Genetic Testing of Patients with Inherited Retinal Diseases in the European Countries. An International Survey by the European Vision Institute.

INTRODUCTION: The purpose of this project was to explore the current standards of clinical care genetic testing and counseling for patients with inherited retinal diseases (IRDs) from the perspective of leading experts in selected European countries. Also, to gather opinions on current bottlenecks and future solutions to improve patient care. METHODS: On the initiative of the European Vision Institute, a survey questionnaire with 41 questions was designed and sent to experts in the field from ten European countries. Each participant was asked to answer with reference to the situation in their own country. RESULTS: Sixteen questionnaires were collected by November 2023. IRD genetic tests are performed in clinical care settings for 80% or more of tested patients in 9 countries, and the costs of genetic tests in clinical care are covered by the public health service to the extent of 90% or more in 8 countries. The median proportion of patients who are genetically tested, the median rate of genetically solved patients among those who are tested, and the median proportion of patients receiving counseling are 51-70%, 61-80% and 61-80%, respectively. Improving the education of healthcare professionals who facilitate patient referrals to specialised centres, improving access of patients to more thorough genotyping, and increasing the number of available counselors were the most advocated solutions. CONCLUSION: There is a significant proportion of IRD patients who are not genetically tested, whose genetic testing is inconclusive, or who do not receive counseling. Educational programs, greater availability of state-of-the-art genotyping and genetic counselors could improve healthcare for IRD patients.

Characterising the refractive error in paediatric patients with congenital stationary night blindness: a multicentre study.

BACKGROUND/AAIMS: Congenital stationary night blindness (CSNB) is an inherited retinal disease that is often associated with high myopia and can be caused by pathological variants in multiple genes, most commonly CACNA1F, NYX and TRPM1. High myopia is associated with retinal degeneration and increased risk for retinal detachment. Slowing the progression of myopia in patients with CSNB would likely be beneficial in reducing risk, but before interventions can be considered, it is important to understand the natural history of myopic progression. METHODS: This multicentre, retrospective study explored CSNB caused by variants in CACNA1F, NYX or TRPM1 in patients who had at least 6 measurements of their spherical equivalent of refraction (SER) before the age of 18. A mixed-effect model was used to predict progression of SER overtime and differences between genotypes were evaluated. RESULTS: 78 individuals were included in this study. All genotypes showed a significant myopic predicted SER at birth (-3.076D, -5.511D and -5.386D) for CACNA1F, NYX and TRPM1 respectively. Additionally, significant progression of myopia per year (-0.254D, -0.257D and -0.326D) was observed for all three genotypes CACNA1F, NYX and TRPM1, respectively. CONCLUSIONS: Patients with CSNB tend to be myopic from an early age and progress to become more myopic with age. Patients may benefit from long-term myopia slowing treatment in the future and further studies are indicated. Additionally, CSNB should be considered in the differential diagnosis for early-onset myopia.

Adaptive optics retinal imaging in patients with usher syndrome.

Purpose: To determine the structure of the cone photoreceptor mosaic in the macula in eyes with retinitis pigmentosa related to Usher syndrome using adaptive optics fundus (AO) imaging and to correlate these findings with those of the standard clinical diagnostics. Methods: Ten patients with a genetically confirmed retinitis pigmentosa in Usher syndrome due to biallelic variants in MYO7A or USH2A were enrolled in the study. All patients underwent a complete ophthalmological examination including best corrected visual acuity (BCVA), spectral-domain optical coherence tomography (SD-OCT) with fundus autofluorescence photography (FAF), full-field (ffERG) and multifocal electroretinography (mfERG) and Adaptive Optics Flood Illuminated Ophthalmoscopy (AO, rtx1™, Imagine Eyes, Orsay, France). The cone density was assessed centrally and at each 0.5 degree horizontally and vertically from 1-4 degree of eccentricity. Results: In the AO images, photoreceptor cell death was visualized as a disruption of the cone mosaic and low cone density. In the early stage of the disease, cones were still visible in the fovea, whereas outside the fovea a loss of cones was recognizable by blurry, dark patches. The blurry patches corresponded to the parafoveal hypofluorescent ring in the FAF images and the beginning loss of the IS/OS line and external limiting membrane in the SD-OCT images. FfERGs were non-recordable in 7 patients and reduced in 3. The mfERG was reduced in all patients and correlated significantly (p <0.001) with the cone density. The kinetic visual field area, measured with III4e and I4e, did not correlate with the cone density. Conclusion: The structure of the photoreceptors in Usher syndrome patients were detectable by AO fundus imaging. The approach of using high-resolution technique to assess the photoreceptor structure complements the established clinical examinations and allows a more sensitive monitoring of early stages of retinitis pigmentosa in Usher syndrome.

Defining reference values of arterioles in healthy individuals for studies with adaptive optics imaging.

Purpose: To investigate age-dependent wall to lumen ratio (WLR) reference values for healthy individuals in adaptive optics imaging (AO). WLR serves as an objective, dimensionless parameter for the evaluation of structural changes in vessels caused by conditions like arterial hypertension, diabetes or vascular stenosis. Methods: 50 right eyes of healthy individuals were examined by adaptive optics imaging. The central big arterioles and smaller arterial branches at least one disc diameter away from the optic disc, approximately above or below the macula were measured by the manufacturer's software. The wall-lumen-ratio (WLR), the wall cross-sectional area (WCSA) and lumen diameter (LD) were assessed. Subsequent data analysis was performed with a focus on variables including age, gender and blood pressure. Results: Normative values for WLR, WCSA and LD in 5 different age groups could be established. However, no significant differences between the age groups were found. Intra-subject comparisons revealed significantly higher WLRs on peripheral branches when compared to central arterioles. WLR showed in this normotensive cohort no relevant correlation with the systolic, diastolic and mean blood pressure. Gender and intraocular pressure had no influence on the vascular parameters. Conclusion: AO is capable of examining vascular alterations in arterioles at an almost microscopic level. Age did not seem to alter WLR, normotensive blood pressure parameters showed also no significant impact. AO-based vessel analysis may provide clinically useful biomarkers for cardiovascular health and should be tested in future studies.

A case of AZOOR under immunomodulatory treatment.

PURPOSE: To describe the clinical course and treatment response of a case of Acute Zonal Occult Outer Retinopathy (AZOOR). METHODS: This is an observational case report. The examinations included ophthalmic examination, longitudinal multimodal imaging, visual field testing, electrophysiological recordings, serologic analyses and whole genome sequencing. RESULTS: In this report, the authors present the case of an otherwise healthy 33-year-old female with bilateral AZOOR manifestation. Other possible causes of the observed retinal lesions were ruled out by extensive diagnostic work-up. Treatment with oral prednisolone therapy led to temporal disease control but progression was observed after prednisolone discontinuation. A tapered oral prednisolone therapy in combination with adalimumab initiation prevented further progression for at least 21 months. CONCLUSION: Diagnosis of AZOOR is frequently complicated by the unspecific symptoms of the disease and significant number of differential diagnoses. Complete diagnostic work-up is important to rule out other etiologies. Due to the lack of randomized controlled trials, therapeutic decisions obligatorily rely on empiric treatment decisions in combination with frequent follow-up examinations.

Self-Reported Functional Vision in USH2A-Associated Retinal Degeneration as Measured by the Michigan Retinal Degeneration Questionnaire.

Purpose: The purpose of this study was to evaluate self-reported functional vision (FV) and the impact of vision loss in patients with USH2A-associated retinal degeneration using a patient-reported outcome (PRO) measure, the Michigan Retinal Degeneration Questionnaire (MRDQ), to correlate MRDQ scores with well-established visual function measurements. Design: An observational cross-sectional study (n = 93) of participants who had Usher Syndrome Type 2 (USH2, n = 55) or autosomal recessive non-syndromic retinitis pigmentosa (ARRP; n = 38) associated with biallelic variants in the USH2A gene. Methods: The study protocol was approved by all ethics boards and informed consent was obtained from each participant. Participants completed the MRDQ at the 48-month study follow-up visit. Disease duration was self-reported by participants. One-way ANOVA was used to compare subgroups (clinical diagnosis, age, disease duration, and full-field stimulus threshold [FST] Blue-Red mediation) on mean scores per domain. Spearman correlation coefficients were used to assess associations between MRDQ domains and visual/retinal function assessments. Results: Of the study sample, 58% were female participants and the median disease duration was 13 years. MRDQ domains were sensitive to differences between subgroups of clinical diagnosis, age, disease duration, and FST Blue-Red mediation. MRDQ domains correlated with static perimetry, microperimetry, full-field stimulus testing, and best-corrected visual acuity (BCVA). Conclusions: Self-reported FV measured by the MRDQ, when applied to USH2 and ARRP participants, had good distributional characteristics and correlated well with visual function tests. MRDQ adds a new dimension of understanding on vision-related functioning and establishes this PRO tool as an informative measure in evaluating USH2A outcomes.

Clinical and Genetic Findings in a Cohort of Patients with PRPF31-Associated Retinal Dystrophy.

PURPOSE: The purpose of our study was to assess the phenotypic and genotypic spectrum in a large cohort of patients with PRPF31-associated retinal dystrophy. DESIGN: Retrospective cohort study. METHODS: In this retrospective chart review study, we collected cross-sectional data on the phenotype and genotype of patients with PRPF31-associated retinal dystrophy from the clinics for inherited retinal dystrophies at the University of Tuebingen and the local RetDis database and biobank. Patients underwent thorough ophthalmological examinations and genetic testing. RESULTS: Eighty-six patients from 61 families were available for clinical assessment, while genomic DNA was available for 111 individuals (index patients and family members). Fifty-three different disease-associated variants were observed in our cohort. Point mutations were the most common class. All but two patients exhibited features of a typical Retinitis pigmentosa (RP). One patient showed a cone-rod dystrophy pattern. One mutation carrier revealed no signs of a retinal dystrophy. There was a statistically significant better visual acuity for patients with large deletions in the 20-39 age group. Cystoid macular edema was common in those with preserved central retina and showed an association with female sex. CONCLUSION: Our study confirms high phenotypic variability in disease onset and age at which legal blindness is reached in PRPF31-associated RP. Non-penetrance is commonly documented in family history, although poorly represented in our study, possibly indicating that true asymptomatic mutation carriers are rare if followed-up over lifetime with thorough ophthalmologic workup.

S-cone contribution to oscillatory potentials in patients with blue cone monochromacy.

PURPOSE: The aim of this exploratory study is to investigate the role of S-cones in oscillatory potentials (OPs) generation by individuals with blue-cone monochromacy (BCM), retaining S-cones, and achromatopsia (ACHM), lacking cone functions. METHODS: This retrospective study analyzed data from 39 ACHM patients, 20 BCM patients, and 26 controls. Central foveal thickness was obtained using spectral-domain optical coherence tomography, while amplitude and implicit time (IT) of a- and b-waves were extracted from the ISCEV Standard dark-adapted 3 cd.s.m-2 full-field ERG (ffERG). Time-frequency analysis of the same measurement enabled the extraction of OPs, providing insights into the dynamic characteristics of the recorded signal. RESULTS: Both ACHM and BCM groups showed a significant reduction (p < .00001) of a- and b-wave amplitudes and ITs as well as the power of the OPs compared to the control groups. The comparison between ACHM and BCM didn't show any statistically significant differences in the electrophysiological parameters. The analysis of covariance revealed significantly reduced central foveal thickness in the BCM group compared to ACHM and controls (p < .00001), and in ACHM compared to controls (p < .00001), after age correction and Tukey post-hoc analysis. CONCLUSIONS: S-cones do not significantly influence OPs, and the decline in OPs' power is not solely due to a reduced a-wave. This suggests a complex non-linear network influenced by photoreceptor inputs. Morphological changes don't correlate directly with functional alterations, prompting further exploration of OPs' function and physiological role.

Comprehensive analysis of two hotspot codons in the TUBB4B gene and associated phenotypes.

Our purpose was to elucidate the genotype and ophthalmological and audiological phenotype in TUBB4B-associated inherited retinal dystrophy (IRD) and sensorineural hearing loss (SNHL), and to model the effects of all possible amino acid substitutions at the hotspot codons Arg390 and Arg391. Six patients from five families with heterozygous missense variants in TUBB4B were included in this observational study. Ophthalmological testing included best-corrected visual acuity, fundus examination, optical coherence tomography, fundus autofluorescence imaging, and full-field electroretinography (ERG). Audiological examination included pure-tone and speech audiometry in adult patients and auditory brainstem response testing in a child. Genetic testing was performed by disease gene panel analysis based on genome sequencing. The molecular consequences of the substitutions of residues 390 and 391 on TUBB4B and its interaction with α-tubulin were predicted in silico on its three-dimensional structure obtained by homology modelling. Two independent patients had amino acid exchanges at position 391 (p.(Arg391His) or p.(Arg391Cys)) of the TUBB4B protein. Both had a distinct IRD phenotype with peripheral round yellowish lesions with pigmented spots and mild or moderate SNHL, respectively. Yet the phenotype was milder with a sectorial pattern of bone spicules in one patient, likely due to a genetically confirmed mosaicism for p.(Arg391His). Three patients were heterozygous for an amino acid exchange at position 390 (p.(Arg390Gln) or p.(Arg390Trp)) and presented with another distinct retinal phenotype with well demarcated pericentral retinitis pigmentosa. All showed SNHL ranging from mild to severe. One additional patient showed a variant distinct from codon 390 or 391 (p.(Tyr310His)), and presented with congenital profound hearing loss and reduced responses in ERG. Variants at codon positions 390 and 391 were predicted to decrease the structural stability of TUBB4B and its complex with α-tubulin, as well as the complex affinity. In conclusion, the twofold larger reduction in heterodimer affinity exhibited by Arg391 substitutions suggested an association with the more severe retinal phenotype, compared to the substitution at Arg390.

Comparison of Full-Field Stimulus Threshold Measurements in Patients With Retinitis Pigmentosa and Healthy Subjects With Dilated and Nondilated Pupil.

Purpose: The common protocol of full-field stimulus threshold (FST) testing recommends pupil dilation. The aim of this study is to investigate the difference between FST measurements with dilated and nondilated pupils in healthy subjects and patients with retinitis pigmentosa (RP). Methods: Twenty healthy subjects and 20 RP patients were selected. One pupil of each subject was dilated; the other eye was measured in physiological width of the pupil. The FST was conducted using Diagnosys Espion E2/E3 with white, blue, and red stimuli. Statistical analysis was conducted with a mixed-model analysis of variance and a paired t-test. Results: The statistical analysis revealed a significant difference between measurements of dilated and nondilated pupils with the following: blue stimuli for all subjects and groups except those with highly progressed RP; white stimuli for all tested subjects in total, for RP patients with better-preserved visual field (VF), and rod-mediated FST response; and red stimuli for RP patients with better-preserved VF and rod-mediated FST response. On average, the difference between the FST values for RP patients were -3.2 ± 3 dB for blue, -2.3 ± 2.9 dB for white, and -0.83 ± 3 dB for red stimuli. The correlation between the FST values of dilated and nondilated pupils with all three stimuli was linear. Conclusions: Current recommendations are to perform FST with dilated pupils. However, based on this study's findings, pupil dilation can be omitted for clinical diagnostics or rough follow-ups. Translational Relevance: Our data provide useful information for the clinical use of FST.

RPE65-Associated Retinal Dystrophies: Phenotypes and Treatment Effects with Voretigene Neparvovec.

Retinal dystrophies linked to the RPE65 gene are mostly fast-progressing retinal diseases, with childhood onset of night blindness and progressive visual loss up to the middle adult age. Rare phenotypes linked to this gene are known with congenital stationary night blindness or slowly progressing retinitis pigmentosa, as well as an autosomal dominant c.1430A>G (p.Asp477Gly) variant. This review gives an overview of the current knowledge of the clinical phenotypes, as well as experience with the efficacy and safety of the approved gene augmentation therapy voretigene neparvovec.

Influence of open-source virtual-reality based gaze training on navigation performance in Retinitis pigmentosa patients in a crossover randomized controlled trial.

METHODS: A group of RP patients (n = 8, aged 20-60) participated in a study consisting of two 4-week-phases, both carried out by the same patient group in randomized order: In the 'training phase', participants carried out a Virtual-Reality gaze training for 30 minutes per day; In the 'control phase', no training occurred. Before and after each phase, participants were tasked to move through a randomized real-world obstacle course. Navigation performance in the obstacle course as well as eye-tracking data during the trials were evaluated. The study is registered at the German Clinical Trials Register (DRKS) with the ID DRKS00032628. RESULTS: On average, the time required to move through the obstacle course decreased by 17.0% after the training phase, the number of collisions decreased by 50.0%. Both effects are significantly higher than those found in the control phase (p < 0.001 for required time, p = 0.0165 for number of collisions), with the required time decreasing by 5.9% and number of collisions decreasing by 10.4% after the control phase. The average visual area observed by participants increases by 4.41% after training, however the effect is not found to be significantly higher than in the control phase (p = 0.394). CONCLUSION: The performance increase over the training phase significantly surpasses the natural learning effect found in the control phase, suggesting that Virtual-Reality based gaze training can have a positive effect on real-world navigation tasks for patients with RP. The training is available as work-in-progress open-source software.

Chorioretinal Atrophy Growth After Voretigene Neparvovec Retinotopically Is Connected to Retinal Functional Rescue.

Purpose: Chorioretinal atrophy growth after voretigene neparvovec has been reported recently with its positive correlation with successful treatment. This finding raised the question on long-term effects and the etiology of the chorioretinal atrophy. Methods: Using local retinal functional diagnostics, we tested whether the atrophy growth is connected to the initial local functional improvement after the therapy. Results: The results describe factors predicting the development of atrophy. First, the atrophy emerges after approximately 3 months in an area with local functional rescue before. The areas of the greatest gain in the number of functionally rescued rods are prone to be the initial spots of atrophy growth in almost one-half of the cases and the retinotopy corresponds with the area of a high number of post-treatment functioning rods. Second, the dark-adapted perimetry shows that the atrophy growth is in the area with functioning rescued rods. However, the rods with the greatest sensitivity gain are not the parts of the growing atrophy in the first 2 years after intervention. This preservation of rods with the greatest sensitivity seems to explain the excellent profile of rods rescue over the long term measured by full-field stimulus threshold and reported earlier. Conclusions: A disbalance between the increase of functional rods and their threshold shortly after treatment could be an indicator for a metabolic origin of chorioretinal atrophy after voretigene neparvovec. Translational Relevance: A basic understanding of the photoreceptor rescue aspects after gene therapy can demonstrate a metabolic causal influence of the efficacy on the development of side effects, such as chorioretinal atrophy.

Splicing defects and CRISPR-Cas9 correction in isogenic homozygous photoreceptor precursors harboring clustered deep-intronic ABCA4 variants.

Splicing defects from deep-intronic variants significantly contribute to the mutational spectrum in ABCA4-associated inherited retinal diseases, necessitating functional validation for their pathological classification. Typically, minigene assays in HEK293(T) can qualitatively assess splicing defects, yet they often fail to quantitatively reproduce the resulting mis-splicing patterns, leaving uncertainty on severity and pathogenicity. As a potential cellular model derived from patient cells, photoreceptor precursor cells (PPCs) play a pivotal role in assessing the severity of specific splicing mutations. Nevertheless, the accessibility of biosamples is commonly constrained, and their establishment is costly and laborious. In this study, we combined and investigated the use of a minigene assay and isogenic PPCs, as superior qualitative and more accessible cellular models for the assessment of splicing defects. Specifically, we focused on the clustered c.5196+1013A>G, c.5196+1056A>G, and c.5196+1216C>A deep-intronic variants in intron 36 of ABCA4, comparing their resulting (mis)splicing patterns in minigene-transfected cells and isogenic CRISPR-Cas9-knocked-in PPCs harboring these pathogenic variants in homozygous state. Moreover, we demonstrate the successful correction of these three splicing defects in homozygous mutant PPCs using a single pair of guide RNAs to target Cas9 cleavage, thereby identifying an efficient gene editing strategy for therapeutic applications.

Multi-luminance mobility testing after gene therapy in the context of retinal functional diagnostics.

BACKGROUND: Voretigene neparvovec (Luxturna®) is the first approved gene therapy for RPE65-linked Leber congenital amaurosis (LCA). Though individual effects are highly variable, most recipients report improved vision in everyday life. To describe such effects, visual navigation tests are now frequently used in clinical trials. However, it is still unclear how their results should be interpreted compared to conventional parameters of visual function. METHODS: Seven LCA patients underwent a multi-luminance visual navigation test (Ora-VNCTM) before and 3 months after receiving Luxturna gene therapy. Their performance was rated based on the luminance level at which they passed the course. Differences between the first and second test were correlated to changes in visual acuity, full-field stimulus thresholds, chromatic pupil campimetry, and dark-adapted perimetry. RESULTS: A few patients displayed notable improvements in conventional measures of visual function whereas patients with advanced retinal degeneration showed no relevant changes. Independent of these results, almost all participants improved in the visual navigation task by one or more levels. The improvement in the mobility test was best correlated to the change in full-field stimulus thresholds. Other measures of visual functions showed no clear correlation with visual navigation. DISCUSSION: In patients who passed the test's more difficult levels, improved visual navigation can be attributed to the reactivation of rods. However, the performance of patients with low vision seemed to depend much more on confounding factors in the easier levels. In sum, such tests might only be meaningful for patients with better preserved visual functions.

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.

A morphometric analysis of the retinal arterioles with adaptive optics imaging in RPE65-associated retinal dystrophy after treatment with voretigene neparvovec.

PURPOSE: To investigate the changes in retinal arterial architecture after treatment with voretigene neparvovec in patients with retinal dystrophy caused by bi-allelic mutations in the RPE65 gene. METHODS: Sixteen eyes treated with voretigene neparvovec at the University Eye Clinic in Tuebingen, Germany, underwent adaptive optics ophthalmoscopy (AO) imaging at baseline and 2 weeks, 1, 3, 6 and 12 months after treatment. Follow-up was performed in six eyes of four patients. For each eye, five different positions at arterial vessels were selected and the wall-to-lumen ratio (WLR), the lumen diameter (LD) and the wall cross-sectional area (WCSA) were measured by the manufacturer's software over the observational period. RESULTS: Vast retinal atrophy dominated all gained AO images. WLR fluctuated in the observation period without statistically significant change. LD and WCSA changed significantly after 2 weeks from the baseline examination and returned to values similar to baseline thereafter. There were no signs of inflammation such as macrophages or perivascular accumulated fluid visible. CONCLUSION: AO imaging of the retinal vessels in RPE65-associated retinal dystrophies (IRD) is challenging. There was no change in the retinal arterial vasculature over the observation period of 12 months that would indicate inflammatory changes. Decrease of the LD and WCSA shortly after treatment might be caused by the perioperative prednisolone intake. AO of retinal vessels can be used as a diagnostic module to complement monitoring the disease and effects of genetic treatments if the acquisition is possible in selected cases.