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Purpose: To investigate the histology of Bruch’s membrane (BM) calcification in pseudoxanthoma elasticum (PXE) and correlate this to clinical retinal imaging. Design: Experimental study with clinicopathological correlation. Subjects and Controls: Six postmortem eyes from 4 PXE patients and 1 comparison eye from an anonymous donor without PXE. One of the eyes had a multimodal clinical image set for comparison. Methods: Calcification was labeled with OsteSense 680RD, a fluorescent dye specific for hydroxyapatite, and visualized with confocal microscopy. Scanning electron microscopy coupled with energy-dispersive x-ray spectroscopy (SEM-EDX) and time-of-flight secondary ion mass spectrometry (TOF-SIMs) were used to analyze the elemental and ionic composition of different anatomical locations. Findings on cadaver tissues were compared with clinical imaging of 1 PXE patient. Main Outcome Measures: The characteristics and topographical distribution of hydroxyapatite in BM in eyes with PXE were compared with the clinical manifestations of the disease. Results: Analyses of whole-mount and sectioned PXE eyes revealed an extensive, confluent OsteoSense labeling in the central and midperipheral BM, transitioning to a speckled labeling in the midperiphery. These areas corresponded to hyperreflective and isoreflective zones on clinical imaging. Scanning electron microscopy coupled with energy-dispersive x-ray spectroscopy and TOF-SIMs analyses identified these calcifications as hydroxyapatite in BM of PXE eyes. The confluent fluorescent appearance originates from heavily calcified fibrous structures of both the collagen and the elastic layers of BM. Calcification was also detected in an aged comparison eye, but this was markedly different from PXE eyes and presented as small snowflake-like deposits in the posterior pole. Conclusions: Pseudoxanthoma elasticum eyes show extensive hydroxyapatite deposition in the inner and outer collagenous and elastic BM layers in the macula with a gradual change toward the midperiphery, which seems to correlate with the clinical phenotype. The snowflake-like calcification in BM of an aged comparison eye differed markedly from the extensive calcification in PXE. Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Inherited optic neuropathies (IONs) are rare genetic diseases characterized by progressive visual loss due the atrophy of optic nerves. The standard diagnostic workup involving next-generation sequencing panels has a diagnostic yield of about forty percent. In the other 60% of the patients with a clinical diagnosis of ION, the underlying genetic variants remain unknown. In this case study, we describe a potentially new disease-associated gene, NSUN3, for IONs. The proband was a young woman with consanguineous parents. She presented with bilateral optic atrophy and nystagmus at the age of seven years. Genetic testing revealed the homozygous variant c.349_352dup p.(Ala118Glufs*45) in NSUN3, with a segregation in the family compatible with autosomal recessive inheritance. Additional functional analysis showed decreased NSUN3 mRNA levels, slightly diminished mitochondrial complex IV levels, and decreased cell respiration rates in patient fibroblasts compared to healthy controls. In conclusion, pathogenic variants in NSUN3 can cause optic neuropathy. Trio whole-exome sequencing should be considered as a diagnostic strategy in ION cases where standard diagnostic analysis does not reveal disease-causing variants.

Within the last decade, a wide variety of protocols have emerged for the generation of retinal organoids. A subset of studies have compared protocols based on stem cell source, the physical features of the microenvironment, and both internal and external signals, all features that influence embryoid body and retinal organoid formation. Most of these comparisons have focused on the effect of signaling pathways on retinal organoid development. In this study, our aim is to understand whether starting cell conditions, specifically those involved in embryoid body formation, affect the development of retinal organoids in terms of differentiation capacity and reproducibility. To investigate this, we used the popular 3D floating culture method to generate retinal organoids from stem cells. This method starts with either small clumps of stem cells generated from larger clones (clumps protocol, CP) or with an aggregation of single cells (single cells protocol, SCP). Using histological analysis and gene-expression comparison, we found a retention of the pluripotency capacity on embryoid bodies generated through the SCP compared to the CP. Nonetheless, these early developmental differences seem not to impact the final retinal organoid formation, suggesting a potential compensatory mechanism during the neurosphere stage. This study not only facilitates an in-depth exploration of embryoid body development but also provides valuable insights for the selection of the most suitable protocol in order to study retinal development and to model inherited retinal disorders in vitro.

X-linked juvenile retinoschisis (XLRS) is an early-onset progressive inherited retinopathy affecting males. It is characterized by abnormalities in the macula, with formation of cystoid retinal cavities, frequently accompanied by splitting of the retinal layers, impaired synaptic transmission of visual signals, and associated loss of visual acuity. XLRS is caused by loss-of-function mutations in the retinoschisin gene located on the X chromosome (RS1, MIM 30083). While proof-of-concept studies for gene augmentation therapy have been promising in in vitro and rodent models, clinical trials in XLRS patients have not been successful thus far. We performed a systematic literature investigation using search strings related to XLRS and gene therapy in in vivo and in vitro models. Three rounds of screening (title/abstract, full text and qualitative) were performed by two independent reviewers until consensus was reached. Characteristics related to study design and intervention were extracted from all studies. Results were divided into studies using (1) viral and (2) non-viral therapies. All in vivo rodent studies that used viral vectors were assessed for quality and risk of bias using the SYRCLE’s risk-of-bias tool. Studies using alternative and non-viral delivery techniques, either in vivo or in vitro, were extracted and reviewed qualitatively, given the diverse and dispersed nature of the information. For in-depth analysis of in vivo studies using viral vectors, outcome data for optical coherence tomography (OCT), immunohistopathology and electroretinography (ERG) were extracted. Meta-analyses were performed on the effect of recombinant adeno-associated viral vector (AAV)-mediated gene augmentation therapies on a- and b-wave amplitude as well as the ratio between b- and a-wave amplitudes (b/a-ratio) extracted from ERG data. Subgroup analyses and meta-regression were performed for model, dose, age at injection, follow-up time point and delivery method. Between-study heterogeneity was assessed with a Chi-square test of homogeneity (I2). We identified 25 studies that target RS1 and met our search string. A total of 19 of these studies reported rodent viral methods in vivo. Six of the 25 studies used non-viral or alternative delivery methods, either in vitro or in vivo. Of these, five studies described non-viral methods and one study described an alternative delivery method. The 19 aforementioned in vivo studies were assessed for risk of bias and quality assessments and showed inconsistency in reporting. This resulted in an unclear risk of bias in most included studies. All 19 studies used AAVs to deliver intact human or murine RS1 in rodent models for XLRS. Meta-analyses of a-wave amplitude, b-wave amplitude, and b/a-ratio showed that, overall, AAV-mediated gene augmentation therapy significantly ameliorated the disease phenotype on these parameters. Subgroup analyses and meta-regression showed significant correlations between b-wave amplitude effect size and dose, although between-study heterogeneity was high. This systematic review reiterates the high potential for gene therapy in XLRS, while highlighting the importance of careful preclinical study design and reporting. The establishment of a systematic approach in these studies is essential to effectively translate this knowledge into novel and improved treatment alternatives.

Summary: A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis.One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells.Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment.We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.

Abstract Background To investigate whether copy number variations (CNVs) are implicated in molecular mechanisms underlying primary open-angle glaucoma (POAG), we used genotype data of POAG individuals and healthy controls from two case-control studies, AGS (n = 278) and GLGS-UGLI (n = 1292). PennCNV, QuantiSNP, and cnvPartition programs were used to detect CNV. Stringent quality controls at both sample and marker levels were applied. The identified CNVs were intersected in CNV region (CNVR). After, we performed burden analysis, CNV-genome-wide association analysis, gene set overrepresentation and pathway analysis. In addition, in human eye tissues we assessed the expression of the genes lying within significant CNVRs. Results We reported a statistically significant greater burden of CNVs in POAG cases compared to controls (p-value = 0,007). In common between the two cohorts, CNV-association analysis identified statistically significant CNVRs associated with POAG that span 11 genes (APC, BRCA2, COL3A1, HLA-DRB1, HLA-DRB5, HLA-DRB6, MFSD8, NIPBL, SCN1A, SDHB, and ZDHHC11). Functional annotation and pathway analysis suggested the involvement of cadherin, Wnt signalling, and p53 pathways. Conclusions Our data suggest that CNVs may have a role in the susceptibility of POAG and they can reveal more information on the mechanism behind this disease. Additional genetic and functional studies are warranted to ascertain the contribution of CNVs in POAG.

Retinitis pigmentosa (RP) comprises a group of inherited retinal dystrophies characterized by the degeneration of rod photoreceptors, followed by the degeneration of cone photoreceptors. As a result of photoreceptor degeneration, affected individuals experience gradual loss of visual function, with primary symptoms of progressive nyctalopia, constricted visual fields and, ultimately, central vision loss. The onset, severity and clinical course of RP shows great variability and unpredictability, with most patients already experiencing some degree of visual disability in childhood. While RP is currently untreatable for the majority of patients, significant efforts have been made in the development of genetic therapies, which offer new hope for treatment for patients affected by inherited retinal dystrophies. In this exciting era of emerging gene therapies, it remains imperative to continue supporting patients with RP using all available options to manage their condition. Patients with RP experience a wide variety of physical, mental and social-emotional difficulties during their lifetime, of which some require timely intervention. This review aims to familiarize readers with clinical management options that are currently available for patients with RP.

Zinc supplementation has been shown to be beneficial to slow the progression of age-related macular degeneration (AMD). However, the molecular mechanism underpinning this benefit is not well understood. This study used single-cell RNA sequencing to identify transcriptomic changes induced by zinc supplementation. Human primary retinal pigment epithelial (RPE) cells could mature for up to 19 weeks. After 1 or 18 weeks in culture, we supplemented the culture medium with 125 µM added zinc for one week. RPE cells developed high transepithelial electrical resistance, extensive, but variable pigmentation, and deposited sub-RPE material similar to the hallmark lesions of AMD. Unsupervised cluster analysis of the combined transcriptome of the cells isolated after 2, 9, and 19 weeks in culture showed considerable heterogeneity. Clustering based on 234 pre-selected RPE-specific genes divided the cells into two distinct clusters, we defined as more and less differentiated cells. The proportion of more differentiated cells increased with time in culture, but appreciable numbers of cells remained less differentiated even at 19 weeks. Pseudotemporal ordering identified 537 genes that could be implicated in the dynamics of RPE cell differentiation (FDR < 0.05). Zinc treatment resulted in the differential expression of 281 of these genes (FDR < 0.05). These genes were associated with several biological pathways with modulation of ID1/ID3 transcriptional regulation. Overall, zinc had a multitude of effects on the RPE transcriptome, including several genes involved in pigmentation, complement regulation, mineralization, and cholesterol metabolism processes associated with AMD.

Background and purpose: Primary open-angle glaucoma (POAG) is an optic neuropathy characterized by death of retinal ganglion cells and atrophy of the optic nerve head. The susceptibility of the optic nerve to damage has been shown to be mediated by mitochondrial dysfunction. In this study, we aimed to determine a possible association between mitochondrial SNPs or haplogroups and POAG.Methods: Mitochondrial DNA single nucleotide polymorphisms (mtSNPs) were genotyped using the Illumina Infinium Global Screening Array-24 (GSA) 700K array set. Genetic analyses were performed in a POAG case-control study involving the cohorts, Groningen Longitudinal Glaucoma Study-Lifelines Cohort Study and Amsterdam Glaucoma Study, including 721 patients and 1951 controls in total. We excluded samples not passing quality control for nuclear genotypes and samples with low call rate for mitochondrial variation. The mitochondrial variants were analyzed both as SNPs and haplogroups. These were determined with the bioinformatics software HaploGrep, and logistic regression analysis was used for the association, as well as for SNPs.Results: Meta-analysis of the results from both cohorts revealed a significant association between POAG and the allele A of rs2853496 [odds ratio (OR) = 0.64; p = 0.006] within the MT-ND4 gene, and for the T allele of rs35788393 (OR = 0.75; p = 0.041) located in the MT-CYB gene. In the mitochondrial haplogroup analysis, the most significant p-value was reached by haplogroup K (p = 1.2 × 10−05), which increases the risk of POAG with an OR of 5.8 (95% CI 2.7–13.1).Conclusion: We identified an association between POAG and polymorphisms in the mitochondrial genes MT-ND4 (rs2853496) and MT-CYB (rs35788393), and with haplogroup K. The present study provides further evidence that mitochondrial genome variations are implicated in POAG. Further genetic and functional studies are required to substantiate the association between mitochondrial gene polymorphisms and POAG and to define the pathophysiological mechanisms of mitochondrial dysfunction in glaucoma.

Background: Primary open-angle glaucoma (POAG) is the most prevalent glaucoma subtype, but its exact etiology is still unknown. In this study, we aimed to prioritize the most likely ‘causal’ genes and identify functional characteristics and underlying biological pathways of POAG candidate genes. Methods: We used the results of a large POAG genome-wide association analysis study from GERA and UK Biobank cohorts. First, we performed systematic gene-prioritization analyses based on: (i) nearest genes; (ii) nonsynonymous single-nucleotide polymorphisms; (iii) co-regulation analysis; (iv) transcriptome-wide association studies; and (v) epigenomic data. Next, we performed functional enrichment analyses to find overrepresented functional pathways and tissues. Results: We identified 142 prioritized genes, of which 64 were novel for POAG. BICC1, AFAP1, and ABCA1 were the most highly prioritized genes based on four or more lines of evidence. The most significant pathways were related to extracellular matrix turnover, transforming growth factor-β, blood vessel development, and retinoic acid receptor signaling. Ocular tissues such as sclera and trabecular meshwork showed enrichment in prioritized gene expression (>1.5 fold). We found pleiotropy of POAG with intraocular pressure and optic-disc parameters, as well as genetic correlation with hypertension and diabetes-related eye disease. Conclusions: Our findings contribute to a better understanding of the molecular mechanisms underlying glaucoma pathogenesis and have prioritized many novel candidate genes for functional follow-up studies.

Purpose: The lack of suitable animal models for (dry) age-related macular degeneration (AMD) has hampered therapeutic research into the disease, so far. In this study, pigmented rats and mice were systematically injected with various doses of sodium iodate (SI). After injection, the retinal structure and visual function were non-invasively characterized over time to obtain in-depth data on the suitability of these models for studying experimental therapies for retinal degenerative diseases, such as dry AMD. Methods: SI was injected into the tail vein (i.v.) using a series of doses (0–70 mg/kg) in adolescent C57BL/6J mice and Brown Norway rats. The retinal structure and function were assessed non-invasively at baseline (day 1) and at several time points (1–3, 5, and 10-weeks) post-injection by scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), and electroretinography (ERG). Results: After the SI injection, retinal degeneration in mice and rats yielded similar results. The lowest dose (10 mg/kg) resulted in non-detectable structural or functional effects. An injection with 20 mg/kg SI did not result in an evident retinal degeneration as judged from the OCT data. In contrast, the ERG responses were temporarily decreased but returned to baseline within two-weeks. Higher doses (30, 40, 50, and 70 mg/kg) resulted in moderate to severe structural RPE and retinal injury and decreased the ERG amplitudes, indicating visual impairment in both mice and rat strains. Conclusions: After the SI injections, we observed dose-dependent structural and functional pathological effects on the retinal pigment epithelium (RPE) and retina in the pigmented mouse and rat strains that were used in this study. Similar effects were observed in both species. In particular, a dose of 30 mg/kg seems to be suitable for future studies on developing experimental therapies. These relatively easily induced non-inherited models may serve as useful tools for evaluating novel therapies for RPE-related retinal degenerations, such as AMD.

This study investigated the phenotypic spectrum of PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and early-onset cataract) syndrome caused by biallelic variants in the ABHD12 gene. A total of 15 patients from 12 different families were included, with a mean age of 36.7 years (standard deviation [SD] ± 11.0; range from 17.5 to 53.9) at the most recent examination. The presence and onset of neurological, audiological and ophthalmic symptoms were variable, with no evident order of symptom appearance. The mean best-corrected visual acuity was 1.1 logMAR (SD ± 0.9; range from 0.1 to 2.8; equivalent to 20/250 Snellen) and showed a trend of progressive decline. Different types of cataract were observed in 13 out of 15 patients (87%), which also included congenital forms of cataract. Fundus examination revealed macular involvement in all patients, ranging from alterations of the retinal pigment epithelium to macular atrophy. Intraretinal spicular hyperpigmentation was observed in 7 out of 15 patients (47%). From an ophthalmic perspective, clinical manifestations in patients with PHARC demonstrate variability with regard to their onset and severity. Given the variable nature of PHARC, an early multidisciplinary assessment is recommended to assess disease severity.

Purpose: We developed and phenotyped a pigmented knockout rat model for lecithin retinol acyltransferase (LRAT) using CRISPR/Cas9. The introduced mutation (c.12delA) is based on a patient group harboring a homologous homozygous frameshift mutation in the LRAT gene (c.12delC), causing a dysfunctional visual (retinoid) cycle. Methods: The introduced mutation was confirmed by DNA and RNA sequencing. The expression of Lrat was determined on both the RNA and protein level in wildtype and knockout animals using RT-PCR and immunohistochemistry. The retinal structure and function, as well as the visual behavior of the Lrat−/− and control rats, were characterized using scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), electroretinography (ERG) and vision-based behavioral assays. Results: Wildtype animals had high Lrat mRNA expression in multiple tissues, including the eye and liver. In contrast, hardly any expression was detected in Lrat−/− animals. LRAT protein was abundantly present in wildtype animals and absent in Lrat−/− animals. Lrat−/− animals showed progressively reduced ERG potentials compared to wildtype controls from two weeks of age onwards. Vison-based behavioral assays confirmed reduced vision. Structural abnormalities, such as overall retinal thinning, were observed in Lrat−/− animals. The retinal thickness in knockout rats was decreased to roughly 80% by four months of age. No functional or structural differences were observed between wildtype and heterozygote animals. Conclusions: Our Lrat−/− rat is a new animal model for retinal dystrophy, especially for the LRAT-subtype of early-onset retinal dystrophies. This model has advantages over the existing mouse models and the RCS rat strain and can be used for translational studies of retinal dystrophies.

A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A. Seven unexplained cases were selected for future analysis with whole genome sequencing.

Early in vivo embryonic retinal development is a well-documented and evolutionary conserved process. The specification towards eye development is temporally controlled by consecutive activation or inhibition of multiple key signaling pathways, such as the Wnt and hedgehog signaling pathways. Recently, with the use of retinal organoids, researchers aim to manipulate these pathways to achieve better human representative models for retinal development and disease. To achieve this, a plethora of different small molecules and signaling factors have been used at various time points and concentrations in retinal organoid differentiations, with varying success. Additions differ from protocol to protocol, but their usefulness or efficiency has not yet been systematically reviewed. Interestingly, many of these small molecules affect the same and/or multiple pathways, leading to reduced reproducibility and high variability between studies. In this review, we make an inventory of the key signaling pathways involved in early retinogenesis and their effect on the development of the early retina in vitro. Further, we provide a comprehensive overview of the small molecules and signaling factors that are added to retinal organoid differentiation protocols, documenting the molecular and functional effects of these additions. Lastly, we comparatively evaluate several of these factors using our established retinal organoid methodology.

The retinal pigment epithelium (RPE) and the adjacent light-sensitive photoreceptors form a single functional unit lining the back of the eye. Both cell layers are essential for normal vision. RPE degeneration is usually followed by photoreceptor degeneration and vice versa. There are currently almost no effective therapies available for RPE disorders such as Stargardt disease, specific types of retinitis pigmentosa, and age-related macular degeneration. RPE replacement for these disorders, especially in later stages of the disease, may be one of the most promising future therapies. There is, however, no consensus regarding the optimal RPE source, delivery strategy, or the optimal experimental host in which to test RPE replacement therapy. Multiple RPE sources, delivery methods, and recipient animal models have been investigated, with variable results. So far, a systematic evaluation of the (variables influencing) efficacy of experimental RPE replacement parameters is lacking. Here we investigate the effect of RPE transplantation on vision and vision-based behavior in animal models of retinal degenerated diseases. In addition, we aim to explore the effect of RPE source used for transplantation, the method of intervention, and the animal model which is used. Methods: In this study, we systematically identified all publications concerning transplantation of RPE in experimental animal models targeting the improvement of vision (e.g., outcome measurements related to the morphology or function of the eye). A variety of characteristics, such as species, gender, and age of the animals but also cell type, number of cells, and other intervention characteristics were extracted from all studies. A risk of bias analysis was performed as well. Subsequently, all references describing one of the following outcomes were analyzed in depth in this systematic review: a-, b-, and c-wave amplitudes, vision-based, thickness analyses based on optical coherence tomography (OCT) data, and transplant survival based on scanning laser ophthalmoscopy (SLO) data. Meta-analyses were performed on the a- and b-wave amplitudes from electroretinography (ERG) data as well as data from vision-based behavioral assays. Results: original research articles met the inclusion criteria after two screening rounds. Overall, most studies were categorized as unclear regarding the risk of bias, because many experimental details were poorly reported. Twenty-three studies reporting one or more of the outcome measures of interest were eligible for either descriptive (thickness analyses based on OCT data; n = 2) or meta-analyses. RPE transplantation significantly increased ERG a-wave (Hedges’ g 1.181 (0.471–1.892), n = 6) and b-wave (Hedges’ g 1.734 (1.295–2.172), n = 42) amplitudes and improved vision-based behavior (Hedges’ g 1.018 (0.826–1.209), n = 96). Subgroup analyses revealed a significantly increased effect of the use of young and adolescent animals compared to adult animals. Moreover, transplanting more cells (in the range of 105 versus in the range of 104) resulted in a significantly increased effect on vision-based behavior as well. The origin of cells mattered as well. A significantly increased effect was found on vision-based behavior when using ARPE-19 and OpRegen® RPE. Conclusions: This systematic review shows that RPE transplantation in animal models for retinal degeneration significantly increases a- and b- wave amplitudes and improves vision-related behavior. These effects appear to be more pronounced in young animals, when the number of transplanted cells is larger and when ARPE-19 and OpRegen® RPE cells are used. We further emphasize that there is an urgent need for improving the reporting and methodological quality of animal experiments, to make such studies more comparable.

This study describes the clinical, genetic, and histopathological features in patients with RPGR-associated retinal dystrophies. Nine male patients from eight unrelated families underwent a comprehensive ophthalmic examination. Additionally, the histopathology of the right eye from a patient with an end-stage cone-rod-dystrophy (CRD)/sector retinitis pigmentosa (RP) phenotype was examined. All RPGR mutations causing a CRD phenotype were situated in exon ORF15. The mean best-corrected visual acuity (BCVA, decimals) was 0.58 (standard deviation (SD)): 0.34; range: 0.05−1.13); and the mean spherical refractive error was −4.1 D (SD: 2.11; range: −1.38 to −8.19). Hyperautofluorescent rings were observed in six patients. Full-field electroretinography responses were absent in all patients. The visual field defects ranged from peripheral constriction to central islands. The mean macular sensitivity on microperimetry was 11.6 dB (SD: 7.8; range: 1.6−24.4) and correlated significantly with BCVA (r = 0.907; p = 0.001). A histological examination of the donor eye showed disruption of retinal topology and stratification, with a more severe loss found in the peripheral regions. Reactive gliosis was seen in the inner layers of all regions. Our study demonstrates the highly variable phenotype found in RPGR-associated retinal dystrophies. Therapies should be applied at the earliest signs of photoreceptor degeneration, prior to the remodeling of the inner retina.

The retinal pigment epithelium (RPE) is a neural monolayer lining the back of the eye. Degeneration of the RPE leads to severe vision loss in, so far incurable, diseases such as age-related macular degeneration and some forms of retinitis pigmentosa. A promising future replacement therapy may be autologous iris epithelial cell transdifferentiation into RPE in vitro and, subsequently, transplantation. In this study we compared the gene expression profiles of the iris epithelium (IE) and the RPE.We collected both primary RPE- and IE cells from 5 freshly frozen human donor eyes, using respectively laser dissection microscopy and excision. We performed whole-genome expression profiling using 44k Agilent human microarrays. We investigated the gene expression profiles on both gene and functional network level, using R and the knowledge database Ingenuity.The major molecular pathways related to the RPE and IE were quite similar and yielded basic neuro-epithelial cell functions. Nonetheless, we also found major specific differences: For example, genes and molecular pathways, related to the visual cycle and retinol biosynthesis are significantly higher expressed in the RPE than in the IE. Interestingly, Wnt and aryl hydrocarbon receptor (AhR-) signaling pathways are much higher expressed in the IE than in the RPE, suggesting, respectively, a possible pluripotent and high detoxification state of the IE.This study provides a valuation of the similarities and differences between the expression profiles of the RPE and IE. Our data combined with that of the literature, represent a most comprehensive perspective on transcriptional variation, which may support future research in the development of therapeutic transplantation of IE.

The human retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age related macular degeneration (AMD). AMD is the leading cause of blindness worldwide. There is currently no effective treatment available. Preclinical studies in AMD mouse models are essential to develop new therapeutics. This requires further in-depth knowledge of the similarities and differences between mouse and human RPE.We performed a microarray study to identify and functionally annotate RPE specific gene expression in mouse and human RPE. We used a meticulous method to determine C57BL/6J mouse RPE signature genes, correcting for possible RNA contamination from its adjacent layers: the choroid and the photoreceptors. We compared the signature genes, gene expression profiles and functional annotations of the mouse and human RPE.We defined sets of mouse (64), human (171) and mouse-human interspecies (22) RPE signature genes. Not unexpectedly, our gene expression analysis and comparative functional annotation suggested that, in general, the mouse and human RPE are very similar. For example, we found similarities for general features, like "organ development" and "disorders related to neurological tissue". However, detailed analysis of the molecular pathways and networks associated with RPE functions, suggested also multiple species-specific differences, some of which may be relevant for the development of AMD. For example, CFHR1, most likely the main complement regulator in AMD pathogenesis was highly expressed in human RPE, but almost absent in mouse RPE. Furthermore, functions assigned to mouse and human RPE expression profiles indicate (patho-) biological differences related to AMD, such as oxidative stress, Bruch's membrane, immune-regulation and outer blood retina barrier.These differences may be important for the development of new therapeutic strategies and translational studies in age-related macular degeneration.

Purpose: Albinism refers to a group of genetic disorders typically characterized by a loss/reduction of melanin in the hair, skin and eyes of affected patients. Apart from pigment changes, all albinism patients present with foveal hypoplasia and optic nerve misrouting, and have blurred vision. The molecular mechanisms that link this lack of pigment with neural retinal development are poorly understood, with foveal and optic tract development being difficult to model. To advance our knowledge, we developed a novel retinal organoid model of albinism, and characterized the development and outgrowth of retinal ganglion cells affected during albinism as a model for future studies., Methods: Human oculocutaneous albinism 1 (OCA1) patient-derived stem cells were differentiated alongside controls into retinal organoids, as published previously 1,2 . Early retinal ganglion cells develop in the first 4 weeks of differentiation, at which point whole organoids could be plated to allow for optic nerve-like outgrowth. Whole organoids were also fixed and analysed with immunohistochemistry (IHC) to visualize contralateral and ipsilateral ganglion cells present in the organoid., Results: IHC analysis showed differences in the number of ipsilateral and contralateral retinal ganglion cells between the healthy control and albinism organoids, in line with in vivo observations. Further, optic nerve-like outgrowth could be achieved with both models, allowing for future research into optic nerve misrouting in albinism., Conclusions: We generated a novel retinal organoid model of oculocutaneous albinism, and characterized the retinal ganglion cell development and outgrowth. This will allow us in the future to study a different and sometimes overlooked aspect of albinism; optic nerve misrouting. References 1. Wagstaff, P. E., Ten Asbroek, A., Ten Brink, J. B., Jansonius, N. M. & Bergen, A. A. B. An alternative approach to produce versatile retinal organoids with accelerated ganglion cell development. Sci Rep 11, 1101, doi:10.1038/s41598-020-79651-x (2021). 2. Ohlemacher, S. K. et al. Stepwise Differentiation of Retinal Ganglion Cells from Human Pluripotent Stem Cells Enables Analysis of Glaucomatous Neurodegeneration. Stem Cells 34, 1553-1562, doi:10.1002/stem.2356 (2016)., (© 2022 The Authors Acta Ophthalmologica © 2022 Acta Ophthalmologica Scandinavica Foundation.)

Retinal photoreceptor outer segments (POS) are renewed daily through phagocytosis by the adjacent retinal pigment epithelial (RPE) monolayer. Phagocytosis is mainly driven by the RPE circadian clock but the underlying molecular mechanisms remain elusive. Using ARPE-19 (human RPE cell-line) dispersed and monolayer cell cultures, we investigated the influence of cellular organization on the RPE clock and phagocytosis genes. PCR analysis revealed rhythmic expression of clock and phagocytosis genes in all ARPE-19 cultures. Monolayers had a tendency for higher amplitudes of clock gene oscillations. In all conditions ARNTL, CRY1, PER1-2, REV-ERBα, ITGB5, LAMP1 and PROS1 were rhythmically expressed with REV-ERBα being among the clock genes whose expression showed most robust rhythms in ARPE-19 cells. Using RPE-choroid explant preparations of the mPer2 Luc knock-in mice we found that Rev-Erbα deficiency induced significantly longer periods and earlier phases of PER2-bioluminescence oscillations. Furthermore, early phagocytosis factors β 5 -Integrin and FAK and the lysosomal marker LAMP1 protein levels are rhythmic. Finally, POS incubation affects clock and clock-controlled phagocytosis gene expression in RPE monolayers in a time-dependent manner suggesting that POS can reset the RPE clock. These results shed some light on the complex interplay between POS, the RPE clock and clock-controlled phagocytosis machinery which is modulated by Rev-Erbα.

The European Eye Epidemiology (E3) consortium is a recently formed consortium of 29 groups from 12 European countries. It already comprises 21 population-based studies and 20 other studies (case-control, cases only, randomized trials), providing ophthalmological data on approximately 170,000 European participants. The aim of the consortium is to promote and sustain collaboration and sharing of data and knowledge in the field of ophthalmic epidemiology in Europe, with particular focus on the harmonization of methods for future research, estimation and projection of frequency and impact of visual outcomes in European populations (including temporal trends and European subregions), identification of risk factors and pathways for eye diseases (lifestyle, vascular and metabolic factors, genetics, epigenetics and biomarkers) and development and validation of prediction models for eye diseases. Coordinating these existing data will allow a detailed study of the risk factors and consequences of eye diseases and visual impairment, including study of international geographical variation which is not possible in individual studies. It is expected that collaborative work on these existing data will provide additional knowledge, despite the fact that the risk factors and the methods for collecting them differ somewhat among the participating studies. Most studies also include biobanks of various biological samples, which will enable identification of biomarkers to detect and predict occurrence and progression of eye diseases. This article outlines the rationale of the consortium, its design and presents a summary of the methodology.

Coxsackievirus type B3 (CVB3) is a cardiotropic enterovirus. Infection causes cardiomyocyte necrosis and myocardial inflammation. The damaged tissue that results is replaced with fibrotic or calcified tissue, which can lead to permanently altered cardiac function. The extent of pathogenesis among individuals exposed to CVB3 is dictated by a combination of host genetics, viral virulence, and the environment. Here, we aimed to identify genes that modulate cardiopathology following CVB3 infection. 129S1 mice infected with CVB3 developed increased cardiac pathology compared to 129X1 substrain mice despite no difference in viral burden. Linkage analysis identified a major locus on chromosome 7 (LOD: 8.307, P<0.0001) that controlled the severity of cardiac calcification and necrosis following infection. Sub-phenotyping and genetic complementation assays identified Abcc6 as the underlying gene. Microarray expression profiling identified genotype-dependent regulation of genes associated with mitochondria. Electron microscopy examination showed elevated deposition of hydroxyapatite-like material in the mitochondrial matrices of infected Abcc6 knockout (Abcc6-/-) mice but not in wildtype littermates. Cyclosporine A (CsA) inhibits mitochondrial permeability transition pore opening by inhibiting cyclophilin D (CypD). Treatment of Abcc6 -/- mice with CsA reduced cardiac necrosis and calcification by more than half. Furthermore, CsA had no effect on the CVB3-induced phenotype of doubly deficient CypD-/-Abcc6-/- mice. Altogether, our work demonstrates that mutations in Abcc6 render mice more susceptible to cardiac calcification following CVB3 infection. Moreover, we implicate CypD in the control of cardiac necrosis and calcification in Abcc6-deficient mice, whereby CypD inhibition is required for cardioprotection.

Importance: New funduscopic findings in patients with enhanced S-cone syndrome (ESCS) may help clinicians in diagnosing this rare autosomal recessive retinal dystrophy., Objective: To expand the clinical spectrum of ESCS due to mutations in the NR2E3 gene., Design: Retrospective, noncomparative case series of 31 patients examined between 1983 and 2012., Setting: Academic and private ophthalmology practices specialized in retinal dystrophies., Participants: A cohort of patients diagnosed with ESCS and harboring known NR2E3 mutations., Intervention: Patients had ophthalmic examinations including visual function testing that led to the original diagnosis., Main Outcomes and Measures: New fundus features captured with imaging modalities., Results: New clinical observations in ESCS include (1) torpedo-like, deep atrophic lesions with a small hyperpigmented rim, variably sized and predominantly located along the arcades; (2) circumferential fibrotic scars in the posterior pole with a spared center and large fibrotic scars around the optic nerve head; and (3) yellow dots in areas of relatively normal-appearing retina., Conclusions and Relevance: Enhanced S-cone syndrome has more pleiotropy than previously appreciated. While the nummular type of pigmentation at the level of the retinal pigment epithelium and cystoid or schisis-like maculopathy with typical functional findings remain classic hallmarks of the disease, changes such as circumferential fibrosis of the macula or peripapillary area and "torpedo-like" lesions along the vascular arcades may also direct the clinical diagnosis and focus on screening the NR2E3 gene for a molecular diagnosis.

Background: Functional studies in patients with autosomal dominant optic atrophy (ADOA) are usually confined to analysis of physiological and clinical impact at the ganglion cell (GG) and post GC levels. Here we aimed to investigate the impact of the disease at a pre-GC level and its correlation with GC/post-GC related measures., Methods: Visual function was assessed in a population of 22 subjects (44 eyes) from 13 families with ADOA submitted to OPA1 mutation analysis. Quantitative psychophysical methods were used to assess konio and parvocellular chromatic pathways (Cambridge Colour Test) and distinct achromatic spatial frequency channels (Metropsis Contrast Sensitivity Test). Preganglionic and GC measures were assessed with the Multifocal Electroretinogram (mfERG) and Pattern Electroretinogram (PERG) respectively. Global Pattern and Multifocal VEP (visual evoked potentials) were used to assess retinocortical processing, in order to characterize impaired processing at the post GC level. Perimetric sensitivity, retinal and ganglion cell nerve fibre layer (RNFL) thickness measurements were also obtained., Results: Chromatic thresholds were significantly increased for protan, deutan and tritan axes (p < < 0.001 for all comparisons) and achromatic contrast sensitivity (CS) was reduced for all studied six spatial frequency channels (p < < 0.001). We observed significant decreases in peripapillary (p ≤ 0.0008), macular (ring2: p = 0.02; ring 3: p < 0.0001) RNFL, as well as in overall retinal thickness (p < 0.0001 in all regions, except the central one). Interestingly, we found significant decreases in pre-ganglionic multifocal ERG response amplitudes (P1-wave: p ≤ 0.005) that were correlated with retinal thickness (ring 2: r = 0.512; p = 0.026/ring 3: r = 0.583; p = 0.011) and visual acuity (r = 0.458; p = 0.03, central ring 1). Reductions in GC and optic nerve responses amplitude (PERG: p < 0.0001, P50 and N95 components; Pattern VEP: p < 0.0001, P100) were accompanied by abnormalities of the MfVEP, primarily in central locations (ring 1: p = 0.0007; ring 2: p = 0.012)., Conclusions: In the ADOA model of ganglion cell damage, parvo-, konio- and magnocellular pathways are concomitantly affected. Structural changes and physiological impairment also occurs at a preganglionic level, suggesting a retrograde damage mechanism with a significant clinical impact on visual function, as shown by correlation analysis. Cortical impairment is only moderately explained by the retinal phenotype, suggesting additional damage mechanisms at the cortical level.

Leber congenital amaurosis (LCA) is the earliest and most severe retinal degeneration (RD), and the most common cause of incurable blindness diagnosed in children. It is occasionally the presenting symptom of multisystemic ciliopathies which diagnosis will require a specific care of patients. Nineteen LCA genes are currently identified and three of them account for both non-syndromic and syndromic forms of the disease. RD3 (LCA12) was implicated as a LCA gene based on the identification of homozygous truncating mutations in two LCA families despite the screening of large cohorts of patients. Here we provide a comprehensive survey of RD3 mutations and of their clinical expression through the screening of a cohort of 852 patients originating worldwide affected with LCA or early-onset and severe RD. We identified three RD3 mutations in seven unrelated consanguineous LCA families - i.e., a 2 bp deletion and two nonsense mutations - predicted to cause complete loss of function. Five families originating from the Southern Shores of the Mediterranean segregated a similar mutation (c.112C>T, p.R38*) suggesting that this change may have resulted from an ancient founder effect. Considering the low frequency of RD3 carriers, the recurrence risk for LCA in non-consanguineous unions is negligible for both heterozygote and homozygote RD3 individuals. The LCA12 phenotype in our patients is highly similar to those of patients with mutant photoreceptor-specific guanylate cyclase (GUCY2D/LCA1). This observation is consistent with the report of the role of RD3 in trafficking of GUCYs and gives further support to a common mechanism of photoreceptor degeneration in LCA12 and LCA1, i.e., inability to increase cytoplasmic cGMP concentration in outer segments and thus to recover the dark-state. Similar to LCA1, LCA12 patients have no extraocular symptoms despite complete inactivation of both RD3 alleles, supporting the view that extraocular investigations in LCA infants with RD3 mutations should be avoided.

The circadian expression patterns of genes encoding for proteins that make up the core of the circadian clock were measured in rat retina using real-time quantitative PCR (qPCR). Transcript levels of several genes previously used for normalization of qPCR assays were determined and the effect of ischemia-reperfusion on the expression of clock genes was studied. Statistically significant circadian changes in transcript levels were found for: Per2, Per3, Cry2, Bmal1, Rora, Rorb, and Rorc with changes ranging between 1.6- and 2.6-fold. No changes were found for Per1, Cry1, Clock, Rev-erb alpha, and Rev-erb beta. Significant differences in transcript levels were observed for several candidate reference genes: HPRT, GAPDH, rhodopsin, and Thy1 and, consequently, the use of these genes for normalization purposes in qPCR or Northern blots may lead to erroneous conclusions. Ischemia-reperfusion leads to a persistent decrease of Per1 and Cry2, which may be related to the selective degeneration of amacrine and ganglion cells. We conclude that while all clock genes are expressed in the retina, only a few show a clear circadian pattern.