Genetics of Retinitis Pigmentosa and Other Hereditary Retinal Disorders in Western Switzerland.

INTRODUCTION: Mutational screening of inherited retinal disorders is prerequisite for gene targeted therapy. Our aim was to report and analyze the proportions of mutations in inherited retinal disease (IRD)-causing genes from a single center in Switzerland in order to describe the distribution of IRDs in Western Switzerland. METHODS: We conducted a retrospective study of patient records. Criteria for inclusion were residence in Western Switzerland for patients and relatives presenting a clinical diagnosis of IRDs and an established molecular diagnosis managed by the genetics service of the Jules-Gonin Eye Hospital (JGEH) of Lausanne between January 2002 and December 2022. We initially investigated the IRD phenotypes in all patients (full cohort) with a clinical diagnosis, then calculated the distribution of IRD gene mutations in the entire cohort (genetically determined cohort). We analyzed a sub-group that comprised pediatric patients (≤18 years of age). In addition, we calculated the distribution of gene mutations within the most represented IRDs. Comprehensive gene screening was performed using a combined approach of different generation of DNA microarray analysis, direct sequencing, and Sanger sequencing. RESULTS: The full cohort comprised 899 individuals from 690 families with a clinical diagnosis of IRDs. We identified 400 individuals from 285 families with an elucidated molecular diagnosis (variants in 84 genes) in the genetically determined cohort. The pediatric cohort included 89 individuals from 65 families with an elucidated molecular diagnosis. The molecular diagnosis rate for the genetically determined cohort was 58.2% (family ratio) and the 5 most frequently implicated genes per family were ABCA4 (11.6%), USH2A (7.4%), EYS (6.7%), PRPH2 (6.3%), and BEST1 (4.6%). The pediatric cohort had a family molecular diagnosis rate of 64.4% and the 5 most common mutated genes per family were RS1 (9.2%), ABCA4 (7.7%), CNGB3 (7.7%), CACNA1F (6.2%), CEP290 (4.6%). CONCLUSIONS: This study describes the genetic mutation landscape of IRDs in Western Switzerland in order to quantify their disease burden and contribute to a better orientation of the development of future gene targeted therapies.

Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure.

BACKGROUND: Clustering of microglia around the vasculature has been reported in the retina and the brain after systemic administration of lipopolysaccharides (LPS) in mice. LPS acts via activation of Toll-like receptor 4 (TRL4), which is expressed in several cell types including microglia, monocytes and vascular endothelial cells. The purpose of this study was to investigate the effect of systemic LPS in the pigmented mouse retina and the involvement of endothelial TLR4 in LPS-induced retinal microglia activation. METHODS: C57BL/6J, conditional knockout mice that lack Tlr4 expression selectively on endothelial cells (TekCre-posTlr4loxP/loxP) and TekCre-negTlr4loxP/loxP mice were used. The mice were injected with 1 mg/kg LPS via the tail vein once per day for a total of 4 days. Prior to initiation of LPS injections and approximately 5 h after the last injection, in vivo imaging using fluorescein angiography and spectral-domain optical coherence tomography was performed. Immunohistochemistry, flow cytometry, electroretinography and transmission electron microscopy were utilized to investigate the role of endothelial TLR4 in LPS-induced microglia activation and retinal function. RESULTS: Activation of microglia, infiltration of monocyte-derived macrophages, impaired ribbon synapse organization and retinal dysfunction were observed after the LPS exposure in C57BL/6J and TekCre-negTlr4loxP/loxP mice. None of these effects were observed in the retinas of conditional Tlr4 knockout mice after the LPS challenge. CONCLUSIONS: The findings of the present study suggest that systemic LPS exposure can have detrimental effects in the healthy retina and that TLR4 expressed on endothelial cells is essential for retinal microglia activation and retinal dysfunction upon systemic LPS challenge. This important finding provides new insights into the role of microglia-endothelial cell interaction in inflammatory retinal disease.

Clinical Heterogeneity in Two Siblings Harbouring a Heterozygous PRPH2 Pathogenic Variant.

PURPOSE: The aim of the study was to describe the clinical and genetic correlation of a c.469 G>A p.(Asp157Asn) heterozygous pathogenic variant in PRPH2 in two siblings of Italian origin. PATIENTS AND METHODS: Both patients underwent ophthalmic examination, electrophysiological testing, autofluorescence imaging, and optical coherence tomography (OCT). Screening for pathogenic variants of the obtained DNA from the family members was carried out. RESULTS: The 52-year-old (♀, index patient) and 50-year-old (♂) siblings had BCVA (OD and OS) of 20/20 and 20/16 (♀) and 20/25 and 20/40 (♂), respectively, and suffered increased sensitivity to glare. Yellow irregular macular deposits, numerous small irregular hypo- and hyperreflective spots at the posterior pole, a patchy loss of photoreceptors, and retinal pigment epithelium (RPE) in the perifoveal region were seen. Electrophysiology showed dysfunction of rods and cones, with more affected cone dysfunction in the index patient, contrary to the generalised rod dysfunction in the brother of the index patient. The clinical, electrophysiological, and multimodal imaging findings of both siblings pointed towards Stargardt retinopathy with heterogenic presentation. The DNA analysis identified an autosomal dominant c.469 G>A p.(Asp157Asn) heterozygous pathogenic variant in PRPH2 associated with autosomal dominant cone-rod dystrophy and rod-cone dystrophy. PRPH2 codes for peripherin-2, a membrane protein that consists of 346 amino acids. CONCLUSIONS: Our findings confirm a heterogeneity in clinical presentation associated with pathogenic variants in PRPH2. It may follow either an autosomal dominant or an autosomal recessive mode of inheritance and show a very heterogeneous clinical manifestation of retinal degeneration, e.g., autosomal dominant retinitis pigmentosa (♂ sibling; II-3) and autosomal dominant cone-rod dystrophy (index ♀ sibling; II-2), autosomal dominant macular dystrophy, and also autosomal recessive retinitis pigmentosa.

Fluorescence Lifetime Patterns of Retinal Pigment Epithelium Atrophy in Patients with Stargardt Disease and Age-Related Macular Degeneration.

PURPOSE: To investigate whether autofluorescence lifetime patterns within retinal pigment epithelium (RPE) atrophy differ between age-related macular degeneration (AMD) and Stargardt disease (STGD). METHODS: Mean retinal autofluorescence lifetimes were measured in a short and a long spectral channel (SSC: 498-560 nm; LSC: 560-720 nm). Mean retinal fluorescence lifetimes were analyzed with corresponding clinical features, fundus images, fundus autofluorescence intensity images, and optical coherence tomography. Mean fluorescence lifetime values of atrophic areas were compared between the two cohorts and within the same patient to adjacent nonatrophic regions. RESULTS: Mean fluorescence lifetimes within areas with RPE atrophy of 13 patients with STGD (mean age ± SEM 43.7 ± 5 years) and 30 patients with geographic atrophy (mean age: 78 ± 2 years) were analyzed and compared to age-matched healthy participants. The mean area of RPE atrophy in STGD and AMD was 6.6 ± 2.3 mm2 (range: 0.66-33.17 mm2) and 17.5 ± 3.8 mm2 (range: 0.58-50.02 mm2), respectively. In patients with AMD, atrophic areas revealed significantly longer mean fluorescence lifetime values as compared with patients with STGD (SSC: 997 ± 60 vs. 363 ± 26 ps; LSC: 880 ± 46 vs. 393 ± 23 ps; p < 0.0001). CONCLUSIONS: This study established that RPE atrophy in patients secondary to STGD and AMD display distinctive mean fluorescence lifetime characteristics. As retinal fluorescence lifetimes within areas of RPE atrophy were significantly longer in AMD patients, the analysis of specific lifetime patterns may provide additional insight into the disease processes and the pathogenetic mechanisms in the development of atrophic patches in AMD and STGD.

RETINAL FLECKS IN STARGARDT DISEASE REVEAL CHARACTERISTIC FLUORESCENCE LIFETIME TRANSITION OVER TIME.

PURPOSE: Stargardt disease is the most common inherited juvenile macular dystrophy and is characterized by yellowish flecks across the posterior pole. The purpose of this study was to investigate fluorescence lifetime changes of retinal flecks over time using fluorescence lifetime imaging ophthalmoscopy. METHODS: Longitudinal fluorescence lifetime data of 12 patients with Stargardt disease (mean age ± SEM, 42.25 ± 2.1 years; range, 28-58 years) were acquired using a fluorescence lifetime imaging ophthalmoscope based on a Heidelberg Engineering Spectralis system. Retinal autofluorescence was excited with a 470-nm laser. The emitted fluorescence was detected in two wavelength channels: a short spectral channel (498-560 nm) and a long spectral channel (560-720 nm). The mean retinal autofluorescence lifetimes were calculated and further analyzed with corresponding color fundus images, autofluorescence intensity images, and spectral domain optical coherence tomography. Patients were classified into three subtypes. RESULTS: All patients with Stargardt disease displayed characteristic autofluorescence lifetime patterns. Mean fluorescence lifetime values within areas of yellow flecks were significantly prolonged (long spectral channel 484 ps) compared with the surrounding tissue (long spectral channel 297 ps). In 91.6% of the eyes, flecks with short fluorescence lifetimes (long spectral channel 255 ps) were identified. Short lifetime flecks progressed to flecks with characteristic long lifetimes in 75.1% of eyes within a mean interval of 29.2 months (range 3-45 months). Between baseline and follow-up, the rate of newly developed short lifetime flecks (number/per year) based on subtypes was 2.62 in Group 1, 1.43 in Group 2, and 0.81 in Group 3. CONCLUSION: Recent onset flecks in Stargardt disease display short fluorescence lifetimes and convert into longer fluorescence lifetime flecks over time. This transition may represent a change in the composition of retinal deposits with accumulation of lipofuscin and retinoid by-products from the visual cycle. With emerging treatment options, these findings may prove useful to monitor disease progression and therapeutic effects.

The nuclear hormone receptor gene Nr2c1 (Tr2) is a critical regulator of early retina cell patterning.

Nuclear hormone receptors play a major role in the development of many tissues. This study uncovers a novel role for testicular receptor 2 (Tr2, Nr2c1) in defining the early phase of retinal development and regulating normal retinal cell patterning and topography. The mammalian retina undergoes an overlapping yet biphasic period of development to generate all seven retinal cell types. We discovered that Nr2c1 expression coincides with development of the early retinal cells. Loss of Nr2c1 causes a severe vision deficit and impacts early, but not late retina cell types. Retinal cone cell topography is disrupted with an increase in displaced amacrine cells. Additionally, genetic background significantly impacts phenotypic outcome of cone photoreceptor cells but not amacrine cells. Chromatin-IP experiments reveal NR2C1 regulates early cell transcription factors that regulate retinal progenitor cells during development, including amacrine (Satb2) and cone photoreceptor regulators thyroid and retinoic acid receptors. This study supports a role for Nr2c1 in defining the biphasic period of retinal development and specifically influencing the early phase of retinal cell fate.

Colony-stimulating factor 1 receptor inhibition prevents disruption of the blood-retina barrier during chronic inflammation.

BACKGROUND: Microglia-associated inflammation is closely related to the pathogenesis of various retinal diseases such as uveitis and diabetic retinopathy, which are associated with increased vascular permeability. In this study, we investigated the effect of systemic lipopolysaccharide (LPS) exposure to activation and proliferation of retinal microglia /macrophages. METHODS: Balb/c and Cx3cr1gfp/+ mice were challenged with LPS (1 mg/kg) daily for four consecutive days. For microglia depletion, mice were treated with colony-stimulating factor 1 receptor (CSF-1R) inhibitor PLX5622 1 week before the first LPS challenge and until the end of the experiment. In vivo imaging of the retina was performed on days 4 and 7 after the first LPS challenge, using optical coherence tomography and fluorescein angiography. Flow cytometry analysis, retinal whole mount, and retinal sections were used to investigate microglia and macrophage infiltration and proliferation after LPS challenge. Cytokines were analyzed in the blood as well as in the retina. Data analysis was performed using unpaired t tests, repeated measures one-way ANOVA, or ordinary one-way ANOVA followed by Tukey's post hoc analysis. Kruskal-Wallis test followed by Dunn's multiple comparison tests was used for the analysis of non-normally distributed data. RESULTS: Repeated LPS challenge led to activation and proliferation of retinal microglia, infiltration of monocyte-derived macrophages into the retina, and breakdown of the blood-retina barrier (BRB) accompanied by accumulation of sub-retinal fluid. Using in vivo imaging, we show that the breakdown of the BRB is highly reproducible but transitory. Acute but not chronic systemic exposure to LPS triggered a robust release of inflammatory mediators in the retina with minimal effects in the blood plasma. Inhibition of the CSF-1R by PLX5622 resulted in depletion of retinal microglia, suppression of cytokine production in the retina, and prevention of BRB breakdown. CONCLUSIONS: These findings suggest that microglia/macrophages play an important role in the pathology of retinal disorders characterized by breakdown of the BRB, and suppression of their activation may be a potential therapeutic target for such retinopathies.

Coupling ex vivo electroporation of mouse retinas and luciferase reporter assays to assess rod-specific promoter activity.

Ex vivo electroporation of mouse retinas is an established tool to modulate gene expression and to study cell type-specific gene expression. Here we coupled ex vivo electroporation to luciferase reporter assays to facilitate the study of rod-photoreceptor-specific gene promoters. The activity of the rod-specific proximal bovine rhodopsin promoter was significantly increased in C57BL/6J wild-type retinas at postnatal days 1 and 7 by 3.4-fold and 8.7-fold respectively. In C57BL/6J Nr2e3(rd7/rd7) retinas, where the rod photoreceptor-specific nuclear receptor Nr2e3 is not expressed, a significant increase by 2.5-fold was only observed at postnatal day 7. Cone-specific S-opsin promoter activity was not modulated in C57BL/6J wild-type and Nr2e3(rd7/rd7) retinas. Taken together, we describe an easily implementable protocol to assess rod-specific promoter activity in a physiological context resembling that of the developing postnatal mouse retina.

Differential dimerization of variants linked to enhanced S-cone sensitivity syndrome (ESCS) located in the NR2E3 ligand-binding domain.

NR2E3 encodes the photoreceptor-specific nuclear hormone receptor that acts as a repressor of cone-specific gene expression in rod photoreceptors, and as an activator of several rod-specific genes. Recessive variants located in the ligand-binding domain (LBD) of NR2E3 cause enhanced short wavelength sensitive- (S-) cone syndrome (ESCS), a retinal degeneration characterized by an excess of S-cones and non-functional rods. We analyzed the dimerization properties of NR2E3 and the effect of disease-causing LBD missense variants by bioluminescence resonance energy transfer (BRET(2) ) protein interaction assays. Homodimerization was not affected in presence of p.A256V, p.R039G, p.R311Q, and p.R334G variants, but abolished in presence of p.L263P, p.L336P, p.L353V, p.R385P, and p.M407K variants. Homology modeling predicted structural changes induced by NR2E3 LBD variants. NR2E3 LBD variants did not affect interaction with CRX, but with NRL and rev-erbα/NR1D1. CRX and NRL heterodimerized more efficiently together, than did either with NR2E3. NR2E3 did not heterodimerize with TLX/NR2E1 and RXRα/NR2C1. The identification of a new compound heterozygous patient with detectable rod function, who expressed solely the p.A256V variant protein, suggests a correlation between LBD variants able to form functional NR2E3 dimers and atypical mild forms of ESCS with residual rod function.

IROme, a new high-throughput molecular tool for the diagnosis of inherited retinal dystrophies-a price comparison with Sanger sequencing.

The molecular diagnosis of retinal dystrophies (RD) is difficult because of genetic and clinical heterogeneity. Previously, the molecular screening of genes was done one by one, sometimes in a scheme based on the frequency of sequence variants and the number of exons/length of the candidate genes. Payment for these procedures was complicated and the sequential billing of several genes created endless paperwork. We therefore evaluated the costs of generating and sequencing a hybridization-based DNA library enriched for the 64 most frequently mutated genes in RD, called IROme, and compared them to the costs of amplifying and sequencing these genes by the Sanger method. The production cost generated by the high-throughput (HT) sequencing of IROme was established at CHF 2,875.75 per case. Sanger sequencing of the same exons cost CHF 69,399.02. Turnaround time of the analysis was 3 days for IROme. For Sanger sequencing, it could only be estimated, as we never sequenced all 64 genes in one single patient. Sale cost for IROme calculated on the basis of the sale cost of one exon by Sanger sequencing is CHF 8,445.88, which corresponds to the sale price of 40 exons. In conclusion, IROme is cheaper and faster than Sanger sequencing and therefore represents a sound approach for the diagnosis of RD, both scientifically and economically. As a drop in the costs of HT sequencing is anticipated, target resequencing might become the new gold standard in the molecular diagnosis of RD.

App-based assessment of patient-reported outcomes in the Molecular Tumor Board in the Center for Personalized Medicine-(TRACE).

Background: Biomarker-based therapies are increasingly used in cancer patients outside clinical trials. Systematic assessment of patient-reported outcomes (PRO) is warranted to take patients' perspectives during biomarker-based therapies into consideration. We assessed the feasibility of an electronic PRO assessment via a smartphone application. Methods: An interdisciplinary expert panel developed a smartphone application based on symptom burden and health-related quality of life (HRQoL) metrics reported in a retrospective analysis of 292 neuro-oncological patients. The app included validated assessments of health-related quality of life (HRQoL), the burden of symptoms, and psychological stress. Feasibility and usability were tested in a pilot study. Semi-structured interviews with patients and health care professionals (HCP) were conducted, transcribed, and analyzed according to Mayring´s qualitative content analysis. Furthermore, we assessed compliance and descriptive data of ePROs. Results: A total of 14 patients have been enrolled, (9 female, 5 male). A total of 4 HCPs, 9 patients, and 1 caregiver were interviewed regarding usability/feasibility. The main advantages were the possibility to complete questionnaires at home and comfortable implementation in daily life. Compliance was high, for example, 82% of the weekly distributed NCCN distress thermometer questionnaires were answered on time, however, with interindividual variability. We observed a median distress score of 5 (range 0-10, 197 results, n = 12, weekly assessed) and a median Global health score of 58.3 according to the EORTC QLQ-C30 instrument (range 16.7-100, 77 results, n = 12, monthly assessed). Conclusions: This pilot study proved the feasibility and acceptance of the app. We will therefore expand its application during biomarker-guided therapies to enable systematic PRO assessments.

Retinal pigment epithelium protein of 65 kDA gene-linked retinal degeneration is not modulated by chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C/ chondroitin sulfate proteoglycan 5.

PURPOSE: To analyze in vivo the function of chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C (gene symbol: Cspg5) during retinal degeneration in the Rpe65⁻/⁻ mouse model of Leber congenital amaurosis. METHODS: We resorted to mice with targeted deletions in the Cspg5 and retinal pigment epithelium protein of 65 kDa (Rpe65) genes (Cspg5⁻/⁻/Rpe65⁻/⁻). Cone degeneration was assessed with cone-specific peanut agglutinin staining. Transcriptional expression of rhodopsin (Rho), S-opsin (Opn1sw), M-opsin (Opn1mw), rod transducin α subunit (Gnat1), and cone transducin α subunit (Gnat2) genes was assessed with quantitative PCR from 2 weeks to 12 months. The retinal pigment epithelium (RPE) was analyzed at P14 with immunodetection of the retinol-binding protein membrane receptor Stra6. RESULTS: No differences in the progression of retinal degeneration were observed between the Rpe65⁻/⁻ and Cspg5⁻/⁻/Rpe65⁻/⁻ mice. No retinal phenotype was detected in the late postnatal and adult Cspg5⁻/⁻ mice, when compared to the wild-type mice. CONCLUSIONS: Despite the previously reported upregulation of Cspg5 during retinal degeneration in Rpe65⁻/⁻ mice, no protective effect or any involvement of Cspg5 in disease progression was identified.

High-Resolution Optical Coherence Tomography in Healthy Individuals Provides Resolution at the Cellular and Subcellular Levels.

Purpose: To assess the clinical resolution capacities of a novel high-resolution optical coherence tomography (High-Res OCT). Methods: Eight healthy volunteers were included in this observational study. Using the SPECTRALIS High-Res OCT device (Heidelberg Engineering, Heidelberg) macular b-scans were taken and compared with b-scans acquired with a SPECTRALIS HRA+OCT device (Heidelberg Engineering, Heidelberg). High-Res OCT scans were also compared with hematoxylin and eosin-stained sections from a human donor retina. Results: High-Res OCT allowed identification of several retinal structures at the cellular and subcellular levels, namely, cell nuclei of ganglion cells, displaced amacrine cells, cone photoreceptors and retinal pigment epithelial cells compared with the commercial device. Rod photoreceptor nuclei were partially detectable. Localization of cell type-specific nuclei were confirmed by histological sections of human donor retina. Additionally, all three plexus of the retinal vasculature could be visualized. Conclusions: SPECTRALIS High-Res OCT device provides improved resolution compared with the conventional SPECTRALIS HRA+OCT device and allows to identify structures at the cellular level, similar to histological sections. Translational Relevance: High-Res OCT shows improved visualization of retinal structures in healthy individuals and can be used to assess individual cells within the retina.

Expanding Genotype/Phenotype Correlation in 2p11.2-p12 Microdeletion Syndrome.

Chromosomal abnormalities on the short arm of chromosome 2 in the region p11.2 have been associated with developmental delay, intellectual disability, facial anomalies, abnormal ears, skeletal and genital malformations. Here we describe a patient with a de novo interstitial heterozygous microdeletion on the short arm of chromosome 2 in the region p11.2-p12. He presents with facial dysmorphism characterized by a broad and low root of the nose and low-set protruding ears. Clinical examinations during follow-up visits revealed congenital pendular nystagmus, decreased visual acuity and psychomotor development disorder including intellectual disability. The heterozygous 5 Mb-microdeletion was characterized by an array CGH (Comparative Genomic Hybridization) analysis. In the past two decades, nine patients with microdeletions in this region have been identified by array CGH analysis and were reported in the literature. All these patients show psychomotor development disorder and outer and/or inner ear anomalies. In addition, most of the patients have mild to severe intellectual disability and show facial malformations. We reviewed the literature on PubMed and OMIM using the gene/loci names as search terms in an attempt to identify correlations between genes located within the heterozygous microdeletion and the clinical phenotype of the patient, in order to define a recognizable phenotype for the 2p11.2p12 microdeletion syndrome. We discuss additional symptoms that are not systematically present in all patients and contribute to a heterogeneous clinical presentation of this microdeletion syndrome.

Mutations in CNNM4 cause recessive cone-rod dystrophy with amelogenesis imperfecta.

Cone-rod dystrophies are inherited dystrophies of the retina characterized by the accumulation of deposits mainly localized to the cone-rich macular region of the eye. Dystrophy can be limited to the retina or be part of a syndrome. Unlike nonsyndromic cone-rod dystrophies, syndromic cone-rod dystrophies are genetically heterogeneous with mutations in genes encoding structural, cell-adhesion, and transporter proteins. Using a genome-wide single-nucleotide polymorphism (SNP) haplotype analysis to fine map the locus and a gene-candidate approach, we identified homozygous mutations in the ancient conserved domain protein 4 gene (CNNM4) that either generate a truncated protein or occur in highly conserved regions of the protein. Given that CNNM4 is implicated in metal ion transport, cone-rod dystrophy and amelogenesis imperfecta may originate from abnormal ion homeostasis.