Leber Hereditary Research Articles

The m.11778 A > G variant associated with the coexistence of Leber's hereditary optic neuropathy and multiple sclerosis-like illness dysregulates the metabolic interplay between mitochondrial oxidative phosphorylation and glycolysis

Little is known about the molecular mechanism of the rare coexistence of Leber's Hereditary Optic Neuropathy (LHON) and multiple sclerosis (MS), also known as the Harding's syndrome. In this study, we provide novel evidence that the m.11778A > G variant causes a defective metabolic interplay between mitochondrial oxidative phosphorylation and glycolysis. We used dermal fibroblasts derived from a female proband exhibiting clinical symptoms compatible with LHON-MS due to the presence of the pathogenic m.11778A > G variant at near homoplasmic levels. Our mitochondrial morphometric analysis reveals abnormal cristae architecture. Live-cell respiratory studies show stunted metabolic potential and spare respiratory capacity, vital for cell survival upon a sudden energy demand. The m.11778 A > G variant also alters glycolytic activities with a diminished compensatory glycolysis, thereby preventing an efficient metabolic reprogramming during a mitochondrial ATP crisis. Our collective results provide evidence of limited bioenergetic flexibility in the presence of the m.11778 A > G variant. Our study sheds light on the potential pathophysiologic mechanism of the m.11778 A > G variant leading to energy crisis in this patient with the LHON-MS disease.

Gene therapy in optic nerve disease.

Highlight some of the recent advances in gene therapy and gene modification for optic nerve disease to promote axon regeneration, neuroprotection, and increased visual functioning. Visual loss secondary to optic nerve damage occurs in numerous ophthalmologic and neurologic conditions. Damaged retinal ganglion cells (RGCs) do not regenerate once they undergo apoptosis after injury. Gene therapy has been studied to replace gene mutations in disorders affecting the optic nerve as well as to alter genes responsible for suppressing or activating pathways of optic nerve growth and regeneration. Recent clinical trials for Leber's Hereditary Optic Neuropathy have demonstrated safety and feasibility as potential future treatment. Animal studies utilizing gene therapy for optic nerve regeneration have shown various degrees of RGC axon regrowth and target reinnervation. Some studies have also successfully demonstrated a state of neuroprotection in RGCs allowing them to survive in greater numbers following injury. Additional studies will have to evaluate long-term efficacy and safety of these potential treatments, as well as the consequences of manipulating tumor suppressor genes and oncogenes.

The Photopic Negative Response: An Objective Measure of Retinal Ganglion Cell Function in Patients With Leber's Hereditary Optic Neuropathy.

The photopic negative response (PhNR) is a slow negative component of a flash photopic full-field ERG that has been shown to be specific for retinal ganglion cell (RGC) activity. Direct evaluation of RGC function is desirable in patients with Leber's hereditary optic neuropathy (LHON) in which the loss of central acuity can make it difficult to monitor patients with standard metrics. The purpose of this study was to evaluate the use of PhNR as an objective noninvasive clinical metric in LHON. Full-field photopic ERG recordings were collected in subjects with the mt.11778G>A/ND4 LHON mutation using a red on blue stimulus. The PhNR was identified using a computer-based automated detection system, and data were manually examined to remove movement artifacts. The PhNR amplitude was compared between controls (n = 13), carriers (n = 17), and affected (n = 6). Mean PhNR amplitude decreased significantly across groups (P < 0.0001). Post hoc Tukey's test revealed a significant decrease in PhNR amplitude between carriers and controls (P < 0.05) and between carriers and affected (P < 0.01). We are able to demonstrate that the PhNR amplitude is significantly decreased in patients affected by LHON compared to carriers in a well-described pedigree. Surprisingly, there was also a decrease in PhNR in carriers, suggesting potential subclinical RGC dysfunction in some carriers. This is important in patients affected with LHON who typically have a dense central scotoma. The PhNR may be a useful objective outcome measure for future clinical trials.

Increased Mortality and Comorbidity Associated With Leber's Hereditary Optic Neuropathy: A Nationwide Cohort Study

Leber's hereditary optic neuropathy (LHON) is a mitochondrial genetic disease in which optic neuropathy is considered a key feature. Several other manifestations of LHON have been reported; however, only little is known of their incidence and the life expectancy in LHON patients. This study, based on Danish nationwide health registries, included 141 patients diagnosed with LHON and 297 unaffected family members in the maternal line. The incidence of comorbidities and mortality for patients with LHON and unaffected family members was compared with that in the general population. Having LHON was associated with an almost 2-fold risk of mortality with a rate ratio (RR) of 1.95 (95% confidence interval [CI]: 1.47-2.59; P < 0.001). The incidence of several diseases was increased for LHON patients, but not for family members. The incidence of stroke was 5.73 per 1000 patient-years for LHON patients compared to 2.33 for the general population, and the RR was 2.38 (95% CI: 1.58-3.58; P < 0.001). The incidence of demyelinating disorders was 2.24 compared to 0.21 for the general population; RR was 12.89 (95% CI: 6.70-24.77; P < 0.001). A 4-fold risk of dementia was seen for LHON patients (RR: 4.26, 95% CI: 1.91-9.48; P < 0.001), incidence 1.45 for LHON and 0.37 for the general population. Moreover, LHON patients had an increased risk of epilepsy, atherosclerosis, nerve symptoms, neuropathy, and alcohol-related disorders. The manifestation of LHON was associated with increased mortality and increased incidence of several disorders including stroke, demyelinating disorder, dementia, and epilepsy.

LHON: A look into nuclear and environmental factors; What is “sufficient”?

PurposeIn Leber's Hereditary Optic Neuropathy (LHON) mutations of the mtDNA, are necessary but not sufficient for visual loss. We studied nuclear/environmental interactions in patients and in the Wallace LHON mouse model, to consider what is “sufficient”. The LHON mutation causes a modest decrease in ATP production, but a large increase in ROS production. Nuclear genetic and environmental factors may play upon these factorsMethodsWe noted in the data of Kirkman et al (Brain 2009) that heavy smoking mitigated visual loss by delaying disease onset. We reproduced these peculiar results (Carelli et al, Brain 2013). We also studied the ND6 mutant Wallace mouse model of LHON. 30 mice of 12 groups were sacrificed at 23 months including ND6 mutant vs ND6 wildtype and nicotinamide nucleotide transhydrogenase‐NNT +/+ vs ‐/‐ (NNT deletion), under different environments of air and smoke. 10 experiments compared the axonal nerve fiber spectrums.ResultsIn our patient pedigrees, heavy smoking also mitigated visual loss, but we saw in this, the existence of two subtypes of LHON. Type I, with an abrupt and severe loss of vision in the teens, was characterized by severe losses of both structure and function. Type II occured decades later with a more insidious onset, with less structural than functional loss. Type II patients were more likely to smoke. In the mice, we found the predominant loss of small fibers in NNT −/− mice carrying the ND6 mutation which were exposed to smoke.ConclusionsBoth nuclear modifiers and environmental factors in combination with LHON mutations can lead to vision loss. NNT is a transhydrogenase that couples hydride transfer from NAD(H) to NADP(+) and proton translocation across the inner mitochondrial membrane. NNT is potentially an important nuclear modifying gene for LHON.

Neurological involvement in mitochondrial eye diseases

SummaryThe most frequent mitochondrial eye diseases are Leber's Hereditary Optic Neuropathy (LHON) and Dominant Optic Atrophy (DOA). They are both characterized by optic atrophy due to retinal ganglion cell loss. Extra‐ocular features have been described in both diseases. In particular, neurological involvement has been reported even though, for some of the neurological features in LHON, the possibility of a chance association is still debated.In LHON the main neurological features are: 1) Multiple Sclerosis “like” phenotype 2) Movement disorders including dystonia, parkinsonism and myoclonus 3) Peripheral neuropathy 5) Migraine 6) Hearing loss 6) “LHON‐MELAS‐Leigh” overlap.In DOA, the “plus” subtype is a well‐recognized entity characterized by the occurrence, over the decades, of optic atrophy and sensorineural deafness, polyneuropathy and myopathy with chronic external ophthalmoplegia, and in some families Parkinsonism and dementia. DOA “plus” is typically associated with missense mutations affecting the OPA1 GTPase domain, and skeletal muscle is remarkable for COX deficient fibers and mtDNA multiple deletions. More recently, a Multiple Sclerosis “like” phenotype has been described also in DOA.

Clinical trials for inherited optic neuropathies

SummaryThe best scientific evidence for use of a clinical intervention is based upon data obtained from Phase III clinical trials. The robustness of a clinical trial is dependent on the quality of the metrics collected which in turn is dependent on the design of the study. Clinical trials involving inherited optic neuropathies such as Leber's Hereditary Optic Neuropathy (LHON) encounter some unique challenges which can be divided into two broad groups: patient‐centric factors and test‐centric factors. Patient‐centric factors include the heterogeneous phenotypic presentations in LHON with variable rates of spontaneous recovery and time to nadir and plateau. Test‐centric factors include the subjective nature of most visual metrics; intertest and intratest variability, which is compounded by patient adaptation to visual loss. Collectively these issues must be addressed when designing a clinical trial, especially in rare diseases where patient recruitment can be a challenge

No evidence of association between optic neuritis and secondary LHON mtDNA mutations in patients with multiple sclerosis

Leber's Hereditary Optic Neuropathy (LHON) shares features with Multiple Sclerosis (MS). Both diseases develop optic lesions. Frequent secondary LHON mutations in MS patients may explain the optic damage. Here, we tested the hypothesis that secondary LHON mutations are associated with optic neuritis (ON) in MS patients. We recruited 56 MS subjects with ON and 47 MS subjects without ON. DNA was extracted by salting out, after sampling of peripheral blood from each participant. We completed Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis with appropriate primers and restriction endonucleases for seven secondary LHON mutations. Products were visualized using 3% agarose gel electrophoresis with the aid of DNA safe stain in a UV transilluminator. Accuracy of the genotyping procedure was confirmed by sequencing. Data was analyzed using chi square and Fisher exact tests and logistic regression analysis. There was no significant difference between the numbers of MS subjects with ON and without ON that carried secondary LHON mutations (T4216C [P=0.1], A4917G [P=0.2], G13708A [P=0.6], G15257A [P=1], G15812A [P=0.8], G15927A [P=1], G15928A [P=0.4]). The evidence from the present study are not consistent with the hypothesis that secondary LHON mutations are associated with ON in MS subjects.

Incomplete penetrance in mitochondrial optic neuropathies

Incomplete penetrance characterizes the two most frequent inherited optic neuropathies, Leber's Hereditary Optic Neuropathy (LHON) and dominant optic atrophy (DOA), due to genetic errors in the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA), respectively.For LHON, compelling evidence has accumulated on the complex interplay of mtDNA haplogroups and environmental interacting factors, whereas the nDNA remains essentially non informative. However, a compensatory mechanism of activated mitochondrial biogenesis and increased mtDNA copy number, possibly driven by a permissive nDNA background, is documented in LHON; when successful it maintains unaffected the mutation carriers, but in some individuals it might be hampered by tobacco smoking or other environmental factors, resulting in disease onset. In females, mitochondrial biogenesis is promoted and maintained within the compensatory range by estrogens, partially explaining the gender bias in LHON.Concerning DOA, none of the above mechanisms has been fully explored, thus mtDNA haplogroups, environmental factors such as tobacco and alcohol, and further nDNA variants may all participate as protective factors or, on the contrary, favor disease expression and severity.Next generation sequencing, complemented by transcriptomics and proteomics, may provide some answers in the next future, even if the multifactorial model that seems to apply to incomplete penetrance in mitochondrial optic neuropathies remains problematic, and careful stratification of patients will play a key role for data interpretation. The deep understanding of which factors impinge on incomplete penetrance may shed light on the pathogenic mechanisms leading to optic nerve atrophy, on their possible compensation and, thus, on development of therapeutic strategies.

Editorial introductions

Editorial introductions

Association of Critical Fusion Frequency and Visual Function in Leber's Hereditary Optic Neuropathy

Association of Critical Fusion Frequency and Visual Function in Leber's Hereditary Optic Neuropathy

Changes in Choroidal Thickness follow the RNFL Changes in Leber's Hereditary Optic Neuropathy.

Leber’s hereditary optic neuropathy (LHON) is typically characterized by vascular alterations in the acute phase. The aim of this study was to evaluate choroidal changes occurring in asymptomatic, acute and chronic stages of LHON. We enrolled 49 patients with LHON, 19 with Dominant Optic Atrophy (DOA) and 22 healthy controls. Spectral Domain-Optical Coherence Tomography (SD-OCT) scans of macular and peripapillary regions were performed in all subjects, to evaluate macular and peripapillary choroidal thickness, and retinal nerve fiber layer (RNFL) thicknes. Macular and peripapillary choroidal thicknesses were significantly increased in the acute LHON stage. On the contrary, macular choroidal thickness was significantly reduced in the chronic stage. Furthermore, peripapillary choroidal thickness was decreased in chronic LHON and in DOA. Both RNFL and choroid had the same trend (increased thickness, followed by thinning), but RNFL changes preceded those affecting the choroid. In conclusion, our study quantitatively demonstrated the involvement of the choroid in LHON pathology. The increase in choroidal thickness is a feature of the LHON acute stage, which follows the thickening of RNFL. Conversely, thinning of the choroid is the common outcome in chronic LHON and in DOA.

WN1316, A Novel Anti-Oxidant Compound for the Treatment of Leber's Hereditary Optic Neuropathy (LHON)

WN1316, A Novel Anti-Oxidant Compound for the Treatment of Leber's Hereditary Optic Neuropathy (LHON)

OPA1 gene and mitochondrial optic neuropathy: disease mechanisms and potential therapies

SummaryPrimary inherited optic neuropathies are a group of blinding genetic disorders in which optic atrophy secondary to loss of retinal ganglion cells is a key clinical feature. The commonest causes world‐wide are mutation in mitochondrial DNA (causing Leber's Hereditary Optic Neuropathy) and mutation in the nuclear gene, OPA1 (causing Autosomal Dominant Optic Atrophy: ADOA). 60–75% of patients with autosomal dominant optic atrophy have mutations in the OPA1 gene. The OPA1 protein is targeted to the mitochondria and is involved in regulation of mitochondrial fusion. A better understanding of mitochondrial function, including dynamics, is revealing that functional and structural changes in mitochondrial morphology are important factors in diseases of ageing in the eye and visual system. Key proteins have been discovered which control the balance of mitochondrial fusion and fission and have a range of other functions, such as controlling maintenance of mitochondrial DNA, cell death, autophagy, mitochondrial metabolism and redox signalling. A decline in mitochondrial function plays a role in the ageing process and increases the incidence of age‐related disorders. Mitochondrial optic neuropathies are ‘orphan’ diseases but with the advent of recent trials of novel therapies in patients with the mitochondrial optic neuropathy, Leber's hereditary optic neuropathy, there is the first glimmer of hope for the treatment of this group of patients.

Stem cells in reparing optic nerve damage

SummaryModelling Leber's Hereditary Optic neuropathy using human induced pluripotent stem cells. Human induced pluripotent stem cells (iPSCs) provide an invaluable tool for disease modelling in vitro. Here, we report the generation of iPSCs from LHON patients, their differentiation to retinal ganglion cells (RGCs) for subsequent modelling of phenotypic abnormalities. We also report the manipulation of mitochondrial DNA to correct LHON iPSCs by generation of cybrids. Comparison of LHON iPSCs and their isogenic cybrid control provide novel information on the potential cellular mechanisms leading to RGC death observed in human patients.

The Elusive Pathophysiology of Leber's Hereditary Optic Neuropathy

Leber’s Hereditary Optic Neuropathy (LHON) is a mitochondrially inherited disorder characterized by rapid, subacute vision loss. LHON has historically been a difficult disease to study due to its low incidence. Many questions concerning its pathophysiology have remained unanswered. A significant enigma concerns LHON’s gender bias as represented by the male-to-female ratio of about 4:1. Another source of confusion includes the variable penetrance, since the mitochondrial mutation is necessary, but not sufficient, to cause conversion. A third challenge involves the tissue specificity, since typically only the optic nerve is involved with a specific pattern of optic atrophy due to primary loss of the papillomacular bundle (PMB) in affected individuals. The fourth remaining complexity is the pattern of rapid, subacute vision loss observed in nearly all affected individuals. Several clinical genetic studies, cybrid experiments, and histochemical studies have been conducted to address the three enigmas of gender bias, variable penetrance, and tissue specificity. Despite elucidation of these three aspects of the pathophysiology, the mechanism behind the rapid, significant vision loss remains a mystery.

Retinal Ganglion Cell and Inner Plexiform Layer Loss Correlate with Visual Acuity Loss in LHON: A Longitudinal, Segmentation OCT Analysis.

Describe changes in the retina as vision loss progresses in Leber's Hereditary Optic Neuropathy (LHON) using spectral-domain optical coherence tomography (SD-OCT) autosegmentation, and determine if relationship exists between retinal changes and vision loss. From patient records we identified nine LHON patients who underwent periodic neuro-ophthalmologic examinations and high-resolution SD-OCT as part of their care. We describe the impact of LHON progression on each retinal layer, and the relationship between these structural changes and visual acuity using generalized estimating equations and nonparametric tests. The thickness of the ganglion cell layer (GCL) and inner plexiform layer (IPL) decreased immediately or soon after symptom onset, and this decrease was associated with worsening vision: in the GCL a 1-mm3 volume loss was associated with a 3.2 increase in logMAR visual acuity (95% confidence interval [CI]: 2.1-4.1); in the IPL a 1-mm3 volume loss was associated with a 4.9 increase in visual acuity (95%CI: 6.5-3.2). The retinal nerve fiber layer (RNFL) also thinned, but not until after the GCL and IPL, and only in the papillomacular bundle (PMB) and temporal layers was thinning associated with vision loss. For the first time these analyses describe a structure-function relationship between the retinal changes that occur in LHON patients as their disease progresses and vision worsens. The structural changes in the GCL, IPL, and RNFL preceded structural changes in the other retinal layers. This analysis suggests that the first 6 months after diagnosis define a target for therapeutic intervention, and this can inform treatment guidelines for ongoing therapeutic trials.

Leber’s Hereditary Optic Neuropathy and Ocular Hypertension - a case report

Purpose: to report an unusual case of Leber's Hereditary Optic Neuropathy (LHON) and simultaneous ocular hypertension. Methods: case report of a 40 year-old patient, who complained of recently noticed vision loss in the right eye (OD). Results: OD presented with simultaneous ocular hypertension, optic atrophy and total cupping, leading to the suspicion of glaucomatous optic atrophy. Progressive vision loss was later noticed in the left eye (OS), but no fundoscopic findings were detected in sequential examinations. The patient became increasingly anxious and uncooperative, raising the hypothesis of non-organic visual loss in OS. Several visual evoked potentials were needed to prove reduction of P100 wave. Genetic results for LHON were positive for the 11778 G>A mutation. Conclusions: This case highlights the importance of considering the diagnosis of LHON in all cases of unexplained optic neuropathy, including cases with normal fundoscopic appearance. It also emphasizes that nonorganic vision loss should always be a diagnosis of exclusion in the context of unexplained vision loss. Concomitant LHON and glaucoma have been rarely described and it is uncertain whether LHON-associated mitochondrial DNA mutations are risk factors for glaucoma. However it is hypothesized that the two diseases may have a cumulative effect on retinal ganglion cell damage with the consequent rapid progression of visual impairment.

A multi-parametric workflow for the prioritization of mitochondrial DNA variants of clinical interest.

Assigning a pathogenic role to mitochondrial DNA (mtDNA) variants and unveiling the potential involvement of the mitochondrial genome in diseases are challenging tasks in human medicine. Assuming that rare variants are more likely to be damaging, we designed a phylogeny-based prioritization workflow to obtain a reliable pool of candidate variants for further investigations. The prioritization workflow relies on an exhaustive functional annotation through the mtDNA extraction pipeline MToolBox and includes Macro Haplogroup Consensus Sequences to filter out fixed evolutionary variants and report rare or private variants, the nucleotide variability as reported in HmtDB and the disease score based on several predictors of pathogenicity for non-synonymous variants. Cutoffs for both the disease score as well as for the nucleotide variability index were established with the aim to discriminate sequence variants contributing to defective phenotypes. The workflow was validated on mitochondrial sequences from Leber’s Hereditary Optic Neuropathy affected individuals, successfully identifying 23 variants including the majority of the known causative ones. The application of the prioritization workflow to cancer datasets allowed to trim down the number of candidate for subsequent functional analyses, unveiling among these a high percentage of somatic variants. Prioritization criteria were implemented in both standalone (http://sourceforge.net/projects/mtoolbox/) and web version (https://mseqdr.org/mtoolbox.php) of MToolBox.Electronic supplementary materialThe online version of this article (doi:10.1007/s00439-015-1615-9) contains supplementary material, which is available to authorized users.

Treatment of Leber's hereditary optic neuropathy with EPI‐743: the Brazilian experience

PurposeTo determine the degree of visual improvement in a homogeneous Brazilian cohort of Leber's Hereditary Optic Neuropathy (LHON) 11778 receiving 18–24 months of therapy with the quinone EPI‐743MethodsSix patients with severe vision loss from LHON were enrolled in an open label clinical trial. Each patient received 400 mg TID of EPI‐743. Two patients were treated at the time of conversion, while 4 patients had chronic disease (&gt;5 years). Outcome measures included best corrected visual acuity (BCVA), retinal nerve fiber layer (RNFL) thickness as measured by Optical Coherence Tomography and Humphrey Visual Fields (HVF) mean deviations. Mean deviations were calculated using the HVF algorithm (Stim III) or University of Iowa Visual Field Reading Center Algorithm (Stim V)ResultsVA declined or stabilized in the first 6 months, then improved gradually for all 6 patients and remained stable for 18–24 months with EPI‐743. VF increased marginally in one chronic and both acute patients, while 3 chronic patients remained at baseline. The acute patients showed continued bilateral progression for the first 6 months, consistent with the natural history of LHON. As expected, all eyes showed a decline in RNFL that stabilized after treatmentConclusionsVA improvements continued beyond the initial 1 year period to 18–24 months of treatment, including 4 patients who had lost vision over 5 years prior to start of EPI‐743. This data is consistent with data from the United States where an EPI‐743.