNA fragmentation and cytochrome c release—signs of mitochondrial involvement in the process. However, caspase-3 activation was not observed. More detailed studies revealed that the culture on galactose resulted in a significant reduction in ATP production and apoptotic death via a caspase-independent pathway with the sequential release of cytochrome c, AIF and EndoG from mitochondria.
Cells bearing mutations causing Leber's hereditary optic neuropathy are sensitized to Fas-Induced apoptosis,2001
Leber's hereditary optic neuropathy (LHON) pathogenic mutations induce mitochondrial-dependent apoptotic death in transmitochondrial cells incubated with galactose medium,2002
Apoptotic cell death of cybrid cells bearing Leber's hereditary optic neuropathy mutations is caspase independent,2006
Caspase-independent death of Leber's hereditary optic neuropathy cybrids is driven by energetic failure and mediated by AIF and Endonuclease G,2005
Why the optic, but not any other nerve?
The answer to this fundamental question can lie in the complex structure formed by retinal ganglion cells whose axons constitute the optic nerve. One interesting morphological feature is myelination: it is present in the part of the nerve located behind the lamina, while it is absent in the head of the optic nerve. The mitochondrial content is higher in the unmyelinated part, reflecting the higher energetic requirements needed to sustain the action potential. In such a complicated structure, some of its parts may be more prone to different damaging factors.Unlike any other part of the nervous system, the retina is exposed to light, which can be responsible for increased ROS production. As ROS production is already increased in cells with LHON mutations, additional light-induced ROS production may lead to the damage of very sensitive Muller glial cells, which are responsible for maintenance of RGC homeostasis.Another characteristic of this cells is that they are sensitive to glutamate levels and die when the expression of excitatory amino acid transporter EAAT1 responsible for glutamate uptake is low. At the same time LHON mutations are known to influence the function of this transporter and cells with these mutations show decreased glutamate levels.
Pathogenesis of retinal ganglion cell death in Leber hereditary optic neuropathy (LHON): possible involvement of mitochondria, light and glutamate,2005
Leber hereditary optic neuropathy mtDNA mutations disrupt glutamate transport in cybrid cell lines,2004
Depression of retinal glutamate transporter function leads to elevated intravitreal glutamate levels and ganglion cell death,2000
RISK FACTORS
The primary LHON mutation is a prerequisite, but secondary factors are clearly modulating the risk of visual loss. Their identification has proven challenging, and the accumulated evidence favours a complex disease model, with both genetic and environmental factors interacting to precipitate optic nerve dysfunction.
Mitochondrial genetic factors
Among heteroplasmic LHON carriers, visual loss only occurs if the mutational load exceeds 60%, the threshold required for trigger bioenergetic defect However, incomplete penetrance is still observed among heteroplasmic carriers harbouring supra-threshold mutational levels, and over 80% of all LHON pedigrees are homoplasmic for the primary mtDNA mutation). Another possible mitochondrial modulating factor is the haplogroup background on which the LHON mutation is segregating: there is a significantly increased risk of visual failure when the m.11778G>A and m.14484T>C mutations occurred on a haplogroup J background, whereas m.3460G>A carriers were more likely to experience visual loss if they belonged to haplogroup K. A protective effect is conferred by haplogroup H, but only among m.11778G>A mutational carriers.
However, the link between specific mtDNA haplogroups and the risk of visual failure in LHON is not entirely clear-cut.
Leber hereditary optic neuropathy: Does heteroplasmy influence the inheritance and expression of the G11778A mitochondrial DNA mutation,2010
Heteroplasmy in Leber's hereditary optic neuropathy,1993
Pedigree analysis in Leber hereditary optic neuropathy families with a pathogenic mtDNA mutation,1995
Nuclear genetic factors
The marked male bias seen in LHON can not be explained by mitochondrial genetic factors. An extensive analysis have proposed a two-locus model for visual failure in LHON. The segregation pattern was consistent with a visual-loss susceptibility gene on the X-chromosome, acting in synergy with the primary mtDNA mutation to precipitate visual loss among at risk carriers. Male carriers have only one X-chromosome, and unlike female carriers, they cannot compensate for the inheritance of a putative X-linked visual-loss susceptibility allele . However, the actual gene or genes involved have yet to be identified, and more sophisticated bioinformatic tools are currently being applied for candidate geneanalysis and to narrow down specific areas of interest. LHON could be an even more complex disorder than originally considered and the existence of autosomal nuclear modifiers remains a distinct possibility.
Identification of an X-chromosomal locus and haplotype modulating the phenotype of a mitochondrial DNA disorder,2005
Evidence for a novel x-linked modifier locus for leber hereditary optic neuropathy,2008
Hormonal factors
Although much attention has been focused on possible secondary genetic modifiers in LHON, hormonal factors could also influence phenotypic expression. This hypothesis has recently been investigated “in vitro” studies. Treatment of the cells bearing the LHON mutations with 17b-oestradiol had a mitigating effect on these pathological features, led to increased cellular levels of the anti-oxidant enzyme superoxide dismutase (SOD) and to more efficient mitochondrial biogenesis. These results are very interesting, providing another explanation for the protective effect of female gender on LHON penetrance, and supporting the possible therapeutic use of oestrogen-like compounds in this disorder.
Oestrogens ameliorate mitochondrial dysfunction in Leber's hereditary optic neuropathy,2010
Environmental factors
The role of smoking and alcohol in LHON has been studied in a number of relatively small case-control studies, with contradictory findings. Smoking was strongly associated with an increased risk of visual loss, and interestingly, there was a dose-response relationship, with the risk of visual loss being much greater in heavy smokers compared to light smokers. There was also a trend towards an increased risk of visual failure among heavy drinkers, but this effect was not as strong as smoking. Based on these results, LHON carriers should be strongly advised not to smoke and to moderate their alcohol intake. Although no functional studies were performed, smoking could further impair mitochondrial OXPHOS, either through a direct effect on complex I activity, or by reducing arterial oxygen concentration. Several other environmental triggers have been reported in LHON, includinghead trauma, acute physical illness, psychological stress, occupational exposure to chemical toxins such as 2,5-hexanedione, anti-retroviral drugs, and anti-tuberculous agents.
Gene-environment interactions in Leber hereditary optic neuropathy,2009
Leber hereditary optic neuropathy: bad habits, bad vision?,2009
THERAPY
Despite the advances in the understanding of the molecular mechanisms of LHON, there is not so efficient medical treatment to offer patients with this devastating disease.
Potential pharmacologic agents in LHON may come from several sources.
Antioxidants, substances that prevent or repair oxidative damage of cell constituents, are one major class of candidate drugs for LHON. Vitamins C and E, which act via a free radical scavenger mechanism, are safe antioxidants that have been used extensively in man. Vitamins C and E, along with coenzyme Q10 (ubiquinone), are often given to patients with acute LHON, but their efficacy is questionable. Idebenone is an analog of ubiquinone that has better penetration into the central nervous system.
While antioxidants are theoretically attractive for treatment of LHON, currently available antioxidants may not be optimal. This may explain the lack of efficacy of those tried so far. The process of oxidative stress-induced apoptosis is postulated to be important, though the exact steps underlying the acute neuronal death is unknown. The complex, multi-step nature of this process provides a number of potential targets. Endogenous antioxidant defense systems could be enhanced by the administration of N-acetyl cysteine to increase intracellular glutathione levels. This agent has already been used in clinical trials of neurodegenerative diseases such as Alzheimer’s disease. Coenzyme Q10 serves to enhance electron flow within the inner mitochondrial membrane, in addition to its antioxidant properties. Creatine supplementation increases brain levels of creatine and phosphocreatine, both of which are important in energy metabolism. Creatine and immunophilins (such as cyclosporin A)also inhibit activation of the mitochondrial permeability transition pore complex. This activation is a critical step in mitochondrial-dependent apoptosis. Cyclosporin A has been demonstrated to be protective in cell culture analysis of oxidative stress due to the 11778 LHON-associated mtDNA mutation and of Complex I toxin-induced apoptosis in neurons.
Neuroprotection may play a role in the future treatment of neurodegenerative diseases. Excitotoxic injury to neurons via activation of glutamate receptors can be blocked by a variety of antagonists that bind at different sites and receptors. Memantine, an NMDA-receptor antagonist, is currently being studied as a neuroprotective agent in glaucoma. Riluzole is a glutamate antagonist that has shown minimal efficacy in amyotrophic lateral sclerosis. Topiramate is a newer anti-convulsant drug that has a complex array of mechanisms, including antagonism of the AMPA-class of glutamate receptors. Selegiline(deprenyl), an inhibitor of monamine oxidase isoform B, may have subtle neuroprotective effects in Parkinson’s disease. Modafinil, an amphetamine-like agent that is approved for the treatment of narcolepsy, may have additional neuroprotective properties. Future studies will determine which of these agents, if any, might be appropriate for therapeutic trials in LHON.
Marco Consiglio
Davide Coda
