The Optic Nerve at Stake: Update on Environmental Factors Modulating Expression of Leber's Hereditary Optic Neuropathy.

Optic neuropathies are characterized by the degeneration of the optic nerves and represent a considerable individual and societal burden. Notably, Leber's hereditary optic neuropathy (LHON) is a devastating vision disease caused by mitochondrial gene mutations that hinder oxidative phosphorylation and increase oxidative stress, leading to the loss of retinal ganglion neurons and axons. Loss of vision is rapid and severe, predominantly in young adults. Penetrance is incomplete, and the time of onset is unpredictable. Recent findings revealed that the incidence of genetic LHON susceptibility is around 1 in 1000, much higher than believed till now. Environmental factors are critical in LHON triggering or severity. Families at risk have a very strong demand for how to prevent the onset or limit the severity of the disease. Here, we review recent knowledge of the extrinsic determinants of LHON expression, including lifestyle, dietary supplements, common chemicals, and drugs.

ATYPICAL FOVEAL AND PARAFOVEAL ABNORMALITIES IN SICKLE CELL DISEASE.

PURPOSE: The primary aim was to describe the patterns of paramacular involvement, not yet reported but that optical coherence tomography angiography can now detect in patients with sickle cell disease. The secondary aim was to search arguments concerning the physiopathogeny of paramacular involvement. METHODS: This institutional cohort retrospective study was conducted in a Referral Center for Ophthalmological Rare Diseases. Follow-up included an ophthalmologic examination with optical coherent tomography and optical coherent tomography angiography. RESULTS: One hundred and thirty-two patients with SCD were included. Typical sickle cell maculopathy was observed in temporal area in 84 eyes (40.0%) of SS patients and eight eyes (14.8%) of SC patients ( P < 0.001). Enlargement of the foveal avascular zone was observed in 10 eyes of eight SS patients. Two atypical parafoveal abnormalities were found in SS patients only. The first one consisted of macular thinning with normal vascularization in 15 eyes of 11 patients. The second atypical maculopathy was large areas of loss of vascularization without retinal thinning 10 eyes of six patients. Multivariate analysis did not show a statistically significant relation between the peripheral sickle retinopathy stage and the different type of sickle cell maculopathy ( P = 0.21). CONCLUSION: Those atypical sickle cell maculopathy may correspond to early forms preceding a typical sickle cell disease maculopathy (SCDM). This would point toward several physiopathogenic mechanisms. The first one included the existence of ischemia that can be related to anemia. Presence of retinal thinning without vascular involvement point out to a neurogenic mechanism.

Relationship between paramacular thinning, cerebral vasculopathy, and hematological risk factors in sickle cell disease.

Purpose: To identify risk factors for sickle cell maculopathy due to hematological parameters (especially anemia and hemolysis) or cerebral vasculopathy. Methods: This retrospective study was conducted at a Referral Center. The follow-up included optical coherent tomography/optical coherent tomography angiography, neuro-radiological imaging, and a hematological assessment (hemoglobin, hemoglobin S level, reticulocytes, mean corpuscular volume, bilirubin, and lactate dehydrogenase). Results: Hundred and thirty-two sickle cell patients were included. Maculopathy was observed in 127 eyes of SS patients and 10 eyes of SC patients (p < 0.001), unrelated to peripheral retinopathy. Cerebral vasculopathy was more frequent in SS patients (p < 0.001) and was also associated with the presence of maculopathy (p = 0.049), and it was related to peripheral retinopathy (p < 0.001). All biological parameters significantly differed according to the genotype (p < 0.001) but not according to the presence of cerebral vasculopathy or maculopathy. In the multivariate analysis, reticulocytes and bilirubin were associated with the presence of cerebral vasculopathy and maculopathy. Conclusion: The data obtained were consistent with the role of anemia or hemolysis markers in cerebral vasculopathy and macular involvement. As a trend of hemolysis appears to be a risk factor for these complications, this validates the use of preventive plasmapheresis in these patients.

Case Report: Abnormalities of sperm motility and morphology in a patient with Leber hereditary optic neuropathy: Improvement after idebenone therapy.

Case: We report the sperm characteristics of a male patient who developed, when he was 18 years old, a Leber hereditary optic neuropathy, a hereditary optic neuropathy due to mtDNA mutation as well as variants in the nuclear DNA. At the age of 30 years-old, he complained of infertility lasting for 2 years. Semen analyses showed low motility spermatozoa and a high percentage of morphological or ultrastructural abnormalities. Levels of epididymal markers were strongly atypical. Idebenone was prescribed as treatment of his Leber hereditary optic neuropathy in order to improve his visual acuity. After 5 months of this treatment, motility of spermatozoa increased, and their vitality improved. A natural conception occurred. Outcome: This case is the first description of an anomaly of spermatozoas and of the epididymis epithelium in a patient with Leber hereditary optic neuropathy. It draws attention to sperm pathologies in patients with mitochondrial disorders. The role of the mtDNA mutations must be suspected since it plays an important role in the development and motility of spermatozoa. In addition, idebenone can by-pass the complex I and transfer electrons to complex III. It has been suspected to have a favorable effect on spermatogenesis. Conclusion: This case confirms the possibility of sperm dysfunction in Leber hereditary optic neuropathy and the interest of idebenone as a treatment for infertility due to mtDNA mutations in human.

MCAT Mutations Cause Nuclear LHON-like Optic Neuropathy.

Pathological variants in the nuclear malonyl-CoA-acyl carrier protein transacylase (MCAT) gene, which encodes a mitochondrial protein involved in fatty-acid biogenesis, have been reported in two siblings from China affected by insidious optic nerve degeneration in childhood, leading to blindness in the first decade of life. After analysing 51 families with negative molecular diagnostic tests, from a cohort of 200 families with hereditary optic neuropathy (HON), we identified two novel MCAT mutations in a female patient who presented with acute, sudden, bilateral, yet asymmetric, central visual loss at the age of 20. This presentation is consistent with a Leber hereditary optic neuropathy (LHON)-like phenotype, whose existence and association with NDUFS2 and DNAJC30 has only recently been described. Our findings reveal a wider phenotypic presentation of MCAT mutations, and a greater genetic heterogeneity of nuclear LHON-like phenotypes. Although MCAT pathological variants are very uncommon, this gene should be investigated in HON patients, irrespective of disease presentation.

A plasma metabolomic signature of Leber hereditary optic neuropathy showing taurine and nicotinamide deficiencies.

Leber's hereditary optic neuropathy (LHON) is the most common disorder due to mitochondrial DNA mutations and complex I deficiency. It is characterized by an acute vision loss, generally in young adults, with a higher penetrance in males. How complex I dysfunction induces the peculiar LHON clinical presentation remains an unanswered question. To gain an insight into this question, we carried out a non-targeted metabolomic investigation using the plasma of 18 LHON patients, during the chronic phase of the disease, comparing them to 18 healthy controls. A total of 500 metabolites were screened of which 156 were accurately detected. A supervised Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) highlighted a robust model for disease prediction with a Q2 (cum) of 55.5%, with a reliable performance during the permutation test (cross-validation analysis of variance, P-value = 5.02284e-05) and a good prediction of a test set (P = 0.05). This model highlighted 10 metabolites with variable importance in the projection (VIP) > 0.8. Univariate analyses revealed nine discriminating metabolites, six of which were the same as those found in the Orthogonal Projections to Latent Structures Discriminant Analysis model. In total, the 13 discriminating metabolites identified underlining dietary metabolites (nicotinamide, taurine, choline, 1-methylhistidine and hippurate), mitochondrial energetic substrates (acetoacetate, glutamate and fumarate) and purine metabolism (inosine). The decreased concentration of taurine and nicotinamide (vitamin B3) suggest interesting therapeutic targets, given their neuroprotective roles that have already been demonstrated for retinal ganglion cells. Our results show a reliable predictive metabolomic signature in the plasma of LHON patients and highlighted taurine and nicotinamide deficiencies.

Cyclosporine A does not prevent second-eye involvement in Leber's hereditary optic neuropathy.

BACKRGROUND: Evaluation of the efficacy of oral cyclosporine A as a prophylactic agent in preventing second-eye involvement in Leber's hereditary optic neuropathy (LHON) in a prospective, open-label, non-randomized, multicenter pilot study. Only LHON patients aged 18 years or more, with confirmed primary mitochondrial DNA mutations and strictly unilateral optic neuropathy occurring within 6 months prior to enrolment, were included in the study. All these patients, receiving treatment with oral cyclosporine (Neoral®, Novartis) at 2.5 mg/kg/day, were examined at three-month intervals for a year. The primary endpoint was the best corrected visual acuity in the unaffected eye; the secondary endpoints were the best corrected visual acuity in the first eye affected, the mean visual field defect on automated perimetry, the thickness of the perifoveal retinal ganglion cell inner plexiform layer, and the thickness of the peripapillary retinal nerve fiber layer in both eyes. RESULTS: Among the 24 patients referred to our institution with genetically confirmed LHON, between July 2011 and April 2014, only five patients, four males and one female, fulfilled the inclusion criteria. Age at enrolment ranged from 19 to 42 years (mean: 27.2 years; median: 26 years), four patients harbored the m.11778G > A pathogenic variant, and one the m.14484 T > C pathogenic variant. The time-interval between the onset of symptoms and inclusion in the study ranged from 7 to 17 weeks (mean: 11.8 weeks; median: 9 weeks). Despite treatment with oral cyclosporine A, all patients eventually experienced bilateral eye involvement, occurring within 11-65 weeks after the initiation of treatment. Over the study time period, the average best corrected visual acuity worsened in the first eye affected; by the end of the study, both eyes were equally affected. CONCLUSIONS: Oral cyclosporine, at 2.5 mg/kg/day, did not prevent second-eye involvement in patients with strictly unilateral Leber's hereditary optic neuropathy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02176733 . Registrated June 25, 2014.

Cardiac Disorders in Patients With Leber Hereditary Optic Neuropathy.

BACKGROUND: Cardiac abnormalities have been described in patients with Leber hereditary optic neuropathy (LHON). Some are life-threatening because of the risk of ventricular fibrillation and sudden death. The purpose of our study was to better characterize the cardiac abnormalities in a large patient cohort with LHON. METHODS: A retrospective study of the electrocardiogram (EKG) results performed on all patients with LHON evaluated at The Reference Center for Rare Diseases in Ophthalmology, Paris, France, from January 2015 to June 2017. RESULTS: Our series included 73 patients with LHON (9 women/64 men) with a mean age of 30.29 ± 14.48 years. Although only 1 patient had cardiac complaints, cardiac abnormalities were detected in 17 patients (23.2%): 9 patients had an excitation syndrome, 6 had atrioventricular block, and 2 had repolarization abnormalities. All patients harbored mtDNA point mutations 11778 or 3460. CONCLUSIONS: Cardiac abnormalities occur frequently enough in patients with LHON that a baseline EKG is warranted. However, further studies are needed to determine the true cardiac risk associated with specific LHON mtDNA mutations.

International Consensus Statement on the Clinical and Therapeutic Management of Leber Hereditary Optic Neuropathy.

Leber hereditary optic neuropathy (LHON) is currently estimated as the most frequent mitochondrial disease (1 in 27,000-45,000). Its molecular pathogenesis and natural history is now fairly well understood. LHON also is the first mitochondrial disease for which a treatment has been approved (idebenone-Raxone, Santhera Pharmaceuticals) by the European Medicine Agency, under exceptional circumstances because of the rarity and severity of the disease. However, what remains unclear includes the optimal target population, timing, dose, and frequency of administration of idebenone in LHON due to lack of accepted definitions, criteria, and general guidelines for the clinical management of LHON. To address these issues, a consensus conference with a panel of experts from Europe and North America was held in Milan, Italy, in 2016. The intent was to provide expert consensus statements for the clinical and therapeutic management of LHON based on the currently available evidence. We report the conclusions of this conference, providing the guidelines for clinical and therapeutic management of LHON.

Relevance of Identifying JAG1 Mutations in Patients With Isolated Posterior Embryotoxon.

PURPOSE: Although posterior embryotoxon (PE) has a high incidence in the general population, clinicians should exclude any sign of glaucoma in its presence. This anatomic abnormality is often referred to as "isolated" when the intraocular pressure is normal. Nevertheless, it may be the only sign of Alagille syndrome (AS) that can be clinically heterogenous, as presented here. This possibility must be known, to look for involvement of other organs, and in case of suspicion, mutation of the JAG1 gene must be considered. METHODS: In this case series, we present the observation of a family with 3 individuals from 3 generations, in whom PE was a marker of AS. RESULTS: PE were observed in these 3 patients and considered as "isolated" as the intraocular pressure was normal. The 2 elder patients were also followed for atypical retinal dystrophy with speckling of the retinal pigment and optic disc drusen. AS syndrome was suspected when mild liver dysfunction was detected in the youngest girl. The detection of JAG1 mutation confirmed this diagnosis. CONCLUSIONS: As AS can be clinically heterogenous, it must be considered in case of isolated PE. Involvement of other organs must be looked for to search for mutation of the JAG1 gene in relevant cases.

Recessive and Dominant De Novo ITPR1 Mutations Cause Gillespie Syndrome.

Gillespie syndrome (GS) is a rare variant form of aniridia characterized by non-progressive cerebellar ataxia, intellectual disability, and iris hypoplasia. Unlike the more common dominant and sporadic forms of aniridia, there has been no significant association with PAX6 mutations in individuals with GS and the mode of inheritance of the disease had long been regarded as uncertain. Using a combination of trio-based whole-exome sequencing and Sanger sequencing in five simplex GS-affected families, we found homozygous or compound heterozygous truncating mutations (c.4672C>T [p.Gln1558(∗)], c.2182C>T [p.Arg728(∗)], c.6366+3A>T [p.Gly2102Valfs5(∗)], and c.6664+5G>T [p.Ala2221Valfs23(∗)]) and de novo heterozygous mutations (c.7687_7689del [p.Lys2563del] and c.7659T>G [p.Phe2553Leu]) in the inositol 1,4,5-trisphosphate receptor type 1 gene (ITPR1). ITPR1 encodes one of the three members of the IP3-receptors family that form Ca(2+) release channels localized predominantly in membranes of endoplasmic reticulum Ca(2+) stores. The truncation mutants, which encompass the IP3-binding domain and varying lengths of the modulatory domain, did not form functional channels when produced in a heterologous cell system. Furthermore, ITPR1 p.Lys2563del mutant did not form IP3-induced Ca(2+) channels but exerted a negative effect when co-produced with wild-type ITPR1 channel activity. In total, these results demonstrate biallelic and monoallelic ITPR1 mutations as the underlying genetic defects for Gillespie syndrome, further extending the spectrum of ITPR1-related diseases.

Objective Evaluation of Vergence Disorders and a Research-Based Novel Method for Vergence Rehabilitation.

PURPOSE: We performed video-oculography to evaluate vergence eye movement abnormalities in students diagnosed clinically with vergence disorders. We tested the efficiency of a novel rehabilitation method and evaluated its benefits with video-oculography cross-correlated with clinical tests and symptomatology. METHODS: A total of 19 students (20-27 years old) underwent ophthalmologic, orthoptic examination, and a vergence test coupled with video-oculography. Eight patients were diagnosed with vergence disorders with a high symptomatology score (CISS) and performed a 5-week session of vergence rehabilitation. Vergence and rehabilitation tasks were performed with a trapezoid surface of light emitting diodes (LEDs) and adjacent buzzers (US 8851669). We used a novel Vergence double-step (Vd-s) protocol: the target stepped to a second position before the vergence movement completion. Afterward the vergence test was repeated 1 week and 1 month later. RESULTS: Abnormally increased intertrial variability was observed for many vergence parameters (gain, duration, and speed) for the subjects with vergence disorders. High CISS scores were correlated with variability and increased latency. After the Vd-s, variability of all parameters dropped to normal or better levels. Moreover, the convergence and divergence latency diminished significantly to levels better than normal; benefits were maintained 1 month after completion of Vd-s. CISS scores dropped to normal level, which was maintained up to 1 year. CONCLUSIONS AND TRANSLATIONAL RELEVANCE: Intertrial variability is the major marker of vergence disorders. The Vd-s research-based method leads to normalization of vergence properties and lasting removal of symptoms. The efficiency of the method is due to the spatiotemporal parameters of repetitive trials that stimulate neural plasticity.

Visual Impairment in Pseudoxanthoma Elasticum: A Survey of 40 Patients.

BACKGROUND: Pseudoxanthoma elasticum is an inherited disorder of connective tissue characterized, among other symptoms, by impaired vision. OBJECTIVE: To evaluate the nature and age of onset of ophthalmologic manifestations in pseudoxanthoma elasticum. PATIENTS AND METHODS: Forty consecutive patients affected with pseudoxanthoma elasticum underwent measurements of their refractive error and visual acuity, together with slit-lamp examination. RESULTS: The mean age of the patients (8 M, 32 F) was 43.35 years. Fifty-seven eyes (33 patients, mean age: 40.75 years) had a BCVA >20/50 whereas 23 eyes in 16 patients (mean age: 53.31 years) had ≤20/50. Seven patients (17.50%), all but one over 52 years old, were visually disabled. BCVA ≤20/50 in at least one eye was observed in 73.33% of patients of 52 years old or older and in 20.00% of patients younger than 52, respectively. Angioid streaks were observed in 75 eyes (93.75%) and extended toward the macula in 51 eyes from 29 patients. Macular involvement was observed for the first time at a mean age of 44.28 years. Neovascularization was observed in 28 eyes (17 patients; mean age: 51.70 years), all with poor BCVA. CONCLUSION: Macular choroidal neovascularization is frequent in pseudoxanthoma elasticum, and accounts for the poor ophthalmologic natural history of the disease. Patients should be advised to self-monitor their visual acuity using the Amsler grid. The frequency of choroidal neovascularization appears age-dependent, suggesting that bi-yearly fundus examination is appropriate in young patients whereas patients older than 40 should be examined twice a year.

Saccade-vergence properties remain more stable over short-time repetition under overlap than under gap task: a preliminary study.

Under natural circumstances, saccade-vergence eye movements are among the most frequently occurring. This study examines the properties of such movements focusing on short-term repetition effects. Are such movements robust over time or are they subject to tiredness? 12 healthy adults performed convergent and divergent combined eye movements either in a gap task (i.e., 200 ms between the end of the fixation stimulus and the beginning of the target stimulus) or in an overlap task (i.e., the peripheral target begins 200 ms before the end of the fixation stimulus). Latencies were shorter in the gap task than in the overlap task for both saccade and vergence components. Repetition had no effect on latency, which is a novel result. In both tasks, saccades were initiated later and executed faster (mean and peak velocities) than the vergence component. The mean and peak velocities of both components decreased over trials in the gap task but remained constant in the overlap task. This result is also novel and has some clinical implications. Another novel result concerns the accuracy of the saccade component that was better in the gap than in the overlap task. The accuracy also decreased over trials in the gap task but remained constant in the overlap task. The major result of this study is that under a controlled mode of initiation (overlap task) properties of combined eye movements are more stable than under automatic triggering (gap task). These results are discussed in terms of saccade-vergence interactions, convergence-divergence specificities and repetition versus adaptation protocols.

Differential auditory-oculomotor interactions in patients with right vs. left sided subjective tinnitus: a saccade study.

Subjective tinnitus (ST) is a frequent but poorly understood medical condition. Recent studies demonstrated abnormalities in several types of eye movements (smooth pursuit, optokinetic nystagmus, fixation, and vergence) in ST patients. The present study investigates horizontal and vertical saccades in patients with tinnitus lateralized predominantly to the left or to the right side. Compared to left sided ST, tinnitus perceived on the right side impaired almost all the parameters of saccades (latency, amplitude, velocity, etc.) and noticeably the upward saccades. Relative to controls, saccades from both groups were more dysmetric and were characterized by increased saccade disconjugacy (i.e., poor binocular coordination). Although the precise mechanisms linking ST and saccadic control remain unexplained, these data suggest that ST can lead to detrimental auditory, visuomotor, and perhaps vestibular interactions.

Benefit of bi-ocular visual stimulation for postural control in children with strabismus.

Vision is important for postural control as is shown by the Romberg quotient (RQ): with eyes closed, postural instability increases relative to eyes open (RQ = 2). Yet while fixating at far distance, postural stability is similar with eyes open and eyes closed (RQ = 1). Postural stability can be better with both eyes viewing than one eye, but such effect is not consistent among healthy subjects. The first goal of the study is to test the RQ as a function of distance for children with convergent versus divergent strabismus. The second goal is to test whether vision from two eyes relative to vision from one eye provides better postural stability. Thirteen children with divergent strabismus and eleven with convergent strabismus participated in this study. Posturtography was done with the Techno concept device. Experiment 1, four conditions: fixation at 40 cm and at 200 cm both with eyes open and eyes covered (evaluation of RQ). Experiment 2, six conditions: fixation at 40 cm and at 200 cm, with both eyes viewing or under monocular vision (dominant and non-dominant eye). For convergent strabismus, the groups mean value of RQ was 1.3 at near and 0.94 at far distance; for divergent, it was 1.06 at near and 1.68 at far. For all children, the surface of body sway was significantly smaller under both eyes viewing than monocular viewing (either eye). Increased RQ value at near for convergent and at far for divergent strabismus is attributed to the influence of the default strabismus angle and to better use of ocular motor signals. Vision with the two eyes improves postural control for both viewing distances and for both types of strabismus. Such benefit can be due to complementary mechanisms: larger visual field, better quality of fixation and vergence angle due to the use of visual inputs from both eyes.

Postural control in nonamblyopic children with early-onset strabismus.

PURPOSE: In healthy subjects, the postural stability in orthostatic position is better when fixating at near than at far. Increase in the convergence angle contributes to this effect. Children with strabismus present a deficit in vergence. We evaluated postural control in children with respect to the vergence angle as they fixated at different depths, thereby engaging in active vergence movements. METHODS: A TechnoConcept platform was used to record the postural stability of 11 subjects (mean age 11.18 ± 4.02 years) with convergent strabismus and 13 (mean age 11.31 ± 3.54 years) with divergent strabismus in 3 conditions: fixation at 40 cm, at 2 m, and active vergence movements between 20 and 50 cm. RESULTS: The mediolateral body sway decreased significantly with proximity for convergent strabismus (from 3.78-2.70 mm) but increased significantly for divergent strabismus (from 3.27-3.97). Relative to fixation, vergence eye movements resulted in a statistically significant increase in mediolateral body sway for convergent strabismus (3.55 vs. 2.70) and a decrease for divergent strabismus (3.11 vs. 3.97, P = 0.047). Vergence eye movements were associated with the least variance of speed (99 mm(2)/s(2) for convergent and 117 mm(2)/s(2) for divergent strabismus), so less energy was required to control body sway. CONCLUSIONS: The fixation depth at which postural stability is best is proximal for convergent strabismus and distal for divergent strabismus. Optimal postural stability might be mediated by preponderant eye movement signals related to the angle of strabismus. Reduction of variance of speed in the active vergence condition corroborates our hypothesis.

[Decrease in visual acuity: retinal or optic nerve?]. / Baisse de l'acuité visuelle: rétine ou nerf optique?

Identify the role of the retina or of the optic nerve as an etiology of visual decease is sometimes difficult and requires eliminating obvious causes, including toxic or inherited ones. These two last causes are responsible for bilateral forms, though sometimes asymmetrical. The existence of an abnormality of the retinal examination and pupillary movement is an important diagnostic argument that must be systematically looked for, as well as the existence of an afferent pupillary defect in unilateral visual deficit. It is often required perform complementary examinations and to compare their results. However, in some cases, only the disease progression and the repetition of examinations allow to settle between these two etiologies.

Rare primary mitochondrial DNA mutations and probable synergistic variants in Leber's hereditary optic neuropathy.

BACKGROUND: Leber's hereditary optic neuropathy (LHON) is a maternally inherited blinding disorder, which in over 90% of cases is due to one of three primary mitochondrial DNA (mtDNA) point mutations (m.11778G>A, m.3460G>A and m.14484T>C, respectively in MT-ND4, MT-ND1 and MT-ND6 genes). However, the spectrum of mtDNA mutations causing the remaining 10% of cases is only partially and often poorly defined. METHODOLOGY/PRINCIPAL FINDINGS: In order to improve such a list of pathological variants, we completely sequenced the mitochondrial genomes of suspected LHON patients from Italy, France and Germany, lacking the three primary common mutations. Phylogenetic and conservation analyses were performed. Sixteen mitochondrial genomes were found to harbor at least one of the following nine rare LHON pathogenic mutations in genes MT-ND1 (m.3700G>A/p.A132T, m.3733G>A-C/p.E143K-Q, m.4171C>A/p.L289M), MT-ND4L (m.10663T>C/p.V65A) and MT-ND6 (m.14459G>A/p.A72V, m.14495A>G/p.M64I, m.14482C>A/p.L60S, and m.14568C>T/p.G36S). Phylogenetic analyses revealed that these substitutions were due to independent events on different haplogroups, whereas interspecies comparisons showed that they affected conserved amino acid residues or domains in the ND subunit genes of complex I. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that these nine substitutions are all primary LHON mutations. Therefore, despite their relative low frequency, they should be routinely tested for in all LHON patients lacking the three common mutations. Moreover, our sequence analysis confirms the major role of haplogroups J1c and J2b (over 35% in our probands versus 6% in the general population of Western Europe) and other putative synergistic mtDNA variants in LHON expression.