Genome-wide CNV investigation suggests a role for cadherin, Wnt, and p53 pathways in primary open-angle glaucoma

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-021-07846-1

An alternative approach to produce versatile retinal organoids with accelerated ganglion cell development

Genetically complex ocular neuropathies, such as glaucoma, are a major cause of visual impairment worldwide. There is a growing need to generate suitable human representative in vitro and in vivo models, as there is no effective treatment available once damage has occured. Retinal organoids are increasingly being used for experimental gene therapy, stem cell replacement therapy and small molecule therapy. There are multiple protocols for the development of retinal organoids available, however, one potential drawback of the current methods is that the organoids can take between 6 weeks and 12 months on average to develop and mature, depending on the specific cell type wanted. Here, we describe and characterise a protocol focused on the generation of retinal ganglion cells within an accelerated four week timeframe without any external small molecules or growth factors. Subsequent long term cultures yield fully differentiated organoids displaying all major retinal cell types. RPE, Horizontal, Amacrine and Photoreceptors cells were generated using external factors to maintain lamination.</p

Gene expression-based comparison of the human secretory neuroepithelia of the brain choroid plexus and the ocular ciliary body:potential implications for glaucoma

BACKGROUND: The neuroepithelia of the choroid plexus (CP) in the brain and the ciliary body (CB) of the eye have common embryological origins and share similar micro-structure and functions. The CP epithelium (CPE) and the non-pigmented epithelium (NPE) of the CB produce the cerebrospinal fluid (CSF) and the aqueous humor (AH) respectively. Production and outflow of the CSF determine the intracranial pressure (ICP); production and outflow of the AH determine the intraocular pressure (IOP). Together, the IOP and ICP determine the translaminar pressure on the optic disc which may be involved in the pathophysiology of primary open angle glaucoma (POAG). The aim of this study was to compare the molecular machinery of the secretory neuroepithelia of the CP and CB (CPE versus NPE) and to determine their potential role in POAG. METHODS: We compared the transcriptomes and functional annotations of healthy human CPE and NPE. Microarray and bioinformatic studies were performed using an Agilent platform and the Ingenuity Knowledge Database (IPA). RESULTS: Based on gene expression profiles, we found many similar functions for the CPE and NPE including molecular transport, neurological disease processes, and immunological functions. With commonly-used selection criteria (fold-change > 2.5, p-value < 0.05), 14% of the genes were expressed significantly differently between CPE and NPE. When we used stricter selection criteria (fold-change > 5, p-value < 0.001), still 4.5% of the genes were expressed differently, which yielded specific functions for the CPE (ciliary movement and angiogenesis/hematopoiesis) and for the NPE (neurodevelopmental properties). Apart from a few exceptions (e.g. SLC12A2, SLC4A4, SLC4A10, KCNA5, and SCN4B), all ion transport protein coding genes involved in CSF and AH production had similar expression profiles in CPE and NPE. Three POAG disease genes were expressed significantly higher in the CPE than the NPE, namely CDH1, CDKN2B and SIX1. CONCLUSIONS: The transcriptomes of the CPE and NPE were less similar than we previously anticipated. High expression of CSF/AH production genes and candidate POAG disease genes in the CPE and NPE suggest that both might be involved in POAG

Mitochondrial Genome Study Identifies Association Between Primary Open-Angle Glaucoma and Variants in MT-CYB, MT-ND4 Genes and Haplogroups

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

Gene expression and functional annotation of the human and mouse choroid plexus epithelium

Background: The choroid plexus epithelium (CPE) is a lobed neuro-epithelial structure that forms the outer blood-brain barrier. The CPE protrudes into the brain ventricles and produces the cerebrospinal fluid (CSF), which is crucial for brain homeostasis. Malfunction of the CPE is possibly implicated in disorders like Alzheimer disease, hydrocephalus or glaucoma. To study human genetic diseases and potential new therapies, mouse models are widely used. This requires a detailed knowledge of similarities and differences in gene expression and functional annotation between the species. The aim of this study is to analyze and compare gene expression and functional annotation of healthy human and mouse CPE. Methods: We performed 44k Agilent microarray hybridizations with RNA derived from laser dissected healthy human and mouse CPE cells. We functionally annotated and compared the gene expression data of human and mouse CPE using the knowledge database Ingenuity. We searched for common and species specific gene expression patterns and function between human and mouse CPE. We also made a comparison with previously published CPE human and mouse gene expression data. Results: Overall, the human and mouse CPE transcriptomes are very similar. Their major functionalities included epithelial junctions, transport, energy production, neuro-endocrine signaling, as well as immunological, neurological and hematological functions and disorders. The mouse CPE presented two additional functions not found in the human CPE: carbohydrate metabolism and a more extensive list of (neural) developmental functions. We found three genes specifically expressed in the mouse CPE compared to human CPE, being ACE, PON1 and TRIM3 and no human specifically expressed CPE genes compared to mouse CPE. Conclusion: Human and mouse CPE transcriptomes are very similar, and display many common functionalities. Nonetheless, we also identified a few genes and pathways which suggest that the CPE between mouse and man differ with respect to transport and metabolic functions

RPGR-associated dystrophies: Clinical, genetic, and histopathological features

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

Gene Expression and Functional Annotation of the Human Ciliary Body Epithelia

Purpose: The ciliary body (CB) of the human eye consists of the non-pigmented (NPE) and pigmented (PE) neuro-epithelia. We investigated the gene expression of NPE and PE, to shed light on the molecular mechanisms underlying the most important functions of the CB. We also developed molecular signatures for the NPE and PE and studied possible new clues for glaucoma. Methods: We isolated NPE and PE cells from seven healthy human donor eyes using laser dissection microscopy. Next, we performed RNA isolation, amplification, labeling and hybridization against 44×k Agilent microarrays. For microarray conformations, we used a literature study, RT-PCRs, and immunohistochemical stainings. We analyzed the gene expression data with R and with the knowledge database Ingenuity. Results: The gene expression profiles and functional annotations of the NPE and PE were highly similar. We found that the most important functionalities of the NPE and PE were related to developmental processes, neural nature of the tissue, endocrine and metabolic signaling, and immunological functions. In total 1576 genes differed statistically significantly between NPE and PE. From these genes, at least 3 were cell-specific for the NPE and 143 for the PE. Finally, we observed high expression in the (N)PE of 35 genes previously implicated in molecular mechanisms related to glaucoma. Conclusion: Our gene expression analysis suggested that the NPE and PE of the CB were quite similar. Nonetheless, cell-type specific differences were found. The molecular machineries of the human NPE and PE are involved in a range of neuro-endocrinological, developmental and immunological functions, and perhaps glaucoma

Long-term follow-up of retinal degenerations associated with LRAT mutations and their comparability to phenotypes associated with RPE65 mutations

Purpose: To investigate the natural history in patients with LRAT-associated retinal degenerations (RDs), in the advent of clinical trials testing treatment options. Methods: A retrospective cohort of 13 patients with LRAT-RDs. Results: Twelve patients from a genetic isolate carried a homozygous c.12del mutation. One unrelated patient carried a homozygous c.326G>T mutation. The mean follow-up time was 25.3 years (SD 15.2; range 4.8-53.5). The first symptom was nyctalopia (n = 11), central vision loss (n = 1), or light-gazing (n = 1), and was noticed in the first decade of life. Seven patients (54%) reached low vision (visual acuity < 20/67), four of whom reaching blindness (visual acuity < 20/400), respectively, at mean ages of 49.9 (SE 5.4) and 59.9 (SE 3.1) years. The fundus appearance was variable. Retinal white dots were seen in six patients (46%). Full-field electroretinograms (n = 11) were nondetectable (n = 2; ages 31-60), reduced in a nonspecified pattern (n = 2; ages 11-54), or showed rod-cone (n = 6; ages 38-48) or cone-rod (n = 1; age 29) dysfunction. Optical coherence tomography (n = 4) showed retinal thinning but relative preservation of the (para-)foveal outer retinal layers in the second (n = 1) and sixth decade of life (n = 2), and profound chorioretinal degeneration from the eighth decade of life (n = 1). Conclusions: LRAT-associated phenotypes in this cohort were variable and unusual, but generally milder than those seen in RPE65-associated disease, and may be particularly amenable to treatment. The window of therapeutic opportunity can be extended in patients with a mild phenotype. Translational Relevance: Knowledge of the natural history of LRAT-RDs is essential in determining the window of opportunity in ongoing and future clinical trials for novel therapeutic options

Efficacy of Carbonic Anhydrase Inhibitors on Cystoid Fluid Collections and Visual Acuity in Patients with X-Linked Retinoschisis

Purpose: To date, there is no standard treatment regimen for carbonic anhydrase inhibitors (CAIs) in X-linked retinoschisis (XLRS) patients. This retrospective study aims to evaluate the efficacy of CAIs on visual acuity and cystoid fluid collections (CFC) in XRLS patients in Dutch and Belgian tertiary referral centers. Design: Retrospective cohort study. Participants: Forty-two patients with XLRS. Methods: In total, 42 patients were enrolled. To be included, patients had to have previous treatment with an oral CAI (acetazolamide), a topical CAI (brinzolamide/dorzolamide), or a combination of an oral and a topical CAI for at least 4 consecutive weeks. We evaluated the effect of the CAI on best-corrected visual acuity (BCVA) and central foveal thickness (CFT) on OCT. Main Outcome Measures: Central foveal thickness and BCVA. Results: The median age at the baseline visit of the patients in this cohort study was 14.7 (range, 43.6) years, with a median (interquartile range [IQR]) follow-up period of 4.0 (2.2–5.2) years. During the follow-up period, 25 patients were treated once with an oral CAI (60%), 24 patients were treated once with a topical CAI (57%), and 11 patients were treated once with a combination of both topical and oral CAI (26%). We observed a significant reduction of CFT for oral CAI by 14.37 μm per 100 mg per day (P &lt; 0.001; 95% confidence interval [CI], −19.62 to −9.10 μm) and for topical CAI by 7.52 μm per drop per day (P = 0.017; 95% CI, −13.67 to −1.32 μm). The visual acuity changed significantly while on treatment with oral CAI by −0.0059 logMAR per 100 mg (P = 0.008; 95% CI, −0.010 to −0.0013 logMAR). Seven patients (17%) had side effects leading to treatment discontinuation. Conclusions: Our data indicate that treatment with (oral) CAI may be beneficial for short-term management of CFC in patients with XLRS. Despite a significant reduction in CFT, the change in visual acuity was modest and not of clinical significance. Nonetheless, the anatomic improvement of the central retina in these patients may be of value to create an optimal retinal condition for future potential treatment options such as gene therapy. Financial Disclosure(s): The authors have no proprietary or commercial interest in any materials discussed in this article.</p

Efficacy of Carbonic Anhydrase Inhibitors on Cystoid Fluid Collections and Visual Acuity in Patients with X-Linked Retinoschisis

Purpose: To date, there is no standard treatment regimen for carbonic anhydrase inhibitors (CAIs) in X-linked retinoschisis (XLRS) patients. This retrospective study aims to evaluate the efficacy of CAIs on visual acuity and cystoid fluid collections (CFC) in XRLS patients in Dutch and Belgian tertiary referral centers. Design: Retrospective cohort study. Participants: Forty-two patients with XLRS. Methods: In total, 42 patients were enrolled. To be included, patients had to have previous treatment with an oral CAI (acetazolamide), a topical CAI (brinzolamide/dorzolamide), or a combination of an oral and a topical CAI for at least 4 consecutive weeks. We evaluated the effect of the CAI on best-corrected visual acuity (BCVA) and central foveal thickness (CFT) on OCT. Main Outcome Measures: Central foveal thickness and BCVA. Results: The median age at the baseline visit of the patients in this cohort study was 14.7 (range, 43.6) years, with a median (interquartile range [IQR]) follow-up period of 4.0 (2.2–5.2) years. During the follow-up period, 25 patients were treated once with an oral CAI (60%), 24 patients were treated once with a topical CAI (57%), and 11 patients were treated once with a combination of both topical and oral CAI (26%). We observed a significant reduction of CFT for oral CAI by 14.37 μm per 100 mg per day (P &lt; 0.001; 95% confidence interval [CI], −19.62 to −9.10 μm) and for topical CAI by 7.52 μm per drop per day (P = 0.017; 95% CI, −13.67 to −1.32 μm). The visual acuity changed significantly while on treatment with oral CAI by −0.0059 logMAR per 100 mg (P = 0.008; 95% CI, −0.010 to −0.0013 logMAR). Seven patients (17%) had side effects leading to treatment discontinuation. Conclusions: Our data indicate that treatment with (oral) CAI may be beneficial for short-term management of CFC in patients with XLRS. Despite a significant reduction in CFT, the change in visual acuity was modest and not of clinical significance. Nonetheless, the anatomic improvement of the central retina in these patients may be of value to create an optimal retinal condition for future potential treatment options such as gene therapy. Financial Disclosure(s): The authors have no proprietary or commercial interest in any materials discussed in this article.</p