BBS1 branchpoint variant is associated with non-syndromic retinitis pigmentosa.

BACKGROUND: Inherited retinal diseases (IRDs) can be caused by variants in >270 genes. The Bardet-Biedl syndrome 1 (BBS1) gene is one of these genes and may be associated with syndromic and non-syndromic autosomal recessive retinitis pigmentosa (RP). Here, we identified a branchpoint variant in BBS1 and assessed its pathogenicity by in vitro functional analysis. METHODS: Whole genome sequencing was performed for three unrelated monoallelic BBS1 cases with non-syndromic RP. A fourth case received MGCM 105 gene panel analysis. Functional analysis using a midigene splice assay was performed for the putative pathogenic branchpoint variant in BBS1. After confirmation of its pathogenicity, patients were clinically re-evaluated, including assessment of non-ocular features of Bardet-Biedl syndrome. RESULTS: Clinical assessments of probands showed that all individuals displayed non-syndromic RP with macular involvement. Through detailed variant analysis and prioritisation, two pathogenic variants in BBS1, the most common missense variant, c.1169T>G (p.(Met390Arg)), and a branchpoint variant, c.592-21A>T, were identified. Segregation analysis confirmed that in all families, probands were compound heterozygous for c.1169T>G and c.592-21A>T. Functional analysis of the branchpoint variant revealed a complex splicing defect including exon 8 and exon 7/8 skipping, and partial in-frame deletion of exon 8. CONCLUSION: A putative severe branchpoint variant in BBS1, together with a mild missense variant, underlies non-syndromic RP in four unrelated individuals. To our knowledge, this is the first report of a pathogenic branchpoint variant in IRDs that results in a complex splice defect. In addition, this research highlights the importance of the analysis of non-coding regions in order to provide a conclusive molecular diagnosis.

Anterior segment phenotypic changes in late-onset retinal degeneration with Ser163Arg mutation in CTRP5/C1QTNF5.

PURPOSE: Late-onset retinal degeneration (L-ORD) is a rare retinal dystrophy with anterior segment (AS) abnormalities, including long anterior zonules (LAZ) and iris atrophy. This investigation evaluates AS changes in a L-ORD cohort. METHODS: Prospective, longitudinal study including L-ORD individuals (Ser163Arg) with ocular exam and standard slit-lamp photographs between 2011 and 2022. AS images were merged and assessed for LAZ number and zonule-free zone (ZFZ) radius. Further clinical findings such as iris atrophy patterns were reported descriptively. RESULTS: Twelve eyes of 6 patients (4 males, median age = 60.5 years) were included, showing a median of 160 (11-372) LAZs, mainly localized superiorly (39%) and inferiorly (24%). There was a high inter-ocular correlation (rs = 0.94, p < 0.01), no difference in LAZ count between eyes (p = 0.82), and an inverse relationship between LAZ and age (r = - 0.82; p < 0.05). The ZFZ had median 2.1 mm (1.3-5.4), with no inter-ocular difference (p = 0.31). Iris transillumination defects occurred in 11/12 eyes, with 4 major patterns identified: pupillary ruff rarefaction (10/12), patchy atrophy (6/12), notched defects (6/12), and radial streaks (2/12). In a short-term follow-up of 5.9 years, 4 eyes showed a reduction in LAZ count to median 139.5 (67-169) (p = 0.50) and a concomitant increase in ZFZ measurement to median 2.2 (1.7-2.6) (p = 0.17). CONCLUSION: This study confirms symmetric LAZs count and ZFZ in L-ORD, with ZFZ measurements smaller than in previous cohorts. A reduction in LAZs count and an increase in ZFZ with age were suggested longitudinally, yet findings need further evaluation as follow-up was limited to two cases.

Photoreceptor actin dysregulation in syndromic and non-syndromic retinitis pigmentosa.

Retinitis pigmentosa (RP) is the leading cause of inherited blindness. RP is a genetically heterogeneous disorder, with more than 100 different causal genes identified in patients. Central to disease pathogenesis is the progressive loss of retinal photoreceptors. Photoreceptors are specialised sensory neurons that exhibit a complex and highly dynamic morphology. The highly polarised and elaborated architecture of photoreceptors requires precise regulation of numerous cytoskeletal elements. In recent years, significant work has been placed on investigating the role of microtubules (specifically, the acetylated microtubular axoneme of the photoreceptor connecting cilium) and their role in normal photoreceptor function. This has been driven by the emerging field of ciliopathies, human diseases arising from mutations in genes required for cilia formation or function, of which RP is a frequently reported phenotype. Recent studies have highlighted an intimate relationship between cilia and the actin cystoskeleton. This review will focus on the role of actin in photoreceptors, examining the connection between actin dysregulation in RP.

24-Hour Contact Lens Sensor Monitoring of Intraocular Pressure-Related Profiles in Normal-Tension Glaucoma and Rates of Disease Progression.

PURPOSE: To determine the relationship between the rate of glaucomatous visual field loss and the amplitude of a 24-h intraocular pressure (IOP)-related profile measured using a contact lens sensor (CLS). METHODS: This observational study included 22 patients with glaucoma and an IOP of consistently ≤21 mm Hg during office hours. All subjects underwent Goldmann tonometry, standard automated perimetry (SAP), dilated fundus examination, and had a CLS recording. A cosine function was used to obtain peak (acrophase), trough (bathyphase), and amplitude measurements. Prior rates of change in SAP mean deviation were calculated and compared to CLS parameters. RESULTS: The patients had a mean (± SD) age of 66.6 ± 8.2 years (range 54-89 years). Mean follow-up was 6.6 ± 5.0 years with 8.3 ± 3.2 reliable SAP tests. The mean rate of change in SAP was -0.86 ± 1.0 dB per year (range -0.11 to -2.12 dB). Regression analysis suggested faster rates of prior visual field loss in eyes with higher-amplitude CLS curves, but this did not reach statistical significance (R2 = 0.174, p = 0.053). The CLS accurately identified waking and sleeping periods. 59.1% of eyes had a nocturnal acrophase (peaking between 23:00 and 07:00). There was no significant difference in rates of visual field change between patients with nocturnal or diurnal acrophase (-0.71 ± 1.17 and -1.07 ± 0.84 dB/year, respectively, p = 0.437). CONCLUSION: CLS recordings in patients with normal-tension glaucoma (defined by office hours IOP) indicated that 60% of patients had peak IOP during nocturnal hours, which may not be captured using conventional methods of IOP measurement. Novel parameters obtained using the CLS may provide information for predicting the risk of visual field changes for patients with glaucoma.

Use of induced pluripotent stem-cell technology to understand photoreceptor cytoskeletal dynamics in retinitis pigmentosa.

BACKGROUND: Retinitis pigmentosa, which affects one in 3000 people, causes blindness and has no treatment. Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene cause 20% of all cases. Recent work suggests that RPGR, localised to the photoreceptor connecting cilium, regulates rhodopsin transport to the outer segment through its effect on the turnover of actin. We set out to establish a novel model for RPGR disease to test the hypothesis that RPGR mutations lead to retinal degeneration due to a dysregulation of the actin cytoskeleton. METHODS: Patients with RPGR mutations and their unaffected relatives were recruited and skin biopsy samples taken. Fibroblast lines were established and reprogrammed to generate induced pluripotent stem cell (iPSC) lines. A three-dimensional organogenesis protocol was optimised whereby embryoid bodies were formed and patterned towards an eye field fate in a 100-day retinal differentiation protocol, allowing three-dimensional optic cups to form. RPGR-mutated cultures were compared with their healthy controls. FINDINGS: Mutant and wild-type iPSC lines were generated and characterised. Differentiation of all lines resulted in the generation of optic cups in a self-organising manner after 100 days in culture. These cultures contained mature photoreceptors, as evidenced by morphology and both RNA and protein expression. Photoreceptor cultures from RGPR-mutated iPSCs had increased actin polymerisation compared with controls (mean confocal pixel intensity count 59·02 [SD 16·24] vs 23·70 [8·20], p=0·0081). This finding was confirmed by assessment of F-actin with western blot. Pathways regulating actin turnover were explored; western blot analysis showed a reduction in both Src and ERK phosphorylation in RGPR-mutated photoreceptor cultures. An unbiased protein array confirmed this reduction in ERK and Src activation. Several other pathways were also shown to be dysregulated in the RGPR-mutated photoreceptor cultures. INTERPRETATION: This study supports the hypothesis that RPGR mutations lead to actin dysregulation. We have identified several pathways that are interrupted in RPGR-mutant photoreceptor cultures and could be contributing to disease. This study is the first use, to our knowledge, of human iPSCs with retinitis pigmentosa-causing mutations to look at pathophysiology of disease. FUNDING: Wellcome Trust.

Disease mechanisms of X-linked retinitis pigmentosa due to RP2 and RPGR mutations.

Photoreceptor degeneration is the prominent characteristic of retinitis pigmentosa (RP), a heterogeneous group of inherited retinal dystrophies resulting in blindness. Although abnormalities in many pathways can cause photoreceptor degeneration, one of the most important causes is defective protein transport through the connecting cilium, the structure that connects the biosynthetic inner segment with the photosensitive outer segment of the photoreceptors. The majority of patients with X-linked RP have mutations in the retinitis pigmentosa GTPase regulator (RPGR) or RP2 genes, the protein products of which are both components of the connecting cilium and associated with distinct mechanisms of protein delivery to the outer segment. RP2 and RPGR proteins are associated with severe diseases ranging from classic RP to atypical forms. In this short review, we will summarise current knowledge generated by experimental studies and knockout animal models, compare and discuss the prominent hypotheses about the two proteins' functions in retinal cell biology.

RPGR: Its role in photoreceptor physiology, human disease, and future therapies.

Mammalian photoreceptors contain specialised connecting cilia that connect the inner (IS) to the outer segments (OS). Dysfunction of the connecting cilia due to mutations in ciliary proteins are a common cause of the inherited retinal dystrophy retinitis pigmentosa (RP). Mutations affecting the Retinitis Pigmentosa GTPase Regulator (RPGR) protein is one such cause, affecting 10-20% of all people with RP and the majority of those with X-linked RP. RPGR is located in photoreceptor connecting cilia. It interacts with a wide variety of ciliary proteins, but its exact function is unknown. Recently, there have been important advances both in our understanding of RPGR function and towards the development of a therapy. This review summarises the existing literature on human RPGR function and dysfunction, and suggests that RPGR plays a role in the function of the ciliary gate, which controls access of both membrane and soluble proteins to the photoreceptor outer segment. We discuss key models used to investigate and treat RPGR disease and suggest that gene augmentation therapy offers a realistic therapeutic approach, although important questions still remain to be answered, while cell replacement therapy based on retinal progenitor cells represents a more distant prospect.

Stem cell therapies in the management of diabetic retinopathy.

Diabetic retinopathy is the leading cause of blindness in the working population. We now understand that the pathogenesis of the disease contains both a neurodegenerative and vasodegenerative component. Yet despite this, current treatment is still limited to combating the proliferative end stage component of the disease rather than addressing its underlying causes. In recent years, much basic research has focused on demonstrating the potential that several classes of stem cells have in conferring both neuro- and vasoprotection on the diabetic retina. Further, progress has been made in using stem cells to stimulate both neuro and vascular regeneration. This review will discuss the current understanding as to what mechanisms underlie diabetic retinopathy while highlighting the types of stem cells which offer hope as potential novel therapies for diabetic retinopathy, including those that are now in clinical trial.

Molecular Mechanisms Governing Sight Loss in Inherited Cone Disorders.

Inherited cone disorders (ICDs) are a heterogeneous sub-group of inherited retinal disorders (IRDs), the leading cause of sight loss in children and working-age adults. ICDs result from the dysfunction of the cone photoreceptors in the macula and manifest as the loss of colour vision and reduced visual acuity. Currently, 37 genes are associated with varying forms of ICD; however, almost half of all patients receive no molecular diagnosis. This review will discuss the known ICD genes, their molecular function, and the diseases they cause, with a focus on the most common forms of ICDs, including achromatopsia, progressive cone dystrophies (CODs), and cone-rod dystrophies (CORDs). It will discuss the gene-specific therapies that have emerged in recent years in order to treat patients with some of the more common ICDs.

UVR and RPE - The Good, the Bad and the degenerate Macula.

Ultraviolet Radiation (UVR) has a well-established causative influence within the aetiology of conditions of the skin and the anterior segment of the eye. However, a grounded assessment of the role of UVR within conditions of the retina has been hampered by a historical lack of quantitative, and spectrally resolved, assessment of how UVR impacts upon the retina in terms congruent with contemporary theories of ageing. In this review, we sought to summarise the key findings of research investigating the connection between UVR exposure in retinal cytopathology while identifying necessary avenues for future research which can deliver a deeper understanding of UVR's place within the retinal risk landscape.

Ciliary tip actin dynamics regulate photoreceptor outer segment integrity.

As signalling organelles, cilia regulate their G protein-coupled receptor content by ectocytosis, a process requiring localised actin dynamics to alter membrane shape. Photoreceptor outer segments comprise an expanse of folded membranes (discs) at the tip of highly-specialised connecting cilia, into which photosensitive GPCRs are concentrated. Discs are shed and remade daily. Defects in this process, due to mutations, cause retinitis pigmentosa (RP). Whilst fundamental for vision, the mechanism of photoreceptor disc generation is poorly understood. Here, we show membrane deformation required for disc genesis is driven by dynamic actin changes in a process akin to ectocytosis. We show RPGR, a leading RP gene, regulates actin-binding protein activity central to this process. Actin dynamics, required for disc formation, are perturbed in Rpgr mouse models, leading to aborted membrane shedding as ectosome-like vesicles, photoreceptor death and visual loss. Actin manipulation partially rescues this, suggesting the pathway could be targeted therapeutically. These findings help define how actin-mediated dynamics control outer segment turnover.

Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules.

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type- and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.

Treatment Strategy With Gene Editing for Late-Onset Retinal Degeneration Caused by a Founder Variant in C1QTNF5.

Purpose: Genome editing is an emerging group of technologies with the potential to ameliorate dominant, monogenic human diseases such as late-onset retinal degeneration (L-ORD). The goal of this study was to identify disease stages and retinal locations optimal for evaluating the efficacy of a future genome editing trial. Methods: Twenty five L-ORD patients (age range, 33-77 years; median age, 59 years) harboring the founder variant S163R in C1QTNF5 were enrolled from three centers in the United Kingdom and United States. Patients were examined with widefield optical coherence tomography (OCT) and chromatic perimetry under dark-adapted and light-adapted conditions to derive phenomaps of retinal disease. Results were analyzed with a model of a shared natural history of a single delayed exponential across all subjects and all retinal locations. Results: Critical age for the initiation of photoreceptor loss ranged from 48 years at the temporal paramacular retina to 74 years at the inferior midperipheral retina. Subretinal deposits (sRET-Ds) became more prevalent as critical age was approached. Subretinal pigment epithelial deposits (sRPE-Ds) were detectable in the youngest patients showing no other structural or functional abnormalities at the retina. The sRPE-D thickness continuously increased, reaching 25 µm in the extrafoveal retina and 19 µm in the fovea at critical age. Loss of light sensitivity preceded shortening of outer segments and loss of photoreceptors by more than a decade. Conclusions: Retinal regions providing an ideal treatment window exist across all severity stages of L-ORD.

Centriolar satellites expedite mother centriole remodeling to promote ciliogenesis.

Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by Pericentriolar material 1 (PCM1). To study the requirement for centriolar satellites, we generated mice lacking PCM1, a crucial component of satellites. Pcm1-/- mice display partially penetrant perinatal lethality with survivors exhibiting hydrocephalus, oligospermia, and cerebellar hypoplasia, and variably expressive phenotypes such as hydronephrosis. As many of these phenotypes have been observed in human ciliopathies and satellites are implicated in cilia biology, we investigated whether cilia were affected. PCM1 was dispensable for ciliogenesis in many cell types, whereas Pcm1-/- multiciliated ependymal cells and human PCM1-/- retinal pigmented epithelial 1 (RPE1) cells showed reduced ciliogenesis. PCM1-/- RPE1 cells displayed reduced docking of the mother centriole to the ciliary vesicle and removal of CP110 and CEP97 from the distal mother centriole, indicating compromised early ciliogenesis. Similarly, Pcm1-/- ependymal cells exhibited reduced removal of CP110 from basal bodies in vivo. We propose that PCM1 and centriolar satellites facilitate efficient trafficking of proteins to and from centrioles, including the departure of CP110 and CEP97 to initiate ciliogenesis, and that the threshold to trigger ciliogenesis differs between cell types.

Emerging Gene Manipulation Strategies for the Treatment of Monogenic Eye Disease.

Genetic eye diseases, representing a wide spectrum of simple and complex conditions, are one of the leading causes of visual loss in children and working adults, and progress in the field has led to changes in disease investigation, diagnosis, and management. The past 15 years have seen the emergence of novel therapies for these previously untreatable conditions to the extent that we now have a licensed therapy for one form of genetic eye disease and many more in clinical trial. This is a systematic review of published and ongoing clinical trials of gene therapies for monogenic eye diseases. Databases of clinical trials and the published literature were searched for interventional studies of gene therapies for eye diseases. Standard methodological procedures were used to assess the relevance of search results. A total of 59 registered clinical trials are referenced, showing the significant level of interest in the potential for translation of these therapies from bench to bedside. The breadth of therapy design is encouraging, providing multiple possible therapeutic mechanisms. Some fundamental questions regarding gene therapy for genetic eye diseases remain, such as optimal dosing, the relative benefits of adeno-associated virus (AAV)-packaging and the potential for a significant inflammatory response to the therapy itself. As a result, despite the promise of the eye as a target, it has proven difficult to deliver clinically effective gene therapies to the eye. Despite setbacks, the licensing of Luxturna (voretigene neparvovec, Novartis) for the treatment of RPE65-mediated Leber congenital amaurosis (LCA) is a major advance in efforts to treat these rare, but devastating, causes of visual loss.

Pre-existing diabetic retinopathy as a prognostic factor for COVID-19 outcomes amongst people with diabetes: A systematic review.

AIMS: Certain patients with Diabetes Mellitus (DM) have high risk for complications from COVID-19. We aimed to test the hypothesis that pre-existing diabetic retinopathy (DR), a microvascular disease, is a prognostic indicator for poor COVID-19 outcome in this heterogeneous population. METHODS: Seven databases (including MEDLINE) and grey literature were searched, identifying eligible studies using predetermined selection criteria. The Quality in Prognosis Studies (QUIPS) tool was used for quality assessment, followed by narrative synthesis of included studies. RESULTS: Eight cohort studies were identified. Three showed significant positive associations between DR and poor COVID-19 outcomes. The highest quality study, McGurnaghan, found increased risk of the combined outcome fatal or critical care unit (CCU)-treated COVID-19 with referable-grade DR (OR 1.672, 95% CI 1.38-2.03). Indirectly, four studies reported positive associations with microvascular disease and poorer prognosis. Variability between studies limited comparability. CONCLUSIONS: The current literature suggests an independent association between DR and poorer COVID-19 prognosis in patients with DM after controlling for key variables such as age. The use of standardised methodology in future studies would establish the predictive value of DR with greater confidence. Researchers should consider comparing the predictive value of DR and its severity, to other microvascular complications of DM.

A Systematic Review and Meta-Analyses of Interventional Clinical Trial Studies for Gene Therapies for the Inherited Retinal Degenerations (IRDs).

IRDs are one of the leading causes of visual loss in children and young adults. Mutations in over 271 genes lead to retinal dysfunction, degeneration and sight loss. Though no cure exists, gene augmentation therapy has brought hope to the field. This systematic review sought to assess the efficacy of available gene therapy treatments for IRDs. Databases and public resources were searched for randomised controlled trials (RCTs) and non-randomised studies of interventions (NRSIs). Standard methodological procedures were used, including a risk-of-bias assessment. One RCT and five NRSIs were assessed, all for adeno-associated virus two (AAV2)-mediated treatment of RPE-specific 65 kDa (RPE65)-associated LCA (Leber congenital amaurosis). Five outcomes were reported for meta-analyses. Modest improvements in visual acuity, ambulatory navigation/mobility testing or central retinal thickness was observed. There was significant improvement in red and blue light full-field stimulus testing (FST) (red light risk ratio of 1.89, treated v control, p = 0.04; and blue light risk ratio of 2.01, treated v control, p = 0.001). Study design assessment using a ROBIN-I tool (Cochrane Library) showed risk-of-bias judgement to be "low/moderate", whilst there were "some concerns" for the RCT using a RoB-2 tool (Cochrane Library). Although comparison by meta-analysis is compromised by, amongst other issues, a variable amount of vector delivered in each trial, FST improvements demonstrate a proof-of-principle for treating IRDs with gene therapy.

Mechanisms of Photoreceptor Death in Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is the most common cause of inherited blindness and is characterised by the progressive loss of retinal photoreceptors. However, RP is a highly heterogeneous disease and, while much progress has been made in developing gene replacement and gene editing treatments for RP, it is also necessary to develop treatments that are applicable to all causative mutations. Further understanding of the mechanisms leading to photoreceptor death is essential for the development of these treatments. Recent work has therefore focused on the role of apoptotic and non-apoptotic cell death pathways in RP and the various mechanisms that trigger these pathways in degenerating photoreceptors. In particular, several recent studies have begun to elucidate the role of microglia and innate immune response in the progression of RP. Here, we discuss some of the recent progress in understanding mechanisms of rod and cone photoreceptor death in RP and summarise recent clinical trials targeting these pathways.

Automated detection of age-related macular degeneration in color fundus photography: a systematic review.

The rising prevalence of age-related eye diseases, particularly age-related macular degeneration, places an ever-increasing burden on health care providers. As new treatments emerge, it is necessary to develop methods for reliably assessing patients' disease status and stratifying risk of progression. The presence of drusen in the retina represents a key early feature in which size, number, and morphology are thought to correlate significantly with the risk of progression to sight-threatening age-related macular degeneration. Manual labeling of drusen on color fundus photographs by a human is labor intensive and is where automatic computerized detection would appreciably aid patient care. We review and evaluate current artificial intelligence methods and developments for the automated detection of drusen in the context of age-related macular degeneration.

Posner-Schlossman syndrome.

Posner-Schlossman syndrome, or glaucomatocyclitic crisis, is a unilateral ocular condition characterized by recurrent attacks of nongranulomatous anterior uveitis and raised intraocular pressure that can result in chronic secondary glaucoma. This relatively rare disease is most likely the result of recurrent cytomegalovirus infection and affects predominantly middle-aged males. Diagnosis is largely clinical, with aqueous and blood sampling aiding the identification of any underlying infectious cause. Successful disease management is often achieved by topical treatment, although systemic therapy and even surgical intervention may be required. We discuss our current understanding of Posner-Schlossman syndrome, from its pathophysiology through to recommended treatment options.