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Stem cell-based therapy has gained importance over the past decades due to huge advances in science and technology behind the generation and directed differentiation of pluripotent cells from embryos and adult cells. Preclinical proof-of-concept studies have been followed by clinical trials showing efficacy and safety of transplantation of stem cell-based therapy, which are beginning to establish this as a modality of treatment. Disease candidates of interest are primarily conditions that may benefit from replacing dead or dying cells, including advanced inherited retinal dystrophies and age-related macular degeneration, and predominantly seek to transplant either RPE or photoreceptors, although neurotrophic approaches have also been trialed. Whilst a consensus has yet to be reached about the best stage/type of cells for transplantation (stem cells, progenitor cells, differentiated RPE and photoreceptors) and the methods of implantation (sheet, suspension), several CTs have shown safety. There remain potential concerns regarding tumorigenicity and immune rejection; however, with ongoing improvements in cell generation, selection, and delivery, these can be minimized. Earlier studies showed efficacy with immunosuppressive drugs to prevent rejection, and recent donor-matched transplants have avoided the need for immunosuppression. Retinal regenerative medicine is a challenging field and is in a nascent stage but holds tremendous promise. This narrative review delves into the current understanding of stem cells and the latest clinical trials of retinal cell transplantation. [ABSTRACT FROM AUTHOR]

Macular dystrophies (MDs) constitute a collection of hereditary retina disorders leading to notable visual impairment, primarily due to progressive macular atrophy. These conditions are distinguished by bilateral and relatively symmetrical abnormalities in the macula that significantly impair central visual function. Recent strides in fundus imaging, especially optical coherence tomography (OCT), have enhanced our comprehension and diagnostic capabilities for MD. OCT enables the identification of neurosensory retinal disorganization patterns and the extent of damage to retinal pigment epithelium (RPE) and photoreceptor cells in the dystrophies before visible macular pathology appears on fundus examinations. It not only helps us in diagnostic retinal and choroidal pathologies but also guides us in monitoring the progression of, staging of, and response to treatment. In this review, we summarize the key findings on OCT in some of the most common MD. [ABSTRACT FROM AUTHOR]

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.

To explore fundus autofluorescence (FAF) imaging as an alternative to electroretinography as a noninvasive, quick, and readily interpretable method to predict disease progression in Stargardt disease (STGD). Retrospective case series of patients who attended Moorfields Eye Hospital (London, United Kingdom). Patients with STGD who met the following criteria were included: (1) biallelic disease-causing variants in ABCA4 , (2) electroretinography testing performed in house with an unequivocal electroretinography group classification, and (3) ultrawidefield (UWF) FAF imaging performed up to 2 years before or after the electroretinography. Patients were divided into 3 electroretinography groups based on retinal function and 3 FAF groups according to the extent of hypoautofluorescence and retinal background appearance. Fundus autofluorescence images of 30° and 55° were reviewed subsequently. Electroretinography and FAF concordance and its association with baseline visual acuity (VA) and genetics. Two hundred thirty-four patients were included in the cohort. One hundred seventy patients (73%) were in electroretinography and FAF groups of the same severity, 33 (14%) were in a milder FAF than electroretinography group, and 31 (13%) were in a more severe FAF than electroretinography group. Children < 10 years of age (n = 23) showed the lowest electroretinography and FAF concordance at 57% (9 of the 10 with discordant electroretinography and FAF showed milder FAF than electroretinography), and adults with adult onset showed the highest (80%). In 97% and 98% of patients, 30° and 55° FAF imaging, respectively, matched with the group defined by UWF FAF. We demonstrated that FAF imaging is an effective method to determine the extent of retinal involvement and thereby inform prognostication by comparing FAF with the current gold standard of electroretinography. In 80% of patients in our large molecularly proven cohort, we were able to predict if the disease was confined to the macula or also affected the peripheral retina. Children assessed at a young age, with at least 1 null variant, early disease onset, poor initial VA, or a combination thereof may have wider retinal involvement than predicted by FAF alone, may progress to a more severe FAF phenotype over time, or both. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article. [ABSTRACT FROM AUTHOR]

Stargardt disease is an inherited retinal condition. With an incidence of 8-10 per 100,000 persons, it is the most common juvenile macular degeneration. The majority of cases present in an autosomal recessive fashion and are due to mutations in the ABCA4 gene. The condition tends to manifest at an early age; however, large amounts of heterogeneity are seen in terms of genotype and phenotype thus the condition can have a later onset with variable clinical findings. The purpose of this case report is to present two different phenotypic presentations of Stargardt disease that were diagnosed based on a combination of multimodal imaging and genetic testing. A comprehensive review of the etiology, clinical features, treatment and management of the disease is also presented. [ABSTRACT FROM AUTHOR]

Macular dystrophies (MDs) constitute a collection of hereditary retina disorders leading to notable visual impairment, primarily due to progressive macular atrophy. These conditions are distinguished by bilateral and relatively symmetrical abnormalities in the macula that significantly impair central visual function. Recent strides in fundus imaging, especially optical coherence tomography (OCT), have enhanced our comprehension and diagnostic capabilities for MD. OCT enables the identification of neurosensory retinal disorganization patterns and the extent of damage to retinal pigment epithelium (RPE) and photoreceptor cells in the dystrophies before visible macular pathology appears on fundus examinations. It not only helps us in diagnostic retinal and choroidal pathologies but also guides us in monitoring the progression of, staging of, and response to treatment. In this review, we summarize the key findings on OCT in some of the most common MD.

Macular dystrophies are a group of individually rare but collectively common inherited retinal dystrophies characterised by central vision loss and loss of visual acuity. Single molecule Molecular Inversion Probes (smMIPs) have proved effective in identifying genetic variants causing macular dystrophy. Here, a previously established smMIPs panel tailored for genes associated with macular diseases has been used to examine 57 UK macular dystrophy cases, achieving a high solve rate of 63.2% (36/57). Among 27 bi-allelic STGD1 cases, only three novel ABCA4 variants were identified, illustrating that the majority of ABCA4 variants in Caucasian STGD1 cases are currently known. We examined cases with ABCA4-associated disease in detail, comparing our results with a previously reported variant grading system, and found this model to be accurate and clinically useful. In this study, we showed that ABCA4-associated disease could be distinguished from other forms of macular dystrophy based on clinical evaluation in the majority of cases (34/36) [ABSTRACT FROM AUTHOR]

Stargardt macular dystrophy 3 (STGD3) is caused by dominant mutations in the ELOVL4 gene. Like other macular degenerations, pathogenesis within the retinal pigment epithelium (RPE) appears to contribute to the loss of photoreceptors from the central retina. However, the RPE does not express ELOVL4, suggesting photoreceptor cell loss in STGD3 occurs through two cell nonautonomous events: mutant photoreceptors first affect RPE cell pathogenesis, and then, second, RPE dysfunction leads to photoreceptor cell death. Here, we have investigated how the RPE pathology occurs, using a STGD3 mouse model in which mutant human ELOVL4 is expressed in the photoreceptors. We found that the mutant protein was aberrantly localized to the photoreceptor outer segment (POS), and that resulting POS phagosomes were degraded more slowly in the RPE. In cell culture, the mutant POSs are ingested by primary RPE cells normally, but the phagosomes are processed inefficiently, even by wild-type RPE. The mutant phagosomes excessively sequester RAB7A and dynein, and have impaired motility. We propose that the abnormal presence of ELOVL4 protein in POSs results in phagosomes that are defective in recruiting appropriate motor protein linkers, thus contributing to slower degradation because their altered motility results in slower basal migration and fewer productive encounters with endolysosomes. In the transgenic mouse retinas, the RPE accumulated abnormal-looking phagosomes and oxidative stress adducts; these pathological changes were followed by pathology in the neural retina. Our results indicate inefficient phagosome degradation as a key component of the first cell nonautonomous event underlying retinal degeneration due to mutant ELOVL4.

Stargardt macular dystrophy (STGD1) is the most common form of inherited childhood blindness worldwide and for which no current treatments exist. It is an autosomal recessive disease caused by mutations in ABCA4. To date, a variety of gene supplementation approaches have been tested to create a therapy, with some reaching clinical trials. New technologies, such as CRISPR-Cas based editing systems, provide an exciting frontier for addressing genetic disease by allowing targeted DNA or RNA base editing of pathogenic mutations. ABCA4 has ∼1,200 known pathogenic mutations, of which ∼63% are transition mutations amenable to this editing technology. In this report, we screened the known "pathogenic" and "likely pathogenic" mutations in ABCA4 from available data in gnomAD, Leiden Open Variation Database (LOVD), and ClinVar for potential PAM sites of relevant base editors, including Streptococcus pyogenes Cas (SpCas), Staphylococcus aureus Cas (SaCas), and the KKH variant of SaCas (Sa-KKH). Overall, of the mutations screened, 53% (ClinVar), 71% (LOVD), and 71% (gnomAD), were editable, pathogenic transition mutations, of which 35–47% had "ideal" PAM sites. Of these mutations, 16–20% occur within a range of multiple PAM sites, enabling a variety of editing strategies. Further, in relevant patient data looking at three cohorts from Germany, Denmark, and China, we find that 44–76% of patients, depending on the presence of complex alleles, have at least one transition mutation with a nearby SaCas, SpCas, or Sa-KKH PAM site, which would allow for potential DNA base editing as a treatment strategy. Given the complexity of the genetic landscape of Stargardt, these findings provide a clearer understanding of the potential for DNA base editing approaches to be applied as ABCA4 gene therapy strategies. [ABSTRACT FROM AUTHOR]

Abstract Background Stargardt disease 1 (STGD1; MIM 248200) is a monogenic form of autosomal recessive genetic disease caused by mutation in ABCA4. This gene has a major role in hydrolyzing N-retinylidene-phosphatidylethanolamine to all-trans-retinal and phosphatidylethanolamine. The purpose of this study is to identify the frequency of putative disease-causing mutations associated with Stargardt disease in a South Indian population. Methods A total of 28 clinically diagnosed Stargardt-like phenotype patients were recruited from south India. Ophthalmic examination of all patients was carefully carried out by a retina specialist based on the stages of fundus imaging and ERG grouping. Genetic analysis of ABCA4 was performed for all patients using Sanger sequencing and clinical exome sequencing. Results This study identified disease-causing mutations in ABCA4 in 75% (21/28) of patients, 7% (2/28) exhibited benign variants and 18% (5/28) were negative for the disease-causing mutation. Conclusion This is the first study describing the genetic association of ABCA4 disease-causing mutation in South Indian Stargardt 1 patients (STGD1). Our findings highlighted the presence of two novel missense mutations and an (in/del, single base pair deletion & splice variant) in ABCA4. However, genetic heterogeneity in ABCA4 mutants requires a larger sample size to establish a true correlation with clinical phenotype.

Purpose: To illustrate the potential diagnostic confusion between Batten disease and Stargardt disease created by associated signs and symptoms. Observations: A six-year-old girl with vision loss and prominent behavioral changes and overlooking was presumptively diagnosed as having the Batten disease form of neuronal ceroid lipofuscinosis. However, genetic testing confirmed the diagnosis of Stargardt disease. Conclusions and importance: Behavioral changes and overlooking can accompany the devastating central visual loss of Stargardt disease. In the absence of objective neurologic findings, these features need not signify a potentially life-threatening disease.

Stargardt macular dystrophy (STGD1) is the most common form of inherited childhood blindness worldwide and for which no current treatments exist. It is an autosomal recessive disease caused by mutations in ABCA4. To date, a variety of gene supplementation approaches have been tested to create a therapy, with some reaching clinical trials. New technologies, such as CRISPR-Cas based editing systems, provide an exciting frontier for addressing genetic disease by allowing targeted DNA or RNA base editing of pathogenic mutations. ABCA4 has ∼1,200 known pathogenic mutations, of which ∼63% are transition mutations amenable to this editing technology. In this report, we screened the known “pathogenic” and “likely pathogenic” mutations in ABCA4 from available data in gnomAD, Leiden Open Variation Database (LOVD), and ClinVar for potential PAM sites of relevant base editors, including Streptococcus pyogenes Cas (SpCas), Staphylococcus aureus Cas (SaCas), and the KKH variant of SaCas (Sa-KKH). Overall, of the mutations screened, 53% (ClinVar), 71% (LOVD), and 71% (gnomAD), were editable, pathogenic transition mutations, of which 35–47% had “ideal” PAM sites. Of these mutations, 16–20% occur within a range of multiple PAM sites, enabling a variety of editing strategies. Further, in relevant patient data looking at three cohorts from Germany, Denmark, and China, we find that 44–76% of patients, depending on the presence of complex alleles, have at least one transition mutation with a nearby SaCas, SpCas, or Sa-KKH PAM site, which would allow for potential DNA base editing as a treatment strategy. Given the complexity of the genetic landscape of Stargardt, these findings provide a clearer understanding of the potential for DNA base editing approaches to be applied as ABCA4 gene therapy strategies.

Purpose: To report a case of ABCA4-related retinopathy and potential complications. Methods: The authors describe a case report of intraretinal neovascularization in a patient with ABCA4-related retinopathy and describe the multimodal retinal imaging findings. Results: A 49-year-old woman presents with cystoid macular edema and diffuse intraretinal and perivascular hyperpigmentation in both eyes. Genetic testing confirmed ABCA4-related retinopathy. Fluorescein angiography revealed peripapillary intraretinal neovascularization in the absence of any other identifiable retinal pathology. Conclusion: To the authors' knowledge, this is the first reported case of intraretinal neovascularization--associated ABCA4-related retinopathy. Ancillary testing of ABCA4- related retinopathy with fluorescein angiography or optical coherence tomography angiography may be helpful in identifying this rare complication. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the clinical and genotypic differences in the spectrum of ABCA4-associated retinopathies (ABCA4Rs)., Design: Observational, cross sectional case series., Participants: Sixty-six patients (132 eyes) carrying biallelic ABCA4 variants., Methods: Patients underwent visual acuity measurement and multimodal imaging. Clinical records were reviewed for age at onset, presenting symptoms, genetic variants, and electroretinogram (ERG). Each eye was assigned to a phenotype based on age at onset, imaging and ERG: cone dystrophy-bull's-eye maculopathy (CD-BEM, 40 eyes), cone-rod dystrophy (CRD, 12 eyes), Stargardt disease (SD, 28 eyes), late-onset SD (LO-SD, 38 eyes), and fundus flavimaculatus (14 eyes). Images were analyzed for: peripapillary sparing, retinal pigment epithelium (RPE) atrophy (definitely decreased autofluorescence, DDAF), flecks patterns using autofluorescence; type of atrophy according to Classification of Atrophy Meeting reports, macular and choroidal thickness on OCT; and choriocapillaris flow deficits on OCT angiography., Main Outcome Measures: Primary outcome was to report the demographic, genotypic, and imaging characteristics of the different ABCA4R phenotypes. Secondary objectives included the assessment of imaging biomarkers as outcome measures for clinical trials., Results: Age at onset was lower in CRD (12 ± 8 years) and higher in patients with LO-SD (59 ± 9 years) (all P < 0.01). Central vision loss was a common presenting symptom in CD-BEM and SD, whereas patients with LO-SD primarily complained of difficult dark adaptation. Missense variants were more frequent in CD-BEM, and splice site in CRD and LO-SD (P < 0.05). Peripapillary sparing was absent in 3 eyes with LO-SD (8%). Cone dystrophy-bull's-eye maculopathy eyes typically had complete outer retinal atrophy alterations (98%), whereas CRD and SD eyes showed both complete outer retinal atrophy and complete RPE and outer retinal atrophy (cRORA) (71%-100%). Patients with LO-SD had larger areas of DDAF (100% cRORA) and of choriocapillaris flow deficits (all P < 0.01). Repeatability of DDAF measurements was low for some phenotypes (CD-BEM and CRD) and atrophic areas <7.5 mm 2 . Resorbed flecks were significantly associated with CRD and LO-SD (P < 0.01)., Conclusions: This research provides a thorough evaluation of the spectrum of ABCA4R. Our findings suggest that certain phenotypes show preferential photoreceptor degeneration (e.g., CD-BEM), whereas others have substantial RPE and choriocapillaris alterations (e.g., LO-SD). We recommend that clinical trial end points take into consideration these imaging features to improve the interpretation of their results., Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article., (Copyright © 2023 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

ELOngation of Very Long chain fatty acids-4 (ELOVL4) is an elongase responsible for the biosynthesis of very long chain (VLC, ≥C28) saturated (VLC-SFA) and polyunsaturated (VLC-PUFA) fatty acids in brain, retina, skin, Meibomian glands, and testes. Fascinatingly, different mutations in this gene have been reported to cause vastly different phenotypes in humans. Heterozygous inheritance of seven different mutations in the coding sequence and 5' untranslated region of ELOVL4 causes autosomal dominant Stargardt-like macular dystrophy (STGD3), while homozygous inheritance of three more mutant variants causes severe seizures with ichthyosis, hypertonia, and even death. Some recent studies have described heterozygous inheritance in yet another three mutant ELOVL4 variants, two that cause spinocerebellar ataxia-34 (SCA34) with erythrokeratodermia (EKV) and one that causes SCA34 without EKV. We identified the specific enzymatic reactions catalyzed by ELOVL4 and, using a variety of genetically engineered mouse models, have actively searched for the mechanisms by which ELOVL4 impacts neural function and health. In this review, we critically compare and contrast the various animal model and case studies involving ELOVL4 deficiency via either mutation or deletion, and the resulting consequences on neuronal health and function in both the retina and central nervous system. [ABSTRACT FROM AUTHOR]

Purpose: Automated spectral domain optical coherence tomography (SD-OCT) segmentation algorithms currently do not perform well in segmenting individual intraretinal layers in eyes with Stargardt disease (STGD). We compared selective B-scan segmentation strategies for generating mean retinal layer thickness and preserved area data from SD-OCT scans in patients with STGD1.Methods: Forty-five eyes from 40 Stargardt patients were randomly selected from the ongoing Natural History of the Progression of Atrophy Secondary to Stargardt Disease (ProgStar) study. All eyes underwent SD-OCT using a standard macular volume consisting of 1024 × 49 equally spaced B-scans within a 20 × 20 degree field centered on the fovea. All 49 B-scans were segmented manually to quantify total retina, outer nuclear layer (ONL), photoreceptor inner segments, photoreceptor outer segments (OS), and retinal pigment epithelial layer (RPE). Mean thickness and total area were generated using all 49 B-scans (spaced 122 μm apart), 25 B-scans (every other B-scan, spaced 240 μm apart), 17 B-scans (every third scan, 353 μm apart), and 13 B-scans (every fourth scan, 462 μm apart), as well as by using an "adaptive" method where a subset (minimum 25 B-scans) of B-scans that the grader deemed as significantly different from adjacent B-scans were utilized. Mean absolute and percentage errors were calculated for macular thickness and area of different retinal layers for the different B-scan subset selection strategies relative to using all 49 B-scans, which was considered the reference or ground truth.Results: Mean thickness and area measurements were significantly different for any regularly spaced reduction in B-scan density relative to the ground truth. When an adaptive approach was applied using a minimum of half the scans, the differences relative to ground truth were no longer significantly different. The mean percent differences for the area and thicknesses of the various layers ranged from 0.02 to 33.66 (p < 0.05 for all comparisons) and 0.44 to 7.24 (p > 0.05) respectively.Conclusion: Manual segmentation of a subset of B-scans using an adaptive strategy can yield thickness and area measurements of retinal sublayers comparable to the reference ground truth derived from using all B-scans in the volume. These results may have implications for increasing the efficiency of SD-OCT grading strategies in clinical trials for STGD and other related macular degenerative disorders. [ABSTRACT FROM AUTHOR]

Animals with enhanced dim-light sensitivity are at higher risk of light-induced retinal degeneration when exposed to bright light conditions. 1,2,3,4 This trade-off is mediated by the rod photoreceptor sensory protein, rhodopsin (RHO), and its toxic vitamin A chromophore by-product, all- trans retinal. 5,6,7,8 Rod arrestin (Arr-1) binds to RHO and promotes sequestration of excess all- trans retinal, 9,10 which has recently been suggested as a protective mechanism against photoreceptor cell death. 2,11 We investigated Arr-1 evolution in animals at high risk of retinal damage due to periodic bright-light exposure of rod-dominated retinas. Here, we find the convergent evolution of enhanced Arr-1/RHO all- trans -retinal sequestration in owls and deep-diving whales. Statistical analyses reveal a parallel acceleration of Arr-1 evolutionary rates in these lineages, which is associated with the introduction of a rare Arr-1 mutation (Q69R) into the RHO-Arr-1 binding interface. Using in vitro assays, we find that this single mutation significantly enhances RHO-all- trans -retinal sequestration by ∼30%. This functional convergence across 300 million years of evolutionary divergence suggests that Arr-1 and RHO may play an underappreciated role in the photoprotection of the eye, with potentially vast clinical significance. • Convergent evolution of Q69R in the rod arrestin of owls and deep-diving whales • Q69R occurs in the binding interface of rod arrestin and rhodopsin • This mutation enhances sequestration of toxic all-trans retinal by rhodopsin • Suggests a novel strategy for treating human blindness caused by all-trans retinal Castiglione et al. identify the convergent evolution of a rare mutation in owls and deep-diving whales. These dim-light specialists also regularly encounter bright sunlight, creating elevated risk for blindness. This mutation enhances sequestration of toxic bright-light by-products, suggesting a novel clinical strategy for treating human blindness. [ABSTRACT FROM AUTHOR]

A 30-year-old female presented with recurrent red, painful right eye. Ocular examination revealed nodular scleritis and on complete workup, tuberculosis was found to be the causative agent. Stargardt’s disease in the same patient was noted as an incidental finding. To the best of our knowledge and after extensive literature search on the internet, this is probably the first reported case of both these diseases of total different etiologies occurring together.

The eye uses vitamin A as a cofactor to sense light and, during this process, some vitamin A molecules dimerize, forming vitamin A dimers. A striking chemical signature of retinas undergoing degeneration in major eye diseases such as age-related macular degeneration (AMD) and Stargardt disease is the accumulation of these dimers in the retinal pigment epithelium (RPE) and Bruch’s membrane (BM). However, it is not known whether dimers of vitamin A are secondary symptoms or primary insults that drive degeneration. Here, we present a chromatography-free method to prepare gram quantities of the vitamin A dimer, A2E, and show that intravenous administration of A2E to the rabbit results in retinal degeneration. A2E-damaged photoreceptors and RPE cells triggered inflammation, induced remolding of the choroidal vasculature and triggered a decline in the retina’s response to light. Data suggest that vitamin A dimers are not bystanders, but can be primary drivers of retinal degeneration. Thus, preventing dimer formation could be a preemptive strategy to address serious forms of blindness.

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., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Purpose Despite being the third most common ABCA4 variant observed in patients with Stargardt disease, the functional effect of the intronic ABCA4 variant c.5461-10T>C is unknown. The purpose of this study was to investigate the molecular effect of this variant. Methods Fibroblast samples from patients carrying the ABCA4 variant c.5461-10T>C were analysed by isolating total RNA, followed by real-time polymerase chain reaction (RT-PCR) using specific primers spanning the variant. For detection of ABCA4 protein, fibroblast samples were lysed and analysed by SDS- PAGE followed by immunoblotting using a monoclonal ABCA4 antibody. Results The ABCA4 variant c.5461-10T>C causes a splicing defect resulting in the reduction of full-length mRNA in fibroblasts from patients and the presence of alternatively spliced mRNAs where exon 39-40 is skipped. A reduced level of full-length ABCA4 protein is observed compared to controls not carrying the variant. Conclusions This study describes the functional effect and the molecular mechanism of the pathogenic ABCA4 variant c.5461-10T>C. The variant is functionally important as it leads to splicing defects and a reduced level of ABCA4 protein. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the structural features of the macular region by enface OCT imaging in patients with clinical diagnosis of Stargardt disease, confirmed by the detection of ABCA4 mutations. Methods: Thirty-two STGD patients were included in the study for a total of 64 eyes. All patients received a comprehensive ophthalmological examination, color fundus photography, fundus auto-fluorescence imaging and OCT. Five OCT scans were considered: ILM and RPE scans (both automatically obtained from the instrument), above-RPE slab, photoreceptor slab and sub-RPE slab (these last three manually obtained). Results: ILM scans showed evident radial folds on the retinal surface in 8/64 eyes (12.5 %). In 6 of the 7 patients, these vitreo-retinal interface abnormalities were unilateral. The photoreceptor slab showed some macular alterations ranging from dis-homogeneous, hypo-reflective abnormalities (7/64 eyes, 11 %) to a homogeneous, well-defined, roundish, hypo-reflective area (17/64 eyes, 27 %) in all the eyes. The sub-RPE slab showed a centrally evident, hyper-reflective abnormality in 58/64 eyes (90.6 %). Superimposing the sub-RPE slab over the images corresponding to the photoreceptor slab, the area of the photoreceptor atrophy sharply exceeded that of the RPE atrophy (44/46 eyes, 96 %). Conclusion: Enface OCT proved to be a clinically useful tool for the management of STGD patients, illustrating in vivo the structural abnormalities of the different retinal layers. [ABSTRACT FROM AUTHOR]

The role of central and peripheral vision in the maintenance of upright stance is debated in literature. Stargardt disease causes visual deficits affecting the central field, but leaving unaltered a patient's peripheral vision. Hence, the study of this rare pathology gives the opportunity to selectively investigate the role of central vision in posture. Postural sway in quiet stance was analyzed in 10 Stargardt patients and 10 control subjects, in three different conditions: (1) eyes closed, (2) eyes open, gazing at a fixed target, and (3) eyes open, tracking a moving target. Stargardt patients outperformed controls in the condition with eyes closed, showing a reduced root mean square (RMS) of the medio-lateral COP displacement, while their performance was not significantly different from controls in the antero-posterior direction. There were no significant differences between patients and controls in open eyes conditions. These results suggest that Stargardt patients adapted to a different visual-somatosensory integration, relying less on vision, especially in the medio-lateral direction. Hence, the central vision seems to affect mostly the medio-lateral direction of postural sway. This finding supports the plausibility of the "functional sensitivity hypothesis", that assigns complementary roles to central and peripheral vision in the control of posture. [ABSTRACT FROM AUTHOR]

La maladie de Stardardt est une dystrophie maculaire héréditaire rare d'apparition précoce caractérisée par une perte progressive de la vision centrale, mais avec une vision périphérique intacte, une légère perte de la vision des couleurs, une adaptation à l'obscurité retardée, et une atrophie maculaire avec ou sans taches paramaculaires et une dégénérescence de l'épithélium pigmentaire de la rétine.

ABSTRACT Autosomal-recessive Stargardt disease ( STGD1) is hallmarked by a large proportion of patients with a single heterozygous causative variant in the disease gene ABCA4. Braun et al. () reported deep intronic variants of ABCA4 in STGD1 patients with one coding variant, prompting us to perform an augmented screen in 131 Belgian STGD1 patients with one or no ABCA4 variant to uncover deep intronic causal ABCA4 variants. This revealed a second variant in 28.6% of cases. Twenty-six percent of these carry the same causal variant c.4539+2001 G> A ( V4). Haplotyping in V4 carriers showed a common region of 63 kb, suggestive of a founder mutation. Genotype-phenotype correlations suggest a moderate-to-severe impact of V4 on the STGD1 phenotype. In conclusion, V4 occurs in a high fraction of Belgian STGD1 patients and represents the first deep intronic founder mutation in ABCA4. This emphasizes the importance of augmented molecular genetic testing of ABCA4 in Belgian STGD1. [ABSTRACT FROM AUTHOR]

ABSTRACT Variants in ABCA4 are responsible for autosomal-recessive Stargardt disease and cone-rod dystrophy. Sequence analysis of ABCA4 exons previously revealed one causative variant in each of 45 probands. To identify the 'missing' variants in these cases, we performed multiplex ligation-dependent probe amplification-based deletion scanning of ABCA4. In addition, we sequenced the promoter region, fragments containing five deep-intronic splice variants, and 15 deep-intronic regions containing weak splice sites. Heterozygous deletions spanning ABCA4 exon 5 or exons 20-22 were found in two probands, heterozygous deep-intronic variants were identified in six probands, and a deep-intronic variant was found together with an exon 20-22 deletion in one proband. Based on ophthalmologic findings and characteristics of the identified exonic variants present in trans, the deep-intronic variants V1 and V4 were predicted to be relatively mild and severe, respectively. These findings are important for proper genetic counseling and for the development of variant-specific therapies. [ABSTRACT FROM AUTHOR]

La maladie de Stardardt est une dystrophie maculaire héréditaire rare d'apparition précoce caractérisée par une perte progressive de la vision centrale, mais avec une vision périphérique intacte, une légère perte de la vision des couleurs, une adaptation à l'obscurité retardée, et une atrophie maculaire avec ou sans taches paramaculaires et une dégénérescence de l'épithélium pigmentaire de la rétine. [ABSTRACT FROM AUTHOR]

Background and Objectives: Stargardt disease produces lipofuscin accumulation predisposing to subretinal fibrosis (SRFib) after ocular trauma. Noninvasive imaging techniques allow in vivo assessment. The purpose of this study is to determine the prevalence of SRFib in a cohort of Stargardt patients, the presence of history of ocular trauma, the clinical features and possible genotype-phenotype associations in Stargardt patients with SRFib., Methods: We evaluated retrospectively 106 Stargardt patients and analysed the multimodal imaging and the genotype of patients with SRFib., Results: Six patients exhibited SRFib, three of them with history of ocular trauma. Multimodal imaging showed extensive SRFib principally in the temporal midperipheral retina with no fluid associated. SRFib was better defined by short wavelength autofluorescence and spectral domain optical coherence tomography and appeared clinically stable over time. There was no particular genotype associated to SRFib., Conclusion: SRFib occurs in a significant percentage of patients with Stargardt disease and can be diagnosed through multimodal imaging regardless the history of trauma, further sustaining the importance of an appropriate imaging in such patients. No genotype-phenotype association has been established, supporting the traumatic etiology in half of cases. The remaining cases may be classified as idiopathic or have a minimal trauma occurring early in life that may be not recalled by the patients.

The diagnosis of Leber hereditary optic neuropathy is suspected in the siblings of an affected person that complains of a decrease in visual acuity. It can also be suspected in a young subject, especially a male, with no medical history that presents with an optic neuropathy. Leber hereditary optic neuropathy is a diagnosis of exclusion. The search for differential diagnoses is essential in all cases, even when a mutation of the mitochondrial DNA was found in the patient of in a healthy carrier maternal relative. This is the interest of multimodal imaging and electrophysiology that allow to exclude retinal pathology mimicking optic neuropathy. A neuroradiological assessment must be systematically prescribed to eliminate a compressive lesion and/or intracranial hypertension. This assessment also provides information on a possible hypersignal of the optic nerve, the appearance of which can be an argument for orientation towards different causes of optic neuritis. Finally, a deficiency or toxic cause must be ruled out., (Copyright © 2022 Publié par Elsevier Masson SAS. All rights reserved.)