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.

Long-term outcomes of autologous platelet treatment for optic disc pit maculopathy.

PURPOSE: Optic disc pits (ODPs) are rare congenital cavitary abnormalities of the optic nerve head, which can lead to serous macular detachments. The aim of this study was to evaluate the long-term efficacy of pars plana vitrectomy (PPV) combined with autologous platelet concentrate (APC) for the treatment of optic disc pit maculopathy (ODP-M). METHODS: A retrospective analysis was performed on eleven eyes of ten patients with ODP-M, who received PPV combined with APC. Nine eyes operated primary, four of which had a repeat surgery also with injection of APC and two eyes underwent a rescue surgery, after they have been operated in another eye center without APC. Morphological and functional results were the main outcome parameters, determined by optical coherence tomography (OCT) and best-corrected visual acuity (BCVA), respectively. RESULTS: The mean duration of visual loss before surgery was 4.7 ± 3.89 months (range 0-12 months). The mean BCVA increased significantly from 0.82 ± 0.33 logMAR (range 0.4-1.3) preoperatively to 0.51 ± 0.36 logMAR (range 0-1.2) at the last examination (p = 0.0022). A significant morphological improvement was also noticed with decrease of the mean foveal thickness from 935.82 ± 248.48 µm (range 559-1400 µm) preoperatively to 226.45 ± 76.09 µm (range 110-344 µm) at the final examination (p < 0.0001). The patients were followed-up for a mean 65.36 ± 48.81 months (range 1-144 months). Two eyes developed postoperatively a retinal detachment. Cataract surgery was performed in 5 eyes during the follow-up period. CONCLUSION: Our study demonstrated that PPV with APC can improve functional and morphological outcomes, both as a primary and a rescue therapy, without any recurrence over a long follow-up period. To the best of our knowledge, this was the longest observation period regarding the use of APC in treatment of ODP-M.

Frequency-dependent retinal responsiveness to sinusoidal electrical stimulation in achromatopsia.

Recently, we proposed a method to assess cell-specific retinal functions based on the frequency-dependent responses to sinusoidal transcorneal electrostimulation. In this study, we evaluated the alterations in responsiveness in achromatopsia patients to explore the frequency-selectivity of photoreceptors. The electrical stimulation was applied to one eye of genetically confirmed achromatopsia patients via corneal electrodes. The stimulus was composed of amplitude-modulated sine waves with variable carrier frequencies (4-30 Hz) and a steady low-frequency envelope. The retinal responsiveness across the spectrum was calculated based on the velocity and the synchronicity of the electrically evoked pupillary oscillations. Achromats displayed a characteristic peak in responsiveness in the 6-10 Hz range. In contrast, stimulus frequencies above 16 Hz elicited only weak pupil responses and weak phosphenes. Compared to the tuning curve of the healthy retina, responses to low-frequency stimulation appear to reflect mainly rod activation while higher frequencies seem to activate cones. The possibility to examine cell-specific retinal functions independently from their responses to light may improve our understanding of the structural changes in the retina induced by gene therapy.

Development of retinal atrophy after subretinal gene therapy with voretigene neparvovec.

BACKGROUND/AIMS: Voretigene neparvovec (VN) is the first and only subretinal gene therapy approved by the Food and Drug Administration and European Medicines Agency. Real-world application has started in 2018 in patients with vision impairment due to biallelic retinal pigment epithelium (RPE) 65 mutation-associated inherited retinal degenerations. Herein, we evaluated the development of retinal atrophy within in a single-centre patient cohort treated with VN. METHODS: 13 eyes of eight patients treated with VN were retrospectively analysed for areas of retinal atrophy over a period of 6-24 months following surgery. Ultrawide field images were used to measure the area of atrophy. Fundus autofluorescence imaging is presented as an instrument for early detection of signs of retinal atrophy in these patients. RESULTS: Atrophic changes beyond the retinotomy site were observed in all eyes. Areas of atrophy developed within the area of detachment (bleb) in all eight patients and outside the bleb in three patients. Changes in autofluorescence preceded the development of retinal atrophy and were already evident 2 weeks after surgery in the majority of patients. The areas of atrophy increase with time and progression continued over year 1. Functional outcomes remained stable (VA, FST, visual field). CONCLUSION: Subretinal injection of VN can lead to RPE atrophy with consequent photoreceptor loss in and outside of the bleb area. Fundus autofluorescence is an important tool to monitor atrophic changes in patients after gene therapy. Interestingly, while areas of atrophy also included central areas, the functional benefits of the treatment did not appear to be affected and remained stable.

Biallelic variants in coenzyme Q10 biosynthesis pathway genes cause a retinitis pigmentosa phenotype.

The aim of this study was to investigate coenzyme Q10 (CoQ10) biosynthesis pathway defects in inherited retinal dystrophy. Individuals affected by inherited retinal dystrophy (IRD) underwent exome or genome sequencing for molecular diagnosis of their condition. Following negative IRD gene panel analysis, patients carrying biallelic variants in CoQ10 biosynthesis pathway genes were identified. Clinical data were collected from the medical records. Haplotypes harbouring the same missense variant were characterised from family genome sequencing (GS) data and direct Sanger sequencing. Candidate splice variants were characterised using Oxford Nanopore Technologies single molecule sequencing. The CoQ10 status of the human plasma was determined in some of the study patients. 13 individuals from 12 unrelated families harboured candidate pathogenic genotypes in the genes: PDSS1, COQ2, COQ4 and COQ5. The PDSS1 variant c.589 A > G was identified in three affected individuals from three unrelated families on a possible ancestral haplotype. Three variants (PDSS1 c.468-25 A > G, PDSS1 c.722-2 A > G, COQ5 c.682-7 T > G) were shown to lead to cryptic splicing. 6 affected individuals were diagnosed with non-syndromic retinitis pigmentosa and 7 had additional clinical findings. This study provides evidence of CoQ10 biosynthesis pathway gene defects leading to non-syndromic retinitis pigmentosa in some cases. Intronic variants outside of the canonical splice-sites represent an important cause of disease. RT-PCR nanopore sequencing is effective in characterising these splice defects.

Cell-specific electrical stimulation of human retinal neurons assessed by pupillary response dynamics in vivo.

Studies on the electrical excitability of retinal neurons show that photoreceptors and other cell types can be selectively activated by distinct stimulation frequencies in vitro. Yet, this principle still needs to be validated in humans in vivo. As a first step, this study explored the frequency preferences of human rods by means of transcorneal electrostimulation (TES), using the electrically-elicited pupillary responses (EEPRs) as an objective readout. The stimulation paradigm contained a 1.2 Hz sinusoidal envelope, which was superimposed on variable carrier frequencies (4-30 Hz). These currents were delivered to one of the participant's eyes via a corneal electrode and consensual pupillary reactions were recorded from the contralateral eye. The responsiveness of the retina at each frequency was assessed based on the EEPR dynamics. Differences between healthy participants and patients with retinitis pigmentosa were evaluated to identify the preferred frequency range of rods. The responsiveness of healthy individuals revealed a clear peak around 6-8 Hz. In contrast, the pupillary responses of patients were significantly reduced in the lower frequency range. These findings suggest that the responses in this frequency bin were selectively mediated by rods. This work provides evidence that different retinal cell types can be selectively activated via TES in vivo, and that this effect can be captured noninvasively using EEPRs. This knowledge may be exploited for the diagnostics and therapy of retinal diseases, e.g., to design cell-specific functional tests for the degenerating retina, or to optimize stimulation paradigms which are currently used by retinal prostheses.

Evaluation of Local Rod and Cone Function in Stargardt Disease.

Purpose: In this study, chromatic pupil campimetry (CPC) was used to map local functional degenerative changes of cones and rods in Stargardt disease (STGD1). Methods: 19 patients (age 36 ± 8 years; 12 males) with genetically confirmed ABCA4 mutations and a clinical diagnosis of STGD1 and 12 age-matched controls (age 37 ± 11 years; 2 males) underwent scotopic (rod-favoring) and photopic (cone-favoring) CPC. CPC evaluates the local retinal function in the central 30° visual field via analysis of the pupil constriction to local stimuli in a gaze-corrected manner. Results: Scotopic CPC revealed that the rod function of patients with STGD1 inside the 30° visual field was not impaired when compared with age-matched controls. However, a statistically significant faster pupil response onset time (∼ 40 ms) was observed in the measured area. Photopic CPC showed a significant reduction of the central cone function up to 6°, with a minor, non-significant reduction beyond this eccentricity. The time dynamic of the pupillary response in photopic CPC did not reveal differences between STGD1 and controls. Conclusions: The functional analysis of the macular region in STGD1 disease indicates reduced central cone function, corresponding to photoreceptor degeneration. In contrast, the rod function in the central area was not affected. Nevertheless, some alteration of the time dynamics in the rod system was observed indicating a complex effect of cone degeneration on the functional performance of the rod system. Our results should be considered when interpreting safety and efficacy in interventional trials of STGD1.

Impurities in Drug Vials Intended for Intravitreal Medication.

Sterility is an important prerequisite for minimizing the risk of severe vision loss due to endophthalmitis after intravitreal injections. We describe three cases series of incidents where an unclear contamination of the drug solution or syringe caused the injection process to stop and continue with a new preparation. During a period of 12 months with 30,502 intravitreal injections at a tertiary center, wherein 7,076 were of the drug Aflibercept drawn up from a glass vial, three cases of the critical incident reporting system relating to intravitreal injections were identified: (1) After a typical contact with the filter cannula, the glass of an Aflibercept vial was no longer intact. (2) In the course of another injection, there was a clear deposition of debris on the outer edge of the syringe when removing the attached filter cannula. (3) After inserting the syringe into the rubber top of the vial, a whitish particle of unclear origin was identified within the drug solution. Later, this contamination/particle was identified as part of the greyish rubber that was punched out with the cannula, according to the analyses of the material sent in and the manufacturer's investigations. Thus, even in busy clinics, visual inspection of the injection solution and materials used for impurities, preferably before and after pulling them out of a vial, must be an essential part of the injection process. Even when using ready-to-use prefilled syringes (PFS), vigilance must be kept high, knowing the risk of potential contamination.

Ex vivo biophysical characterization of a hydrogel-based artificial vitreous substitute.

PURPOSE: To characterize the biophysical properties of an artificial vitreous body substitute (VBS), which consists of a biocompatible, cross-linked, hyaluronic acid (HA)-based hydrogel, by analysing the VBS's influence on intraocular pressure (IOP) and retinal integrity in distinct ex vivo eye models in order to evaluate the its potential for in vivo biocompatibility testing. METHODS: Pig eyes were obtained immediately postmortem, and VBS was injected after core-vitrectomy. IOP was followed for 24 h (n = 5). VBS influence on retinal integrity was investigated using isolated bovine retinas superfused with an oxygen saturated nutrient solution. An electroretinogram (ERG) was recorded on explanted bovine retinae using silver/silver chloride electrodes; after application of VBS for 2 min, a washout period of 70 min was employed. The percentage of a-and b-wave reduction at the end of the washout phase was compared to baseline values (n = 5). Data were calculated throughout as the mean and the standard deviation. qRT-PCR (Bax/Bcl-2-ratio, GFAP- and PGP9.5-levels) or western blot analysis was used to test for toxicity of Princess Volume after 24 h (and ß-3 tubulin with GAPDH as a control gene). Significance was estimated by Student´s t-test; p ≤0.05 was considered to be statistically significant. RESULTS: The IOP increased non-significantly by 10% after 24 h. Short-term biocompatibility testing using isolated superfused bovine retinas showed neither significant reductions of the b-wave nor the a-wave amplitudes (b-wave reduction 14.2%, p>0.05; a-wave reduction 23.9%, p>0.05). qRT-PCR and western blot analysis did not reveal significant toxicity after 24 h. CONCLUSIONS: The manufactured HA-based hydrogel showed highly favourable biophysical characteristics in the explored ex vivo models, justifying in vivo studies enabling the assessment of biocompatibility.