Natural history of patients with Leber hereditary optic neuropathy—results from the REALITY study

Background/objectives REALITY is an international observational retrospective registry of LHON patients evaluating the visual course and outcome in Leber hereditary optic neuropathy (LHON). Subjects/methods Demographics and visual function data were collected from medical charts of LHON patients with visual loss. The study was conducted in 11 study centres in the United States of America and Europe. The collection period extended from the presymptomatic stage to at least more than one year after onset of vision loss (chronic stage). A Locally Weighted Scatterplot Smoothing (LOWESS) local regression model was used to analyse the evolution of best-corrected visual acuity (BCVA) over time. Results 44 LHON patients were included; 27 (61%) carried the m.11778G>A ND4 mutation, 8 (18%) carried the m.3460G>A ND1 mutation, and 9 (20%) carried the m.14484T>C ND6 mutation. Fourteen (32%) patients were under 18 years old at onset of vision loss and 5 (11%) were below the age of 12. The average duration of follow-up was 32.5 months after onset of symptoms. At the last observed measure, mean BCVA was 1.46 LogMAR in ND4 patients, 1.52 LogMAR in ND1 patients, and 0.97 LogMAR in ND6 patients. The worst visual outcomes were reported in ND4 patients aged at least 15 years old at onset, with a mean BCVA of 1.55 LogMAR and no tendency for spontaneous recovery. The LOESS modelling curve depicted a severe and permanent deterioration of BCVA. Conclusions Amongst LHON patients with the three primary mtDNA mutations, adult patients with the m.11778G>A ND4 mutation had the worst visual outcomes, consistent with prior reports.


Introduction
Leber hereditary optic neuropathy (LHON) is an inherited optic neuropathy caused by mitochondrial DNA (mtDNA) mutations, which affect complex I subunits of the mitochondrial respiratory chain, impairing mitochondrial respiration and increasing production of reactive oxygen species [1][2][3][4]. Retinal ganglion cells (RGCs) are particularly vulnerable to mitochondrial dysfunction, which may lead to apoptotic cell death and axonal degeneration, ultimately resulting in optic atrophy [5,6]. LHON affects~1 in 30,000 to 1 in 50,000 people [4,7,8], with a peak age of onset between 15 and 35 years, and a large male predominance [3,[9][10][11][12]. LHON is characterised by bilateral painless central vision loss, typically sequential, with the fellow eye undergoing disease conversion weeks to a few months after onset in the first eye [13][14][15]. There is a well-documented incomplete penetrance, implying that the mtDNA mutations are necessary, but not sufficient to precipitate visual loss. Although there is considerable interfamilial variability, Members  approximately 50% of men and 10% of women who carry one of the primary LHON mutations will experience visual loss during their lifetimes, highlighting also a higher male prevalence [8,14,16]. Three mtDNA point mutations account for about 90% of LHON cases: m.11778G>A in ND4, m.3460G>A in ND1, and m.14484T>C in ND6 [3,4]. The m.11778G>A mutation is the most common cause of LHON worldwide and it is known to be a severe mutation with less than 15% of patients experiencing some degree of visual recovery [17,18]. Children have a better visual prognosis, especially when age of onset is before 12 years [10,19]. Treatment options for LHON remain limited, with some improvement demonstrated in subgroups of LHON patients treated with idebenone [20][21][22], and some early promising results with intravitreal gene therapy [23][24][25][26].
The REALITY study is an international multicenter observational retrospective registry of LHON patients designed to evaluate the natural history of the three most common disease-causing mtDNA mutations and the factors influencing the visual outcome.

Study design
Eleven study centres in the US and Europe (France, Italy, Spain, and the United Kingdom) participated in the REA-LITY registry. To qualify for study inclusion, patients had to have a diagnosis of LHON confirmed by genotyping for one of the three primary mutations (m.11778G>A in ND4, m.3460G>A in ND1 and m.14484T>C in ND6). Furthermore, patients were included only if they had undergone at least two visual function assessments, performed at any time between Year 1 and Year 3 after onset of vision loss. There was no restriction on age, and patients could have received idebenone or any other treatment. The primary source of demographic and clinical data was the enroled subjects' medical records.
The relevant local Independent Ethics Committees approved the study protocol before subjects were identified and data collected. For patients under 18 years old, permission from a legal guardian was required to participate in the study. This study was conducted in accordance with the provisions of the Declaration of Helsinki and Good Clinical Practice guidelines.

Best-corrected visual acuity (BCVA) analysis
On-chart BCVA measures, expressed in decimal fraction or Snellen notation, were converted into LogMAR values. Offchart BCVA was assigned the following LogMAR values: 2.0 for count fingers; 2.3 for hand motion based on the Lange scale equivalence [27]; 4.0 for light perception; and 4.5 for no light perception.

Data analysis
The Full Analysis Set included all enroled subjects whose eligibility was confirmed. Subgroup analyses were performed by LHON genotype (ND4, ND1, ND6), age at onset of vision loss (two cut-offs were applied: 12 and 15 years old), and idebenone treatment status. Change in BCVA was evaluated from the presymptomatic phase to the last available clinic observation. Missing data for presymptomatic BCVA were imputed a LogMAR value of 0 (Snellen equivalent 20/20; decimal fraction equivalent 1) [28,29]. No other data imputation were performed for missing data.
A Locally Weighted Scatterplot Smoothing (LOWESS), non-parametric, local regression model was used on the individual BCVA data points of the LHON ND4 patients who were at least 15 years old at onset. The resulting curve depicting the evolution of BCVA over time was based on a series of polynomial regressions around each data point. The regressions used a limited look back and look forward, giving distant points less weight. The starting point of the curve was set at the onset of vision loss and included presymptomatic values (missing presymptomatic data were imputed 0 LogMAR). All computations and generation of analysis datasets and tables were performed using SAS ® software version 9.4 or higher (SAS Institute, Cary, NC, USA).

Study population characteristics and follow-up period
A total of 44 affected LHON patients were included in the REALITY study: 34/44 (77%) from European countries (France, Italy, Spain, and the United Kingdom) and 10/44 (23%) from the United States of America ( Table 1). The majority of enroled patients were male (33/44, 75%) and Caucasian (33/44, 75%) ( Table 1). The mean age at onset of vision loss was 27.9 years (range: 4-71 years), with 30/44 (68%) patients ≥18 years old at the onset of vision loss. The proportion of patients who were at least 15 years old at onset was 82% (36/44). Five (11%) patients were 12 years old or younger at onset.
Three LHON patients had been diagnosed with an additional genetic syndrome: (i) the blepharophimosis, ptosis, and epicanthus inversus syndrome; (ii) the Ehlers-Danlos syndrome; (iii) and the Marcus-Gunn syndrome. In addition, one woman was diagnosed with multiple sclerosis following the onset of visual loss from LHON.
Most patients (27/44, 61%) were seen by an ophthalmologist or a neuro-ophthalmologist within the first 3 months of disease onset. The mean time from onset to the first visit was 5.7 months (range: 0-25.7 months), and the mean duration of follow-up was 32.5 months (range: 14.1-178.3 months) ( Table 2). Seven patients (7/44, 16%) had a follow-up of more than 3 years, 6 of whom carried the m.11778G>A mutation and one the m.14484T>C mutation. The mean number of BCVA visits per patient was 7.9, with a range of 4-28 visits reported per patient.
Date of onset of vision loss in the first eye to be affected was used to calculate times from onset for each patient.  Table S1). For nine of these patients, the mean (SD) cumulative duration of treatment with idebenone was 70.0 (24.9) months. At the last observed measure, the mean (SD) BCVA for the idebenone-treated group was 1.57 (0.62) LogMAR on average 40.5 (38.0) months after onset ( Table 3).
The LOWESS curve depicting the evolution of BCVA over time showed a marked loss of visual acuity in the first 18 months after disease onset (Fig. 2). Following this initial drop of vision, the deterioration of BCVA continued at a slower rate and no trend of visual recovery was observed.

Discussion
The REALITY study cohort is representative of the general LHON patient population with regards to the age of onset (mean of 27.9 years) and gender distribution (75% males) [3,12]. Prior studies have estimated the peak age of onset to be between 15 and 35 years old, although the reported range for age at onset in molecularly-confirmed LHON patients varies from 2 to 87 years of age [3,10,11]. The proportion of patients in REALITY who were less than 15 years old at disease onset was 18% (8/44) and the predominant genotype among all included patients was the m.11778G>A ND4 mutation (61%), also in keeping with the literature. [5,6,15,17,[30][31][32] Half of the patients enroled in REA-LITY had received treatment with idebenone for a cumulative period of more than 4 years.
The genetic syndromes reported in three patients included in REALITY are unlikely to be related to the underlying LHON-causative mtDNA mutation. One additional patient was diagnosed with multiple sclerosis. The co-occurrence of visual loss due to LHON and central nervous system demyelination is often referred to as Harding's disease [33], and this overlap syndrome can occur with all three primary LHON mutations, predominantly in women [15,33,34]. BCVA best-corrected visual acuity; SD standard deviation. a Missing presymptomatic BCVA were assigned a value of 0 LogMAR (normal visual acuity). The two most important predictors of visual outcome in LHON are the underlying causative mtDNA mutation and the age at onset of vision loss. In the REALITY study, LHON patients carrying the m.14484T>C mutation achieved better final vision compared with those carrying the m.11778G>A and m.3460G>A mutations. The literature reports that children who become affected at the age of 12 years or younger achieve significantly better vision compared with adult-onset LHON patients [13,17,19,35]. Accordingly, in the REALITY registry we observed that patients ≤12 years old at onset had a mean final BCVA of 0.65 LogMAR, compared with 1.46 LogMAR for those over 12 years old at onset. Furthermore, the majority of eyes of LHON patients who were below the age of 15 at onset had a BCVA better than 1.0 LogMAR at the last observed measure, and all the ND4 and ND6 patients younger than 15 at onset had on-chart visual acuity in both eyes, confirming the better visual prognosis in these younger groups.
In the LHON ND4 subgroup of patients who were at least 15 years old at onset, the majority of eyes had a BCVA of 1.0 LogMAR or worse at the last observed measure. The m.11778G>A mutation is known to carry a poor prognosis, with <15% of patients experiencing spontaneous partial visual recovery, and few achieving visual acuities better than 20/200. [17] In REALITY, 84.8% (39/46) of eyes from LHON ND4 patients who were at least 15 at onset had BCVA of 20/200 or worse, and 47.8% (22/46) were off-chart at the last observed measure. In this subgroup, a LOWESS model curve showed a decline in two phases: a rapid decline in the first 18 months, followed by a progressive slow decline. A prospective longterm follow-up study of untreated LHON ND4 patients is needed to confirm the projection of this model, which has important implications for the treatment of chronic cases.
The REALITY registry bears a number of limitations due to the retrospective nature of its study design and the relatively small sample size of 44 LHON patients. Nevertheless, the findings of this study confirm the better visual outcome with childhood-onset LHON and the poor visual prognosis for patients with the m.11778G>A mutation who were at least 15 years old at disease onset.

Summary
What was known before • LHON is a rare mitochondrial blinding disease affecting mostly males between 15 and 35 years old.
• Three primary mtDNA mutations are responsible for 90% of the cases.
• Spontaneous partial visual recovery is scarcely reported in the literature.

What this study adds
• This international retrospective registry study provided a better understanding of the evolution of visual acuity in LHON patients, including clinical data from 44 subjects aged 4 to 71 years at onset of vision loss.
• LHON subjects carrying the ND4 mutation did not show a trend for spontaneous improvement over time.
• LHON subjects aged 12 or less at onset showed the best visual prognosis.

Data and materials availability
All data associated with this study are available in the main text or the supplementary materials  Author contributions All authors participated in the conceptualisation, data collection, analysis, interpretation, writing, and critical review of the manuscript.
Funding GenSight Biologics (Paris, France) fully funded and sponsored the study.

Compliance with ethical standards
Conflict of interest PYWM is a consultant for GenSight Biologics and Stealth BioTherapeutics and has received research support from GenSight Biologies and Santhera Pharmaceuticals. NJN is a consultant for GenSight Biologics, Santhera Pharmaceuticals and Stealth Bio-Therapeutics, has received research support from GenSight Biologics and Santhera Pharmaceuticals, served on the Data Safety Monitoring Board for the Quark NAION study and is a medical-legal consultant. VC is a consultant for Santhera Pharmaceuticals, Gen-Sight Biologics, and Stealth BioTherapeutics and has received research support from Santhera Pharmaceuticals and Stealth Bio-Therapeutics. CV-C is a consultant for Santhera Pharmaceuticals and GenSight Biologics. VB is a consultant for GenSight Biologics. MM is a consultant for GenSight Biologics and has received research support from GenSight. AAS is a consultant for Stealth BioTherapeutics. LB and MT are GenSight Biologics employees. JAS is a co-founder and shareholder of GenSight Biologics, and a patent co-author on allotopic transport. Authors declare competing financial interests in relation with the work submitted (see "Acknowledgements" for details).
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