The Genetics of Leber’s Hereditary Optic Neuropathy

The Genetics of Leber’s Hereditary Optic Neuropathy: A Literature Review
Henry Liu, BSc.

Abstract
Leber’s hereditary optic neuropathy (LHON) is a mitochondrial disease that is characterized by a painless, acute loss of central vision with 95% of affected individuals harbouring one of 3 pathogenic mutations mt.11778G>A, mt. 3460G>A and mt. 14484T>C. The purpose of this review is to highlight the distinguishing clinical presentations of the disease with respect to the mutation subtype and present our recent understanding of two unique features of the disease: male predominance and incomplete penetrance. We also review the recent advancements made in the diagnosis and treatment of this rare mitochondrial disease and the implications for genetic counselling.

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Introduction
Leber’s hereditary optic neuropathy (LHON) is a rare mitochondrial disease, predominantly affecting young males, and is characterized by a painless, subacute loss of central vision (1, 2). Over 95% of individuals with LHON harbour one of three pathogenic mutations to complex I of the oxidative phosphorylation chain (G11778A, G3460A, and T14484C) (3). Affected patients typically experience significantly diminished visual acuity, central or cecocentral scotoma, and dyschromatopsia (2). Visual dysfunction in LHON has been attributed to a selective degeneration of retinal ganglion cells (RGCs) and their axons (4). RGCs are vulnerable to mitochondrial dysfunction, particularly the papillomacullar bundle due of their structural and energy constraints (4). Moreover, histopathological studies of patients with LHON show degeneration of myelin and axoplasms at the optic nerve level, but early changes including edema of the peripapillary retinal nerve fiber layer (RNFL) and retinal telangiectasia may be evident on fundoscopic examination (3).

LHON has a prevalence of approximately 1 in 50,000 and is one of the most commonly inherited optic neuropathies (5). The majority of patients are affected prior to the age of 60, with an average age of onset ranging from 15-35 years. During the acute phase of the disease, patients present with painless blurring of vision that can affect both eyes simultaneously (25%) or sequentially (75%, with an inter-eye delay of 8 weeks) (3). Visual acuity typically reaches a nadir at 4-6 weeks post-disease onset and progresses to less than 6/60 (3). Fundoscopic examinations are typically unremarkable in the acute phase, however, it can demonstrate disc pseudoedema and hyperemia, vascular tortuosity and peripapillary telangiectasias (5). The characteristic visual field defect is a central or centrocecal scotoma which may be observed on Goldmann perimetry. In the more chronic stages of the disease, optic atrophy can be evidenced in the form of a pale optic nerve due to death of the RGCs. (6)
Molecular Mechanism
The missense mutation in NADH dehydrogenase, a co-enzyme responsible for the generation of ATP and cellular energy (7). This subsequently results in decreased ATP synthesis, elevated oxidative stress and disruptions to glutamate transport resulting in degeneration of the retinal ganglion cells and eventual cell death via apoptosis (7). However, majority of the proposed mechanisms have been established through mitochondrial cybrid cell lines. One study by Lin et al. using a mouse model of LHON introduced the P25L mutation in ND6, a mutation that results in LHON in humans. They subsequently concluded that oxidative stress is the primary driver for the pathogenesis of the disease in mediating RGC cell death whereas ATP production has been relatively maintained. Mechanistically, it has been observed that the transport of electrons along the ETC can be disrupted as a result of mutated complex I, resulting in more available free electrons to react with molecular oxygen and converting to superoxide which leads to oxidative stress (9). Interestingly, it is rare for patients to have systemic symptoms. Moreover, no other neuronal cell lines, including photoreceptors which have high energy consumption and mitochondrial demand, appear to be affected apart from RGCs (7). A recent study by Levin (2007) examined the role of superoxide production in RGC-5 cell line and found that superoxide production is at significantly lower levels in RGC compared to brain mitochondria (10). This has been hypothesized to be due to the tighter regulatory role of SOD-2, which is an enzyme that converts the toxic superoxide into nontoxic components (10). Pioneering work by the Guy and Qi et al. have demonstrated knockdowns of SOD-2 in RGCs result in optic neuropathy and overexpression of SOD-2 improved optic neuropathy (11, 12). Despite having the SOD-2 system to reduce levels of superoxide production, mutations in complex I, as observed in LHON, could lead to high levels of superoxide production that may overwhelm the regulatory pathway, manifesting in eventual ROS release and cell death (10).
Clinical Characteristics of LHON
Features of Pathogenic Mutations
Although there are a multitude of primary mutations responsible for LHON, more than 95% of the LHON pedigrees harbour one of the three pathogenic mitochondrial mutations at nucleotide positions 3460, 11778 and 14484 (1). The mutations affect respiratory chain complex I genes at the ND1 (3460), ND4 (11778) and ND6 (14484) subcomponent (1).

There are several distinguishable characteristics based on demographic factors and clinical manifestations across the 3 mutations. The G11778A is the most prevalent, reported to account for 70% of cases in the Northern Europeans, and 90% in Asians (1). The T14484C and G3460A are approximately equal in prevalence but there is a high representation of the T14484C mutation in French Canadians as a result of a founder effect (13). With respect to the age of disease onset T14484C mutation has the lowest age at onset (19.1 ± 10.5 years) compared to G11778A (26.2 ± 15.0 years) and G3460A (20.9 ± 14.5 years) (14). Of clinical importance is the fact that the visual prognosis differs according to the mutations. The rate of spontaneous visual recovery is the highest for the T14484C mutation with between 37% up to 58% recovery and lowest with G11778A mutation with 4% recovery (1). The G3460A mutation is also associated with more severe disease phenotype (15).
In a recent investigation by Liu et al. it was observed that the male preponderance is the most prominent in patients with the T14484C mutation (M:F ratio 4.9:1) and less obvious in patients with the G3460A mutation (M:F ratio 1.7:1) (14). This may be related to the differences in the biochemical consequences of the primary mutations. As based on previous respiration and enzymological analysis on cybrid mitochondria from LHON patients, the G3460A mutation is seen to exhibit a relatively more severe biochemical defect with a consistent decrease in complex I activity while the T14484C mutation had the mildest disruption to the bioenergetics of complex I (16). Female carriers of the biochemically most severe G3460A mutation seem to become affected almost as often as males, whereas among carriers of other, less severe mutations, a greater proportion of females remains unaffected.

Even though all three mutations presented with simultaneous mutations the percentage of simultaneous and sequential was significantly different (14). The T14484C mutation exhibited more simultaneous than sequential onsets compared with the other two mutation subtypes. Moreover, T14484C showed a more reduced and restricted distribution (inter-eye onset range = 1–44 weeks) versus G11778A (range = 1–2016 weeks) G3460A (range 2–816 weeks) for sequential presentations (14). This is interesting to observe given that T14484C it is also the mutation that harbours the mildest defect and greatest potential for recovery. Patient population affected at younger age may be more genetically predetermined to becoming affected.
Male Predominance
One unique characteristic of LHON is the male predominance, as previous pedigrees have reported that 80-90% of affected family members are males (1). One hypothesis proposed by Bu et al. has been related to a recessive X-linked susceptibility gene that interacts with the mitochondrial mutation which could account for the increased disease prevalence in males (17). Female carriers who become affecteds are thought to have homozygosity at the X-linked locus or have experienced an X chromosome inactivation. Although recent linkage analyses have identified a region in the long arm of the X chromosome that may contain this susceptibility gene, still the exact gene remains to be identified. Another hypothesis that could account for the disparity in disease representation between males and females is hormonal influences. Specifically, estrogen have been implicated in modifying the severity of mitochondrial dysfunction including oxidative stress, dysregulated ATP synthesis and cellular apoptosis, as estrogen receptors are particularly abundant in the RGCs and their activation have been associated with increased activity of superoxide dismutase and reduction of ROS (18).

Incomplete Penetrance
A second intriguing characteristic of this mitochondrial disease is its marked incomplete penetrance with approximately 50% of males and 10% of female carriers manifesting the disease during their lifetime (2). Genetic factors are unable to provide a complete account of the reduced penetrance. Based on results of 5 reported monozygotic twin studies (19-24), two pairs had one sibling that did not manifest the disease at long-term follow-up (23, 21). It is therefore more likely that LHON is a multifactorial disease whereby the presentation is influenced by environmental interactions. Indeed, several case-control studies suggest an increased rate of disease conversion in LHON carriers with high alcohol and tobacco consumption. The effect of smoking has been examined by Kirkman et al. in a large, multicentre epidemiological study of 196 affected and 206 unaffected carriers (25). Their study demonstrated that not only is smoking associated with increased risk of visual failure in carriers of LHON, but that heavy smokers were more likely to be affected compared to light smokers, lending support for a biologically plausible dose-response relationship. It is suggested that cigarette smoke can reduce the activity of complex I and subsequently increase production of ROS. Several other reports have also noted other precipitating factors that may contribute to disease conversion including trauma, nutritional deprivation, metabolic disturbances, industrial toxin exposure and psychological stress but the strength of the causal relationship has not been well established (25).
Advancements in Diagnostics and Management
Diagnosis
Research in OCT have also yielded changes in the retinal ganglion cell complex (GCC) relative to the retinal nerve fibre layer (RNFL) over time. Barboni’s discovered that there was a thickening of RNFL on OCT in early LHON (<6 months) and severely thinned or atrophic LHON in chronic LHON (>6 months). The temporal fibers of the papillomacular bundle were the first and most severely affected whereas there appears to be nasal fiber sparing until late stage of the disease (26). Savini et al. investigated RNFL thickness in unaffected carriers of LHON and found that there is a thickening of temporal fibers in all subgroups of LHON carriers (27).
Visual electrophysiological recordings such as visual evoked potentials (VEPs) and pattern electroretinograms (PERGs) have led to characteristic findings in patients with LHON and provide an objective and reliable metric in assessing RGC function. For example, it has been well documented that LHON patients exhibit increased VEP latencies, waveform distortions and decreased VEP amplitudes during the acute stage (28-29). Additionally, reductions in PERG amplitude have been noted in LHON carriers despite normal visual acuity, visual field, and RNFL thickness (30). However, VEP elicits potentials from the visual cortex and as a result does not directly measure RGC function. While PERGs have the ability to measure RGC activity, its utility is primarily restricted to the inner retina and necessitates refractive correction and precise foveal placement (31).

Recently, it was shown that RGCs generate a slow negative wave response that is observable on the electroretinogram (ERG) immediately following the b-wave of a cone response (32). This component of the ERG is referred to as the photopic negative response (PhNR). Several studies have shown correlations between the PhNR amplitude and the presence of RGC pathologies including, glaucoma, idiopathic intracranial hypertension, diabetic optic nerve atrophy, optic neuritis and retinal vascular diseases (32-37). In full-field ERG, the PhNR amplitude reflects cone-related RGC function and is significantly reduced with advancing visual field loss and reduced RNFL thickness as seen on OCT (38). The thinning of the RNFL typically follows the decreased PhNR amplitude, suggesting that functional changes precede structural abnormalities in the ganglions cells in LHON (38). In summary, there is good evidence to suggest the use of clinical ERG and ocular imaging such as OCT in aiding the diagnosis and monitoring the severity of disease in patients with LHON.

Treatment
Presently, there is no definitive medical treatment for LHON. Nevertheless, there is theoretical benefit in the use of anti-oxidants including Coenzyme-Q10 in mitigating stress from ROS (39). Recent trials have also been conducted on idebenone, a short-chain benzoquinone related to coenzyme-Q10 which facilitates bypassing of complex I directly to complex III in the electron transport chain (40). A research trial (RHOSOS- Rescue of Hereditary Optic Disease Outpatient Study) recruited 84 patients with primary LHON mutations. Patients were randomized and received high dose idebenone for 24 weeks versus placebo. Post-hoc analyses revealed improvement in visual acuity in the treatment group particularly those with G11778A and G3460A mutations. Investigations are currently being conducted in EPI-743, a more potent para-benzoquinone compared to idebenone which have shown some encouraging results in smaller cohort studies (41).
Gene therapy have become an exciting avenue for current and continued research in advancing treatments for LHON. Exogenous DNA can be inserted into the nuclear DNA through adenovirus-associated virus (AAV) type 2. Given the defect in LHON is within the mitochondrial genome, allotopic rescue have been employed, whereby genetic material is incorporated into the nuclear genome which encodes proteins which are subsequently targeted to translocate to the mitochondria (11). Preliminary results have been demonstrated in a mouse model injected with an AAV vector containing ND4 (the protein that was affected by the LHON 11778 mutation) and was successful in with preserving vision, maintain ATP synthesis, and rescue RGCs from cellular apoptosis (41). Apart from ND4 viral vectors, the NDI1, a yeast nuclear gene which encodes a complex I equivalent, can be delivered directly as demonstrated by a study by Marella et al. (42). The authors delivered NDI1 gene into the optical layer of the superior colliculus in a rotenone induced rat model of LHON and demonstrated that treatment of the animals with the NDI1 gene led to a complete restoration of the vision to the normal level compared to GFP controls.

Additional, gene therapy holds promising potentials and has been a part of a Phase 1 and 2 dose-escalation trials of GS010, an intravitreal gene therapy for LHON (43). The early phase recruited 14 patients with the ND4 mutation who received a one-time dose of GS010 in their worse eye. Follow-up at 96 weeks revealed that the eyes that were treated with AAV improved by an average of 21 ETDRS letters from their initial baseline. The treatment was also found to be most effective in 5 patients who had experienced vision loss within 2 years of before enrollment. Though these findings are promising, continued follow-up is required to assess the long-term outcome of these patients.

Genetic Counselling
In many inherited diseases, with LHON being no exception, the role of genetic counselling is vital for patients to be informed of the risk of disease and chance of development in their children and relatives. Up to 85% of individuals with LHON are homoplasmic, that is all the mitochondria express the pathogenic DNA mutation. Due to the maternal inheritance, men who are carriers of the mitochondrial mutation cannot pass it on to their offspring while all children of a female carrier will harbour the mutation. Nevertheless, LHON demonstrate incomplete penetrance with approximately 50% of male and 10% of female carriers converting to affecteds. However, the difficulty in genetic counselling lies in the fact that genetic testing cannot predict the severity or rate of progression of disease carriers. LHON is a disease that can have significant visual consequences and quality of life, as such a referral to a low vision specialist is warranted to educate on techniques on improving reading and mobility. Lastly, given its multifactorial nature of the disease, it would be beneficial to also counsel on risk factors for disease conversion including tobacco and alcohol use.

Conclusion
Leber’s hereditary optic neuropathy (LHON) is a well-studied mitochondrial disease that results in bilateral central vision loss and is a result of one of 3 pathogenic mutations (G11778A, G3460A, and T14484C). Currently there is no proven treatment. However, given our further understanding of the disease mechanism and ongoing research and trials conducted in idebenone, EPI743 and gene therapy, there remains hope for emerging solutions for this devastating disease.