The Genetic Determinants of Axial Length: From Microphthalmia to High Myopia in Childhood.

The axial length of the eye is critical for normal visual function by enabling light to precisely focus on the retina. The mean axial length of the adult human eye is 23.5 mm, but the molecular mechanisms regulating ocular axial length remain poorly understood. Underdevelopment can lead to microphthalmia (defined as a small eye with an axial length of less than 19 mm at 1 year of age or less than 21 mm in adulthood) within the first trimester of pregnancy. However, continued overgrowth can lead to axial high myopia (an enlarged eye with an axial length of 26.5 mm or more) at any age. Both conditions show high genetic and phenotypic heterogeneity associated with significant visual morbidity worldwide. More than 90 genes can contribute to microphthalmia, and several hundred genes are associated with myopia, yet diagnostic yields are low. Crucially, the genetic pathways underpinning the specification of eye size are only now being discovered, with evidence suggesting that shared molecular pathways regulate under- or overgrowth of the eye. Improving our mechanistic understanding of axial length determination will help better inform us of genotype-phenotype correlations in both microphthalmia and myopia, dissect gene-environment interactions in myopia, and develop postnatal therapies that may influence overall eye growth.

Identification of the regulatory circuit governing corneal epithelial fate determination and disease.

The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells (LSCs), while the nontransparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human LSCs from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors (TFs) that define specific cell fates and established their regulatory hierarchy. Single-cell RNA-seq (scRNA-seq) analyses of the cornea and the epidermis confirmed these shared and cell type-specific TFs. Notably, the shared and LSC-specific TFs can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the LSC fate and its association with corneal opacity.

Increasing the diagnostic yield of childhood glaucoma cases recruited into the 100,000 Genomes Project.

Childhood glaucoma (CG) encompasses a heterogeneous group of genetic eye disorders that is responsible for approximately 5% of childhood blindness worldwide. Understanding the molecular aetiology is key to improving diagnosis, prognosis and unlocking the potential for optimising clinical management. In this study, we investigated 86 CG cases from 78 unrelated families of diverse ethnic backgrounds, recruited into the Genomics England 100,000 Genomes Project (GE100KGP) rare disease cohort, to improve the genetic diagnostic yield. Using the Genomics England/Genomic Medicine Centres (GE/GMC) diagnostic pipeline, 13 unrelated families were solved (13/78, 17%). Further interrogation using an expanded gene panel yielded a molecular diagnosis in 7 more unrelated families (7/78, 9%). This analysis effectively raises the total number of solved CG families in the GE100KGP to 26% (20/78 families). Twenty-five percent (5/20) of the solved families had primary congenital glaucoma (PCG), while 75% (15/20) had secondary CG; 53% of this group had non-acquired ocular anomalies (including iris hypoplasia, megalocornea, ectopia pupillae, retinal dystrophy, and refractive errors) and 47% had non-acquired systemic diseases such as cardiac abnormalities, hearing impairment, and developmental delay. CYP1B1 was the most frequently implicated gene, accounting for 55% (11/20) of the solved families. We identified two novel likely pathogenic variants in the TEK gene, in addition to one novel pathogenic copy number variant (CNV) in FOXC1. Variants that passed undetected in the GE100KGP diagnostic pipeline were likely due to limitations of the tiering process, the use of smaller gene panels during analysis, and the prioritisation of coding SNVs and indels over larger structural variants, CNVs, and non-coding variants.

Loss of the crumbs cell polarity complex disrupts epigenetic transcriptional control and cell cycle progression in the developing retina.

The crumbs cell polarity complex plays a crucial role in apical-basal epithelial polarity, cellular adhesion, and morphogenesis. Homozygous variants in human CRB1 result in autosomal recessive Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP), with no established genotype-phenotype correlation. The associated protein complexes have key functions in developmental pathways; however, the underlying disease mechanism remains unclear. Using the oko meduzym289/m289 (crb2a-/- ) zebrafish, we performed integrative transcriptomic (RNA-seq data) and methylomic [reduced representation bisulphite sequencing (RRBS)] analysis of whole retina to identify dysregulated genes and pathways. Delayed retinal cell specification was identified in both the crb2a-/- zebrafish and CRB1 patient-derived retinal organoids, highlighting the dysfunction of cell cycle modulation and epigenetic transcriptional control. Differential DNA methylation analysis revealed novel hypermethylated pathways involving biological adhesion, Hippo, and transforming growth factor ß (TGFß) signalling. By integrating gene expression with DNA methylation using functional epigenetic modules (FEM), we identified six key modules involving cell cycle control and disturbance of TGFß, bone morphogenetic protein (BMP), Hippo, and SMAD protein signal transduction pathways, revealing significant interactome hotspots relevant to crb2a function and confirming the epigenetic control of gene regulation in early retinal development, which points to a novel mechanism underlying CRB1-retinopathies. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Successful large gene augmentation of USH2A with non-viral episomal vectors.

USH2A mutations are a common cause of autosomal recessive retinitis pigmentosa (RP) and Usher syndrome, for which there are currently no approved treatments. Gene augmentation is a valuable therapeutic strategy for treating many inherited retinal diseases; however, conventional adeno-associated virus (AAV) gene therapy cannot accommodate cDNAs exceeding 4.7 kb, such as the 15.6-kb-long USH2A coding sequence. In the present study, we adopted an alternative strategy to successfully generate scaffold/matrix attachment region (S/MAR) DNA plasmid vectors containing the full-length human USH2A coding sequence, a GFP reporter gene, and a ubiquitous promoter (CMV or CAG), reaching a size of approximately 23 kb. We assessed the vectors in transfected HEK293 cells and USH2A patient-derived dermal fibroblasts in addition to ush2au507 zebrafish microinjected with the vector at the one-cell stage. pS/MAR-USH2A vectors drove persistent transgene expression in patient fibroblasts with restoration of usherin. Twelve months of GFP expression was detected in the photoreceptor cells, with rescue of Usher 2 complex localization in the photoreceptors of ush2au507 zebrafish retinas injected with pS/MAR-USH2A. To our knowledge, this is the first reported vector that can be used to express full-length usherin with functional rescue. S/MAR DNA vectors have shown promise as a novel non-viral retinal gene therapy, warranting further translational development.

Pseudouridylation defect due to DKC1 and NOP10 mutations causes nephrotic syndrome with cataracts, hearing impairment, and enterocolitis.

RNA modifications play a fundamental role in cellular function. Pseudouridylation, the most abundant RNA modification, is catalyzed by the H/ACA small ribonucleoprotein (snoRNP) complex that shares four core proteins, dyskerin (DKC1), NOP10, NHP2, and GAR1. Mutations in DKC1, NOP10, or NHP2 cause dyskeratosis congenita (DC), a disorder characterized by telomere attrition. Here, we report a phenotype comprising nephrotic syndrome, cataracts, sensorineural deafness, enterocolitis, and early lethality in two pedigrees: males with DKC1 p.Glu206Lys and two children with homozygous NOP10 p.Thr16Met. Females with heterozygous DKC1 p.Glu206Lys developed cataracts and sensorineural deafness, but nephrotic syndrome in only one case of skewed X-inactivation. We found telomere attrition in both pedigrees, but no mucocutaneous abnormalities suggestive of DC. Both mutations fall at the dyskerin-NOP10 binding interface in a region distinct from those implicated in DC, impair the dyskerin-NOP10 interaction, and disrupt the catalytic pseudouridylation site. Accordingly, we found reduced pseudouridine levels in the ribosomal RNA (rRNA) of the patients. Zebrafish dkc1 mutants recapitulate the human phenotype and show reduced 18S pseudouridylation, ribosomal dysregulation, and a cell-cycle defect in the absence of telomere attrition. We therefore propose that this human disorder is the consequence of defective snoRNP pseudouridylation and ribosomal dysfunction.

Gene Augmentation of CHM Using Non-Viral Episomal Vectors in Models of Choroideremia.

Choroideremia (CHM) is an X-linked chorioretinal dystrophy leading to progressive retinal degeneration that results in blindness by late adulthood. It is caused by mutations in the CHM gene encoding the Rab Escort Protein 1 (REP1), which plays a crucial role in the prenylation of Rab proteins ensuring correct intracellular trafficking. Gene augmentation is a promising therapeutic strategy, and there are several completed and ongoing clinical trials for treating CHM using adeno-associated virus (AAV) vectors. However, late-phase trials have failed to show significant functional improvements and have raised safety concerns about inflammatory events potentially caused by the use of viruses. Therefore, alternative non-viral therapies are desirable. Episomal scaffold/matrix attachment region (S/MAR)-based plasmid vectors were generated containing the human CHM coding sequence, a GFP reporter gene, and ubiquitous promoters (pS/MAR-CHM). The vectors were assessed in two choroideremia disease model systems: (1) CHM patient-derived fibroblasts and (2) chmru848 zebrafish, using Western blotting to detect REP1 protein expression and in vitro prenylation assays to assess the rescue of prenylation function. Retinal immunohistochemistry was used to investigate vector expression and photoreceptor morphology in injected zebrafish retinas. The pS/MAR-CHM vectors generated persistent REP1 expression in CHM patient fibroblasts and showed a significant rescue of prenylation function by 75%, indicating correction of the underlying biochemical defect associated with CHM. In addition, GFP and human REP1 expression were detected in zebrafish microinjected with the pS/MAR-CHM at the one-cell stage. Injected chmru848 zebrafish showed increased survival, prenylation function, and improved retinal photoreceptor morphology. Non-viral S/MAR vectors show promise as a potential gene-augmentation strategy without the use of immunogenic viral components, which could be applicable to many inherited retinal disease genes.

Foveal Hypoplasia in CRB1-Related Retinopathies.

The CRB1 gene plays a role in retinal development and its maintenance. When disrupted, it gives a range of phenotypes such as early-onset severe retinal dystrophy/Leber congenital amaurosis (EOSRD/LCA), retinitis pigmentosa (RP), cone-rod dystrophy (CORD) and macular dystrophy (MD). Studies in CRB1 retinopathies have shown thickening and coarse lamination of retinal layers resembling an immature retina. Its role in foveal development has not yet been described; however, this retrospective study is the first to report foveal hypoplasia (FH) presence in a CRB1-related retinopathy cohort. Patients with pathogenic biallelic CRB1 variants from Moorfields Eye Hospital, London, UK, were collected. Demographic, clinical data and SD-OCT analyses with FH structural grading were performed. A total of 15 (48%) patients had EOSRD/LCA, 11 (35%) MD, 3 (9%) CORD and 2 (6%) RP. FH was observed in 20 (65%; CI: 0.47-0.79) patients, all of whom were grade 1. A significant difference in BCVA between patients with FH and without was found (p = 0.014). BCVA continued to worsen over time in both groups (p < 0.001), irrespective of FH. This study reports FH in a CRB1 cohort, supporting the role of CRB1 in foveal development. FH was associated with poorer BCVA and abnormal retinal morphology. Nonetheless, its presence did not alter the disease progression.

Clinical and preclinical therapeutic outcome metrics for USH2A-related disease.

USH2A variants are the most common cause of Usher syndrome type 2, characterized by congenital sensorineural hearing loss and retinitis pigmentosa (RP), and also contribute to autosomal recessive non-syndromic RP. Several treatment strategies are under development; however, sensitive clinical trial endpoint metrics to determine therapeutic efficacy have not been identified. In the present study, we have performed longitudinal retrospective examination of the retinal and auditory symptoms in (i) 56 biallelic molecularly confirmed USH2A patients and (ii) ush2a mutant zebrafish to identify metrics for the evaluation of future clinical trials and rapid preclinical screening studies. The patient cohort showed a statistically significant correlation between age and both rate of constriction for the ellipsoid zone length and hyperautofluorescent outer retinal ring area. Visual acuity and pure tone audiograms are not suitable outcome measures. Retinal examination of the novel ush2au507 zebrafish mutant revealed a slowly progressive degeneration of predominantly rods, accompanied by rhodopsin and blue cone opsin mislocalization from 6 to 12 months of age with lysosome-like structures observed in the photoreceptors. This was further evaluated in the ush2armc zebrafish model, which revealed similar changes in photopigment mislocalization with elevated autophagy levels at 6 days post fertilization, indicating a more severe genotype-phenotype correlation and providing evidence of new insights into the pathophysiology underlying USH2A-retinal disease.

Identification of 4 novel human ocular coloboma genes ANK3, BMPR1B, PDGFRA, and CDH4 through evolutionary conserved vertebrate gene analysis.

PURPOSE: Ocular coloboma arises from genetic or environmental perturbations that inhibit optic fissure (OF) fusion during early eye development. Despite high genetic heterogeneity, 70% to 85% of patients remain molecularly undiagnosed. In this study, we have identified new potential causative genes using cross-species comparative meta-analysis. METHODS: Evolutionarily conserved differentially expressed genes were identified through in silico analysis, with in situ hybridization, gene knockdown, and rescue performed to confirm spatiotemporal gene expression and phenotype. Interrogation of the 100,000 Genomes Project for putative pathogenic variants was performed. RESULTS: Nine conserved differentially expressed genes between zebrafish and mouse were identified. Expression of zebrafish ank3a, bmpr1ba/b, cdh4, and pdgfaa was localized to the OF, periocular mesenchyme cells, or ciliary marginal zone, regions traversed by the OF. Knockdown of ank3, bmpr1b, and pdgfaa revealed a coloboma and/or microphthalmia phenotype. Novel pathogenic variants in ANK3, BMPR1B, PDGFRA, and CDH4 were identified in 8 unrelated coloboma families. We showed BMPR1B rescued the knockdown phenotype but variant messenger RNAs failed, providing evidence of pathogenicity. CONCLUSION: We show the utility of cross-species meta-analysis to identify several novel coloboma disease-causing genes. There is a potential to increase the diagnostic yield for new and unsolved patients while adding to our understanding of the genetic basis of OF morphogenesis.

Decision-making, attitudes, and understanding among patients and relatives invited to undergo genome sequencing in the 100,000 Genomes Project: A multisite survey study.

PURPOSE: The purpose of this study was to assess decisions, attitudes, and understanding of participants (patients, parents, relatives) having genome sequencing for rare disease diagnosis. METHODS: This study involved a cross-sectional observational survey with participants in the 100,000 Genomes Project. RESULTS: Survey response rate was 51% (504/978). Most participants self-reported that they had decided to undergo genome sequencing (94%) and that this was an informed decision (84%) with low decisional conflict (95%). Most self-reported that they had chosen to receive additional findings (88%) and that this was an informed decision (89%) with low decisional conflict (95%). Participants were motivated more by the desire to help others via research than by the belief it would help them obtain a diagnosis (Z = 14.23, P = 5.75 × 10-46), although both motivations were high. Concerns were relatively few but, where expressed, were more about the potential psychological impact of results than data sharing/access (Z = 9.61, P = 7.65 × 10-22). Concerns were higher among male, Asian or Asian British, and more religious participants. General and context-specific understanding of genome sequencing were both moderately high (means 5.2/9.0 and 22.5/28.0, respectively). CONCLUSION: These findings are useful to inform consent guidelines and clinical implementation of genome sequencing.

The Natural History of CNGB1-Related Retinopathy: A Longitudinal Phenotypic Analysis.

Cyclic nucleotide-gated channel ß 1 (CNGB1) encodes a subunit of the rod cyclic nucleotide-gated channel. Pathogenic variants in CNGB1 are responsible for 4% of autosomal recessive retinitis pigmentosa (RP). Several treatment strategies show promise for treating inherited retinal degenerations, however relevant metrics of progression and sensitive clinical trial endpoints are needed to assess therapeutic efficacy. This study reports the natural history of CNGB1-related RP with a longitudinal phenotypic analysis of 33 molecularly-confirmed patients with a mean follow-up period of 4.5 ± 3.9 years (range 0-17). The mean best corrected visual acuity (BCVA) of the right eye was 0.31 ± 0.43 logMAR at baseline and 0.47 ± 0.63 logMAR at the final visit over the study period. The ellipsoid zone (EZ) length was measurable in at least one eye of 23 patients and had a mean rate of constriction of 178 ± 161 µm per year (range 1.0-661 µm), with 57% of patients having a decrease in EZ length of greater than 250 µm in a simulated two-year trial period. Hyperautofluorescent outer ring (hyperAF) area was measurable in 17 patients, with 10 patients not displaying a ring phenotype. The results support previous findings of CNGB1-related RP being a slowly progressive disease with patients maintaining visual acuity. Prospective deep phenotyping studies assessing multimodal retinal imaging and functional measures are now required to determine clinical endpoints to be used in a trial.

Investigating Biomarkers for USH2A Retinopathy Using Multimodal Retinal Imaging.

Pathogenic mutations in USH2A are a leading cause of visual loss secondary to non-syndromic or Usher syndrome-associated retinitis pigmentosa (RP). With an increasing number of RP-targeted clinical trials in progress, we sought to evaluate the photoreceptor topography underlying patterns of loss observed on clinical retinal imaging to guide surrogate endpoint selection in USH2A retinopathy. In this prospective cross-sectional study, twenty-five patients with molecularly confirmed USH2A-RP underwent fundus autofluorescence (FAF), spectral-domain optical coherence tomography (SD-OCT) and adaptive optics scanning laser ophthalmoscopy (AOSLO) retinal imaging. Analysis comprised measurement of FAF horizontal inner (IR) and outer (OR) hyperautofluorescent ring diameter; SD-OCT ellipsoid zone (EZ) and external limiting membrane (ELM) width, normalised EZ reflectance; AOSLO foveal cone density and intact macular photoreceptor mosaic (IMPM) diameter. Thirty-two eyes from 16 patients (mean age ± SD, 36.0 ± 14.2 years) with USH2A-associated Usher syndrome type 2 (n = 14) or non-syndromic RP (n = 2) met the inclusion criteria. Spatial alignment was observed between IR-EZ and OR-ELM diameters/widths (p < 0.001). The IMPM border occurred just lateral to EZ loss (p < 0.001), although sparser intact photoreceptor inner segments were detected until ELM disruption. EZ width and IR diameter displayed a biphasic relationship with cone density whereby slow cone loss occurred until retinal degeneration reached ~1350 µm from the fovea, beyond which greater reduction in cone density followed. Normalised EZ reflectance and cone density were significantly associated (p < 0.001). As the strongest correlate of cone density (p < 0.001) and best-corrected visual acuity (p < 0.001), EZ width is the most sensitive biomarker of structural and functional decline in USH2A retinopathy, rendering it a promising trial endpoint.

Synchronized tissue-scale vasculogenesis and ubiquitous lateral sprouting underlie the unique architecture of the choriocapillaris.

The choriocapillaris is an exceptionally high density, two-dimensional, sheet-like capillary network, characterized by the highest exchange rate of nutrients for waste products per area in the organism. These unique morphological and physiological features are critical for supporting the extreme metabolic requirements of the outer retina needed for vision. The developmental mechanisms and processes responsible for generating this unique vascular network remain, however, poorly understood. Here we take advantage of the zebrafish as a model organism for gaining novel insights into the cellular dynamics and molecular signaling mechanisms involved in the development of the choriocapillaris. We show for the first time that zebrafish have a choriocapillaris highly similar to that in mammals, and that it is initially formed by a novel process of synchronized vasculogenesis occurring simultaneously across the entire outer retina. This initial vascular network expands by un-inhibited sprouting angiogenesis whereby all endothelial cells adopt tip-cell characteristics, a process which is sustained throughout embryonic and early post-natal development, even after the choriocapillaris becomes perfused. Ubiquitous sprouting was maintained by continuous VEGF-VEGFR2 signaling in endothelial cells delaying maturation until immediately before stages where vision becomes important for survival, leading to the unparalleled high density and lobular structure of this vasculature. Sprouting was throughout development limited to two dimensions by Bruch's membrane and the sclera at the anterior and posterior surfaces respectively. These novel cellular and molecular mechanisms underlying choriocapillaris development were recapitulated in mice. In conclusion, our findings reveal novel mechanisms underlying the development of the choriocapillaris during zebrafish and mouse development. These results may explain the uniquely high density and sheet-like organization of this vasculature.

Nonsense-mediated mRNA decay efficiency varies in choroideremia providing a target to boost small molecule therapeutics.

Choroideremia (CHM) is an x-linked recessive chorioretinal dystrophy, with 30% caused by nonsense mutations in the CHM gene resulting in an in-frame premature termination codon (PTC). Nonsense-mediated mRNA decay (NMD) is the cell's natural surveillance mechanism that detects and destroys PTC-containing transcripts, with UPF1 being the central NMD modulator. NMD efficiency can be variable amongst individuals with some transcripts escaping destruction, leading to the production of a truncated non-functional or partially functional protein. Nonsense suppression drugs, such as ataluren, target these transcripts and read-through the PTC, leading to the production of a full length functional protein. Patients with higher transcript levels are considered to respond better to these drugs, as more substrate is available for read-through. Using Quantitative reverse transcription PCR (RT-qPCR), we show that CHM mRNA expression in blood from nonsense mutation CHM patients is 2.8-fold lower than controls, and varies widely amongst patients, with 40% variation between those carrying the same UGA mutation [c.715 C>T; p.(R239*)]. These results indicate that although NMD machinery is at work, efficiency is highly variable and not wholly dependent on mutation position. No significant difference in CHM mRNA levels was seen between two patients' fibroblasts and their induced pluripotent stem cell-derived retinal pigment epithelium. There was no correlation between CHM mRNA expression and genotype, phenotype or UPF1 transcript levels. NMD inhibition with caffeine was shown to restore CHM mRNA transcripts to near wild-type levels. Baseline mRNA levels may provide a prognostic indicator for response to nonsense suppression therapy, and caffeine may be a useful adjunct to enhance treatment efficacy where indicated.

Correction: A Newly Developed Web-Based Resource on Genetic Eye Disorders for Users With Visual Impairment (Gene.Vision): Usability Study.

[This corrects the article DOI: 10.2196/19151.].

A Newly Developed Web-Based Resource on Genetic Eye Disorders for Users With Visual Impairment (Gene.Vision): Usability Study.

BACKGROUND: Despite the introduction of the Web Content Accessibility Guidelines and legislations, many websites remain poorly accessible to users with disability, especially those with visual impairment, as the internet has become a more visually complex environment. With increasing reliance on the internet and almost 2 million people in the United Kingdom being affected by vision loss, it is important that they are not overlooked when developing web-based materials. A significant proportion of those affected have irreversible vision loss due to rare genetic eye disorders, and many of them use the internet as a primary source of information for their conditions. However, access to high-quality web-based health information with an inclusive design remains a challenge for many. We have developed a new web-based resource for genetic eye disorders called Gene.Vision thataims to provide a holistic guide for patients, relatives, and health care professionals. OBJECTIVE: Through a usability testing session of our website prototype, this study aims to identify key web-based accessibility features for internet users with vision impairment and to explore whether the contents provided in Gene.Vision are relevant and comprehensible. METHODS: A face-to-face testing session with 8 participants (5 patients, 2 family members, and 1 member of the public) and 8 facilitators was conducted on a prototype website. Remote testing was performed with another patient due to COVID-19 restrictions. Home page design, navigation, content layout and quality, language, and readability were explored through direct observation and task completion using the think-aloud method. A patient focus group was organized to elicit further feedback. Qualitative data were gathered and analyzed to identify core themes through open and axial coding. RESULTS: All participants had good computer literacy; 6 patients with visual impairment used visual aid software including iOS VoiceOver and Speak Screen, iOS Classic Invert, ZoomText 2020, Job Access With Speech, and Nonvisual Desktop Access. The features identified by the participants that will enhance accessibility and usability for users with visual impairment were a consistent website layout, a structured information hierarchy with a clear description of links, good chromatic and luminance contrast, a simple home page with predictable and easy navigation, adaptability to various assistive software, and readable and relevant content. They reported that dynamic content (such as carousels) and large empty spaces reduced accessibility. Information on research, support available, practical advice, and links to charities were incentives for repeated website visits. CONCLUSIONS: We demonstrated the importance of developing a website with a user-based approach. Through end user testing, we identified several key web-based accessibility features for people with visual impairment. Target end users should always be involved early and throughout the design process to ensure their needs are met. Many of these steps can be implemented easily and will aid in search engine optimization.

Involvement of Oxidative and Endoplasmic Reticulum Stress in RDH12-Related Retinopathies.

Retinol dehydrogenase 12 (RDH12) is expressed in photoreceptor inner segments and catalyses the reduction of all-trans retinal (atRAL) to all-trans retinol (atROL), as part of the visual cycle. Mutations in RDH12 are primarily associated with autosomal recessive Leber congenital amaurosis. To further our understanding of the disease mechanisms, HEK-293 cell lines expressing wildtype (WT) and mutant RDH12 were created. The WT cells afforded protection from atRAL-induced toxicity and oxidative stress. Mutant RDH12 cells displayed reduced protein expression and activity, with an inability to protect cells from atRAL toxicity, inducing oxidative and endoplasmic reticulum (ER) stress, with upregulation of sXBP1, CHOP, and ATF4. Pregabalin, a retinal scavenger, attenuated atRAL-induced ER stress in the mutant RDH12 cell lines. A zebrafish rdh12 mutant model (rdh12u533 c.17_23del; p.(Val6AlafsTer5)) was generated through CRISPR-Cas9 gene editing. Mutant fish showed disrupted phagocytosis through transmission electron microscopy, with increased phagosome size at 12 months post-fertilisation. Rhodopsin mislocalisation and reduced expression of atg12 and sod2 indicated early signs of a rod-predominant degeneration. A lack of functional RDH12 results in ER and oxidative stress representing key pathways to be targeted for potential therapeutics.

The Landscape of Non-Viral Gene Augmentation Strategies for Inherited Retinal Diseases.

Inherited retinal diseases (IRDs) are a heterogeneous group of disorders causing progressive loss of vision, affecting approximately one in 1000 people worldwide. Gene augmentation therapy, which typically involves using adeno-associated viral vectors for delivery of healthy gene copies to affected tissues, has shown great promise as a strategy for the treatment of IRDs. However, the use of viruses is associated with several limitations, including harmful immune responses, genome integration, and limited gene carrying capacity. Here, we review the advances in non-viral gene augmentation strategies, such as the use of plasmids with minimal bacterial backbones and scaffold/matrix attachment region (S/MAR) sequences, that have the capability to overcome these weaknesses by accommodating genes of any size and maintaining episomal transgene expression with a lower risk of eliciting an immune response. Low retinal transfection rates remain a limitation, but various strategies, including coupling the DNA with different types of chemical vehicles (nanoparticles) and the use of electrical methods such as iontophoresis and electrotransfection to aid cell entry, have shown promise in preclinical studies. Non-viral gene therapy may offer a safer and effective option for future treatment of IRDs.

Novel Biallelic Variants and Phenotypic Features in Patients with SLC38A8-Related Foveal Hypoplasia.

Biallelic pathogenic variants in solute carrier family 38 member 8, SLC38A8, cause a pan-ocular autosomal recessive condition known as foveal hypoplasia 2, FVH2, characterised by foveal hypoplasia, nystagmus and optic nerve chiasmal misrouting. Patients are often clinically diagnosed with ocular albinism, but foveal hypoplasia can occur in several other ocular disorders. Here we describe nine patients from seven families who had molecularly confirmed biallelic recessive variants in SLC38A8 identified through whole genome sequencing or targeted gene panel testing. We identified four novel sequence variants (p.(Tyr88*), p.(Trp145*), p.(Glu233Gly) and c.632+1G>A). All patients presented with foveal hypoplasia, nystagmus and reduced visual acuity; however, one patient did not exhibit any signs of chiasmal misrouting, and three patients had features of anterior segment dysgenesis. We highlight these findings in the context of 30 other families reported to date. This study reinforces the importance of obtaining a molecular diagnosis in patients whose phenotype overlap with other inherited ocular conditions, in order to support genetic counselling, clinical prognosis and family planning. We expand the spectrum of SLC38A8 mutations which will be relevant for treatment through future genetic-based therapies.