Diagnostic genome sequencing improves diagnostic yield: a prospective single-centre study in 1000 patients with inherited eye diseases.

PURPOSE: Genome sequencing (GS) is expected to reduce the diagnostic gap in rare disease genetics. We aimed to evaluate a scalable framework for genome-based analyses 'beyond the exome' in regular care of patients with inherited retinal degeneration (IRD) or inherited optic neuropathy (ION). METHODS: PCR-free short-read GS was performed on 1000 consecutive probands with IRD/ION in routine diagnostics. Complementary whole-blood RNA-sequencing (RNA-seq) was done in a subset of 74 patients. An open-source bioinformatics analysis pipeline was optimised for structural variant (SV) calling and combined RNA/DNA variation interpretation. RESULTS: A definite genetic diagnosis was established in 57.4% of cases. For another 16.7%, variants of uncertain significance were identified in known IRD/ION genes, while the underlying genetic cause remained unresolved in 25.9%. SVs or alterations in non-coding genomic regions made up for 12.7% of the observed variants. The RNA-seq studies supported the classification of two unclear variants. CONCLUSION: GS is feasible in clinical practice and reliably identifies causal variants in a substantial proportion of individuals. GS extends the diagnostic yield to rare non-coding variants and enables precise determination of SVs. The added diagnostic value of RNA-seq is limited by low expression levels of the major IRD disease genes in blood.

Influence of Patient Age and Presence of Optic Disc Drusen on Fluctuations in Retinal Nerve Fiber Layer Thickness.

BACKGROUND: It is generally believed that optic disc drusen (ODD) change only over long periods of time. Because, in our experience, this does not apply to younger patients, we investigated the natural course of changes of the peripapillary retinal nerve fiber layer (RNFL) in patients with ODD. METHODS: In this retrospective study, 40 eyes with and 40 eyes without ODD were examined, both cohorts were equally subdivided into younger subjects (20 years or younger) and older subjects (21 years or older). Three optical coherence tomography (OCT) scans of the peripapillary RNFL that had an interval of at least 1 month were required for each patient to be included in this study. The largest difference in total RNFL thickness (delta RNFL-t) and in RNFL thickness of the most differing sector (delta RNFL max) measured by OCT was compared. RESULTS: The differences in total RNFL thickness and in the most differing RNFL sector in the group of patients with ODD younger than 21 years were significantly larger than in each of the other 3 groups ( P = 0.0001). The other 3 groups did not differ significantly. CONCLUSIONS: Patients with ODD younger than 21 years have distinct variations in peripapillary RNFL thickness without evidence of increased intracranial pressure. In the absence of further pathological findings or neurological symptoms, an observational approach seems adequate in these patients.

Central retina plays a decisive role in the suppression of pupillary escape.

PURPOSE: To explore the pupil redilation during persistent light exposure (pupillary escape phenomenon) at the macula and periphery with monochromatic light stimuli. METHODS: Forty healthy subjects aged 18-64 years (24 females) were examined by chromatic pupil campimetry (CPC) using red and blue 4-s stimuli of 10° radius at the center and 20°-peripheral locations one per quadrant. One glaucoma patient and one achromatopsia patient served as disease models. For statistical analyses, linear mixed-effects models were performed followed by post hoc t-tests. RESULTS: A distinct pupillary escape could be demonstrated peripherally (blue 0.099%*s, red 0.153%*s); at the central healthy retina, there was no relevant escape, neither for blue nor red stimulation. Comparing central versus peripheral stimulation revealed highly significant differences in the escape (difference blue 0.100 ± 0.013, red 0.144 ± 0.013, < 0.0001, respectively). In the periphery, the escape was significantly more pronounced for red compared with blue stimulation (difference 0.054 ± 0.013; p = 0.0001). Enhanced pupillary escape outside of the 95% confidence interval of the linear mixed-effects model of the healthy population could be exemplarily shown in a patient with glaucomatous ganglion cell damage. In the achromatopsia example, no relevant escape was found for blue stimulation, but for red stimulation in the periphery in a comparable range to healthy controls. CONCLUSION: The results emphasize that an intact inner retinal network of nerve fibers arising from the central macular region is necessary for maintaining pupillary constriction during a bright 4-s light stimulus and preventing increase of pupillary escape. Increasing receptive field sizes towards the periphery on the level of retinal ganglion cells and less input from central 1:1 connections could be one of the driving mechanisms for pupillary escape.

[Optical coherence tomography in the differential diagnostics of important neuro-ophthalmological disease patterns]. / Die optische Kohärenztomographie in der Differenzialdiagnostik wichtiger neuroophthalmologischer Krankheitsbilder.

There are many disease patterns that are treated jointly by neurologists and ophthalmologists, for which optical coherence tomography (OCT) is of important differential diagnostic significance. In this context neurologists are mainly confronted by two patient collectives: patients with an acute ischemic event, who present with an acute but painless monocular visual deterioration (for central retinal artery occlusion) or with a monocular visual field defect (for arterial branch occlusion or anterior ischemic optic neuropathy). The second collective is patients without ophthalmological symptoms but with conspicuous optic nerve findings (papilledema or optic disc drusen). In this overview article both patient collectives are considered separately. In addition, the most important OCT findings for optic neuritis are presented. Before the disease patterns are described in detail, the normal OCT findings and the diagnostic possibilities of OCT are explained.

[Optical coherence tomography in the differential diagnostics of important neuro-ophthalmological disease patterns]. / Die optische Kohärenztomographie in der Differenzialdiagnostik wichtiger neuroophthalmologischer Krankheitsbilder.

There are many disease patterns that are treated jointly by neurologists and ophthalmologists, for which optical coherence tomography (OCT) is of important differential diagnostic significance. In this context neurologists are mainly confronted by two patient collectives: patients with an acute ischemic event, who present with an acute but painless monocular visual deterioration (for central retinal artery occlusion) or with a monocular visual field defect (for arterial branch occlusion or anterior ischemic optic neuropathy). The second collective is patients without ophthalmological symptoms but with conspicuous optic nerve findings (papilledema or optic disc drusen). In this overview article both patient collectives are considered separately. In addition, the most important OCT findings for optic neuritis are presented. Before the disease patterns are described in detail, the normal OCT findings and the diagnostic possibilities of OCT are explained.

DNAJC30 disease-causing gene variants in a large Central European cohort of patients with suspected Leber's hereditary optic neuropathy and optic atrophy.

BACKGROUND: Leber's hereditary optic neuropathy (LHON) has been considered a prototypical mitochondriopathy and a textbook example for maternal inheritance linked to certain disease-causing variants in the mitochondrial genome. Recently, an autosomal recessive form of LHON (arLHON) has been described, caused by disease-causing variants in the nuclear encoded gene DNAJC30. METHODS AND RESULTS: In this study, we screened the DNAJC30 gene in a large Central European cohort of patients with a clinical diagnosis of LHON or other autosomal inherited optic atrophies (OA). We identified likely pathogenic variants in 35/1202 patients, corresponding to a detection rate of 2.9%. The previously described missense variant c.152A>G;p.(Tyr51Cys) accounts for 90% of disease-associated alleles in our cohort and we confirmed a strong founder effect. Furthermore, we identified two novel pathogenic variants in DNAJC30: the nonsense variant c.610G>T;p.(Glu204*) and the in-frame deletion c.230_232del;p.(His77del). Clinical investigation of the patients with arLHON revealed a younger age of onset, a more frequent bilateral onset and an increased clinically relevant recovery compared with LHON associated with disease-causing variants in the mitochondrial DNA. CONCLUSION: This study expands previous findings on arLHON and emphasises the importance of DNAJC30 in the genetic diagnostics of LHON and OA in European patients.

Characteristics of peripapillary retinal nerve fiber layer atrophy in glaucoma, optic nerve sheath meningioma, and sphenoid wing meningioma.

BACKGROUND/OBJECTIVES: The correct classification of a slowly progressing optic atrophy can be challenging. The aim of this work was to find out if the characteristics of peripapillary retinal nerve fiber layer (RNFL) thickness loss differ between open angle glaucoma (POAG), optic nerve sheath meningioma (ONSM), and sphenoid wing meningioma (SWM). METHODS: A total of 45 patients with POAG, ONSM, and SWM were included in the retrospective study. The peripapillary RNFL thickness measured by spectral-domain optical coherence tomography was analyzed using the Heidelberg Engineering glaucoma module©. RESULTS: Each group consisted of 15 patients. The temporal sector of the RNFL thickness showed a median decrease of - 17 µm in glaucoma patients (range + 6/-34 µm), - 43 µm in ONSM (range - 19/ - 52 µm), and - 44 µm in SWM patients (range - 25/ - 52 µm). The RNFL thickness of the temporal sector of glaucoma patients differed significantly from the other groups (p < 0.001). All other sectors showed no significant difference between the 3 groups. CONCLUSION: The peripapillary RNFL thickness of the temporal sector of patients with beginning to moderate POAG is usually inside normal limits or borderline. In contrast, patients with ONSM and SWM are much more likely to show a considerable reduction in RNFL thickness of the temporal sector. RNFL thickness of the temporal sector marked outside normal limits occurred exclusively in meningioma patients. Considering the presence of this condition as a predictor for meningioma, sensitivity and specificity were 0.8 and 1.0, respectively. In patients with significant reduction in RNFL thickness of the temporal sector, magnetic resonance imaging of the head should be considered to rule out compression of the optic nerves.

How lesions at different locations along the visual pathway influence pupillary reactions to chromatic stimuli.

PURPOSE: To examine systematically how prechiasmal, chiasmal, and postchiasmal lesions along the visual pathway affect the respective pupillary responses to specific local monochromatic stimuli. METHODS: Chromatic pupil campimetry (CPC) was performed in three patient groups (10 subjects with status after anterior ischemic optic neuropathy, 6 with chiasmal lesions, and 12 with optic tract or occipital lobe lesions (tumor, ischemia)) using red, low-intensity red, and blue local stimuli within the central 30° visual field. Affected areas - as determined by visual field defects revealed using conventional static perimetry - were compared with non-affected areas. Outcome parameters were the relative maximal constriction amplitude (relMCA) and the latency to constriction onset of the pupillary responses. RESULTS: A statistically significant relMCA reduction was observed in the affected areas of postchiasmal lesions with red (p = 0.004) and low-intensity red stimulation (p = 0.001). RelMCA reduction in the affected areas seemed more pronounced for low-intensity red stimulation (46.5% mean reduction compared to non-affected areas; 36% for red stimulation), however statistically not significant. In prechiasmal lesions, a statistically significant latency prolongation could be demonstrated in the affected areas with low-intensity red stimulation (p = 0.015). CONCLUSION: Our results indicate that the choice of stimulus characteristics is relevant in detecting defects in the pupillary pathway of impairment along the visual pathway, favoring red stimuli of low intensity over blue stimuli. Such knowledge opens the door for further fundamental research in pupillary pathways and is important for future clinical application of pupillography in neuro-ophthalmologic patients.

Mutation spectrum of the OPA1 gene in a large cohort of patients with suspected dominant optic atrophy: Identification and classification of 48 novel variants.

Autosomal dominant optic atrophy is one of the most common inherited optic neuropathies. This disease is genetically heterogeneous, but most cases are due to pathogenic variants in the OPA1 gene: depending on the population studied, 32-90% of cases harbor pathogenic variants in this gene. The aim of this study was to provide a comprehensive overview of the entire spectrum of likely pathogenic variants in the OPA1 gene in a large cohort of patients. Over a period of 20 years, 755 unrelated probands with a diagnosis of bilateral optic atrophy were referred to our laboratory for molecular genetic investigation. Genetic testing of the OPA1 gene was initially performed by a combined analysis using either single-strand conformation polymorphism or denaturing high performance liquid chromatography followed by Sanger sequencing to validate aberrant bands or melting profiles. The presence of copy number variations was assessed using multiplex ligation-dependent probe amplification. Since 2012, genetic testing was based on next-generation sequencing platforms. Genetic screening of the OPA1 gene revealed putatively pathogenic variants in 278 unrelated probands which represent 36.8% of the entire cohort. A total of 156 unique variants were identified, 78% of which can be considered null alleles. Variant c.2708_2711del/p.(V903Gfs*3) was found to constitute 14% of all disease-causing alleles. Special emphasis was placed on the validation of splice variants either by analyzing cDNA derived from patients´ blood samples or by heterologous splice assays using minigenes. Splicing analysis revealed different aberrant splicing events, including exon skipping, activation of exonic or intronic cryptic splice sites, and the inclusion of pseudoexons. Forty-eight variants that we identified were novel. Nine of them were classified as pathogenic, 34 as likely pathogenic and five as variant of uncertain significance. Our study adds a significant number of novel variants to the mutation spectrum of the OPA1 gene and will thereby facilitate genetic diagnostics of patients with suspected dominant optic atrophy.

Dominant ACO2 mutations are a frequent cause of isolated optic atrophy.

Biallelic mutations in ACO2, encoding the mitochondrial aconitase 2, have been identified in individuals with neurodegenerative syndromes, including infantile cerebellar retinal degeneration and recessive optic neuropathies (locus OPA9). By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, we identified 61 cases harbouring variants in ACO2, among whom 50 carried dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy. Analysis of the ophthalmological and clinical data revealed that recessive cases are affected more severely than dominant cases, while not significantly earlier. In addition, 27% of the recessive cases and 11% of the dominant cases manifested with extraocular features in addition to optic atrophy. In silico analyses of ACO2 variants predicted their deleterious impacts on ACO2 biophysical properties. Skin derived fibroblasts from patients harbouring dominant and recessive ACO2 mutations revealed a reduction of ACO2 abundance and enzymatic activity, and the impairment of the mitochondrial respiration using citrate and pyruvate as substrates, while the addition of other Krebs cycle intermediates restored a normal respiration, suggesting a possible short-cut adaptation of the tricarboxylic citric acid cycle. Analysis of the mitochondrial genome abundance disclosed a significant reduction of the mitochondrial DNA amount in all ACO2 fibroblasts. Overall, our data position ACO2 as the third most frequently mutated gene in autosomal inherited optic neuropathies, after OPA1 and WFS1, and emphasize the crucial involvement of the first steps of the Krebs cycle in the maintenance and survival of retinal ganglion cells.

Leber's hereditary optic neuropathy: course of disease in consideration of idebenone treatment and type of mutation.

PURPOSE: In September 2015, the first and so far only medication for treatment of Leber's hereditary optic neuropathy (LHON) was approved in the EU. The drug in question is idebenone (©Raxone) and has been given to all newly diagnosed patients of the University Eye Hospital Tuebingen since the approval of the drug. The aim of the study was to find out whether regular administration of the drug led to an improvement in vision. We retrospectively examined 2 cohorts of consecutive patients with newly occurred visual impairment and LHON diagnosis: One with the initial diagnosis made from January 2010 until April 2014 and a second from October 2015 until January 2020. METHODS: Retrospective, observational cohort study. All electronic medical files of newly diagnosed and genetically confirmed LHON patients of the University Eye Hospital Tuebingen from January 2010 until April 2014 (cohort 1) and October 2015 until January 2020 (cohort 2) with at least 12 months of follow-up examinations have been analyzed. RESULTS: Five patients were included in the first and 7 patients in the second cohort. Patients of cohort 1 received no medication; patients of cohort 2, a daily dose of 900 mg idebenone. The primary visual acuity (VA) ranged between 0.03 and 0.5 in cohort 1 and did not improve during the observation period (median 60 months, range 23-87 months). The patients of cohort 2 have been observed for a median of 23 months (range 12-35 m). The primary VA ranged from 0.01 to 0.16. A recovery in one or both eyes with a final VA from 0.8 to 1.0 was experienced in 3 out of 7 patients. All patients showing a recovery of VA carried the m.11778G>A mutation. CONCLUSION: The observed improvement in the treated cohort may be considered as a hint on the efficacy of idebenone in LHON. The time course of improvement suggests that idebenone should be given 1.5 years in newly diagnosed LHON cases.

Correction to: Retrospective analysis of fractionated intensity-modulated radiotherapy (IMRT) in the interdisciplinary management of primary optic nerve sheath meningiomas.

An amendment to this paper has been published and can be accessed via the original article.

First submicroscopic inversion of the OPA1 gene identified in dominant optic atrophy - a case report.

BACKGROUND: Dominant optic atrophy (DOA) is an inherited optic neuropathy that mainly affects visual acuity, central visual fields and color vision due to a progressive loss of retinal ganglion cells and their axons that form the optic nerve. Approximately 45-90% of affected individuals with DOA harbor pathogenic variants in the OPA1 gene. The mutation spectrum of OPA1 comprises nonsense, canonical and non-canonical splice site, frameshift and missense as well as copy number variants, but intragenic inversions have not been reported so far. CASE PRESENTATION: We report a 33-year-old male with characteristic clinical features of DOA. Whole-genome sequencing identified a structural variant of 2.4 kb comprising an inversion of 937 bp at the OPA1 locus. Fine mapping of the breakpoints to single nucleotide level revealed that the structural variation was an inversion flanked by two deletions. As this rearrangement inverts the entire first exon of OPA1, it was classified as likely pathogenic. CONCLUSIONS: We report the first DOA case harboring an inversion in the OPA1 gene. Our study demonstrates that copy-neutral genomic rearrangements have to be considered as a possible cause of disease in DOA cases.

Optic neuritis in German children: clinical findings and association with multiple sclerosis.

PURPOSE: Analysis of a cohort of pediatric optic neuritis patients concerning the epidemiology, disease progression, and association with multiple sclerosis (MS). METHODS: Retrospective, observational cohort study. From 2004 to 2018, all electronic medical files of patients younger than 18 years referred to a tertiary care clinic in Germany with the diagnosis optic neuritis have been analyzed. RESULTS: Sixty-nine patients were referred in the study period, 16 did not suffer under optic neuritis and were excluded. The median visual acuity of the remaining 53 patients was 0.07 at the baseline examination and 1.0 at the latest follow-up examination (decimal notation, median 2.1 years after baseline). Forty-two percent of the patients developed MS during the study period. Female sex (p = 0.028) as well as higher age (p = 0.0082) proved to be statistically significant risk factors for MS development. CONCLUSION: The prognosis for restoring vision in pediatric optic neuritis was favorable. During the observation period, the risk of developing MS was overall 42% and 8% for patients younger than 11 years. The percentage of MS as underlying cause of optic neuritis does not differ remarkably between children older 10 years and adults.

Retrospective analysis of fractionated intensity-modulated radiotherapy (IMRT) in the interdisciplinary management of primary optic nerve sheath meningiomas.

BACKGROUND: As optic nerve sheath meningiomas (ONSM) are rare, there are no prospective studies. Our retrospective analysis focusses on a cohort of patients with uniform disease characteristics all treated with the same radiotherapy regimen. We describe treatment decision making, radiotherapy planning and detailed neuro-ophthalmological outcome of the patients. METHODS: 26 patients with unilateral ONSM extending only to the orbit and the optic canal were evaluated for neuro-ophthalmological outcome. Radiation treatment was planned in a simultaneous integrated boost approach to gross tumor volume (GTV) + 2 mm / 5 mm to 54 Gy / 51 Gy in 1.8 Gy / 1.7 Gy fractions. Follow-up was done by specialized neuro-ophthalmologists. Visual acuity and visual field defects were evaluated after therapy as well as during follow-up. RESULTS: Interdisciplinary treatment decision for patients with ONSM follows a rather complex decision tree. Radiation treatment planning (equivalent uniform dose (EUD), maximum dose to the optic nerve) improved with experience over time. With this patient selection visual acuity as well as visual field improved significantly at first follow-up after treatment. For visual acuity this also applied to patients with severe defects before treatment. Long term evaluation showed 16 patients with improved visual function, 6 were stable, in 4 patients visual function declined. Interdisciplinary case discussion rated the visual decline as radiation-associated in two patients. CONCLUSIONS: With stringent patient selection radiotherapy for unilateral primary ONSM to 51 Gy / 54 Gy is safe and leads to significantly improved visual function. Interdisciplinary treatment decision and experience of the radiation oncology team play a major role.

[Visual Acuity and Visual Field in Optic Disc Drusen]. / Visus und Gesichtsfeld bei Drusenpapillen.

BACKGROUND: It has been assumed that visual field defects in optic disc drusen slowly increase with age or occur during adolescence and do not change substantially in later years. In our study, we aimed to validate these assumptions. MATERIAL AND METHODS: 255 consecutive cases with optic disc drusen were identified from the patient records of the University Eye Hospital Tübingen; the diagnosis was verified and visual fields were quantified as long as available and of sufficient quality. Additionally, visual acuity was evaluated. RESULTS: In 104 cases, quantifiable visual fields of sufficient quality for both eyes were available. In general, few patients with marked visual field defects could be detected. Only three patients showed visual field defects of ≥ 50% in both eyes. Both eyes were usually involved to approximately the same extent. Older age was correlated with more visual field defects. Only one patient remained below visual acuity of 0.3 in both eyes. DISCUSSION: By means of our patient base, a continuous slight decline in the visual field with age can be assumed. Marked visual field defects were rare. The same was true for visual acuity, which showed some mild decline above the age of 60 years.

[Optic Disc Drusen]. / Drusenpapillen.

With a prevalence of about 2%, drusen papillae are a very frequent papilla anomaly. The pathological mechanism of their origin is unclear. If the ophthalmoscopic image is not unambiguous, it may be helpful to examine relatives, as the heredity exhibits irregular dominance. Calcium deposits are common and can be detected by sonography. Glands can also be detected by OCT in section and by autofluorescence. Precise funduscopy and documentation of the findings and follow-up are very important. There is no therapy for drusen papillae. The internal ocular pressure must be regularly controlled, as glaucoma cannot be identified from the papilla findings. The risk is increased of anterior ischaemia of the optical nerve.