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Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis.

ABSTRACT: X-linked juvenile retinoschisis (XLRS), linked to mutations in the RS1 gene, is a degenerative retinopathy with a retinal splitting phenotype. We generated human induced pluripotent stem cells (hiPSCs) from patients to study XLRS in a 3D retinal organoid in vitro differentiation system. This model recapitulates key features of XLRS including retinal splitting, defective retinoschisin production, outer-segment defects, abnormal paxillin turnover, and impaired ER-Golgi transportation. RS1 mutation also affects the development of photoreceptor sensory cilia and results in altered expression of other retinopathy-associated genes. CRISPR/Cas9 correction of the disease-associated C625T mutation normalizes the splitting phenotype, outer-segment defects, paxillin dynamics, ciliary marker expression, and transcriptome profiles. Likewise, mutating RS1 in control hiPSCs produces the disease-associated phenotypes. Finally, we show that the C625T mutation can be repaired precisely and efficiently using a base-editing approach. Taken together, our data establish 3D organoids as a valid disease model.

SUBMITTER: Huang KC

PROVIDER: S-EPMC6895767 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis

Project description:The goals of this study are to analyze the effects of RS1 mutation on retinal transcriptome profiling during retina differentiation. We used next generation sequencing to analyze the retinal mRNA profiles in a panel of time-course differentiated 3D retinal cups (RCs) from control donor- and XLRS patient-derived iPSCs, as well as 150-day differentiated RCs from CRISPR/Cas9-reparied patient-derived iPSCs. The transcriptomes of control-RCs and patient-RCs considerably diverged along the time course of differentiation, with the difference becoming more pronounced at the later time points. In contrast, analysis of repaired RCs revealed transcriptomes that were highly similar to those of control-RCs and significantly different from those of patient-RCs. Because RS1 is only expressed in later stage of differentiation, we focued on days 90, 120, and 150 post-induction to identify the genes whose expression was reduced at least 2-fold in patient-RCs. Among all of these genes, 326 genes were overexpressed in normal RCs at all of these stages, emphasizing their importance. According to GSEA annotation, nine out of these 326 genes were implicated in eye development. Among these nine genes, the expression of RS1, IQCB1, and OPA1 was rescued in CRISPR repaired RCs. The relative loss of expression became even more prominent in later stages of retinal differentiation. Our transcriptome analysis revealed significant changes in gene expression between patient-derived and control-RCs that become more pronounced at the later stages of development. This implies that RS1 is essential for normal retinal development at the systemic level. These results reflect the progressive nature of the disease phenotypes observed in RS-1-mutant RCs, mirror the observed ciliary phenotype, and reveal potential links of XLRS to other hereditary retinopathies.

2022-09-03 | GSE137910 | GEO

Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis

Project description:Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis

| PRJNA573847 | ENA

Early Developmental Characteristics and Features of a Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis.

Project description:X-linked juvenile retinoschisis (XLRS) is a hereditary retinal degeneration affecting young males caused by mutations in the retinoschisin (RS1) gene. We generated human induced pluripotent stem cells (hiPSCs) from XLRS patients and established three-dimensional retinal organoids (ROs) for disease investigation. This disease model recapitulates the characteristics of XLRS, exhibiting defects in RS1 protein production and photoreceptor cell development. XLRS ROs also revealed dysregulation of Na/K-ATPase due to RS1 deficiency and increased ERK signaling pathway activity. Transcriptomic analyses of XLRS ROs showed decreased expression of retinal cells, particularly photoreceptor cells. Furthermore, relevant recovery of the XLRS phenotype was observed when co-cultured with control ROs derived from healthy subject during the early stages of differentiation. In conclusion, our in vitro XLRS RO model presents a valuable tool for elucidating the pathophysiological mechanisms underlying XLRS, offering insights into disease progression. Additionally, this model serves as a robust platform for the development and optimization of targeted therapeutic strategies, potentially improving treatment outcomes for patients with XLRS.

| S-EPMC11311801 | biostudies-literature

CRX controls retinal expression of the X-linked juvenile retinoschisis (RS1) gene.

Project description:X-linked juvenile retinoschisis is a heritable condition of the retina in males caused by mutations in the RS1 gene. Still, the cellular function and retina-specific expression of RS1 are poorly understood. To address the latter issue, we characterized the minimal promoter driving expression of RS1 in the retina. Binding site prediction, site-directed mutagenesis, and reporter assays suggest an essential role of two nearby cone-rod homeobox (CRX)-responsive elements (CRE) in the proximal -177/+32 RS1 promoter. Chromatin immunoprecipitation associates the RS1 promoter in vivo with CRX, the coactivators CBP, P300, GCN5 and acetylated histone H3. Transgenic Xenopus laevis expressing a green fluorescent protein (GFP) reporter under the control of RS1 promoter sequences show that the -177/+32 fragment drives GFP expression in photoreceptors and bipolar cells. Mutating either of the two conserved CRX binding sites results in strongly decreased RS1 expression. Despite the presence of sequence motifs in the promoter, NRL and NR2E3 appear not to be essential for RS1 expression. Together, our in vitro and in vivo results indicate that two CRE sites in the minimal RS1 promoter region control retinal RS1 expression and establish CRX as a key factor driving this expression.

| S-EPMC2582616 | biostudies-literature

X-linked juvenile retinoschisis: clinical diagnosis, genetic analysis, and molecular mechanisms.

Project description:X-linked juvenile retinoschisis (XLRS, MIM 312700) is a common early onset macular degeneration in males characterized by mild to severe loss in visual acuity, splitting of retinal layers, and a reduction in the b-wave of the electroretinogram (ERG). The RS1 gene (MIM 300839) associated with the disease encodes retinoschisin, a 224 amino acid protein containing a discoidin domain as the major structural unit, an N-terminal cleavable signal sequence, and regions responsible for subunit oligomerization. Retinoschisin is secreted from retinal cells as a disulphide-linked homo-octameric complex which binds to the surface of photoreceptors and bipolar cells to help maintain the integrity of the retina. Over 190 disease-causing mutations in the RS1 gene are known with most mutations occurring as non-synonymous changes in the discoidin domain. Cell expression studies have shown that disease-associated missense mutations in the discoidin domain cause severe protein misfolding and retention in the endoplasmic reticulum, mutations in the signal sequence result in aberrant protein synthesis, and mutations in regions flanking the discoidin domain cause defective disulphide-linked subunit assembly, all of which produce a non-functional protein. Knockout mice deficient in retinoschisin have been generated and shown to display most of the characteristic features found in XLRS patients. Recombinant adeno-associated virus (rAAV) mediated delivery of the normal RS1 gene to the retina of young knockout mice result in long-term retinoschisin expression and rescue of retinal structure and function providing a 'proof of concept' that gene therapy may be an effective treatment for XLRS.

| S-EPMC3334421 | biostudies-literature

X-linked juvenile retinoschisis: phenotypic and genetic characterization.

Project description:Juvenile X-linked retinoschisis (XLRS, MIM#312700) belongs to a group of the vitreoretinal dystrophies. We aimed to describe the phenotype-genotype correlation of three XLRS cases in juveniles with different novel mutations from the Lithuanian population. The patients demonstrated macular retinoschisis and typical cyst-like cavities on spectral-domain optical coherence tomography (SD-OCT) images. The mean central foveal thickness was 569.7 µm. Two patients presented with peripheral retinoschisis. Flash electroretinogram demonstrated a reduced b/a ratio (<1.0) in all patients. RS1 (NM_000330.3) gene coding exons Sanger sequencing was performed. RS1 c.599G>T (p.R200L) mutation was detected in one case, showing to be pathogenic in silico analysis. c. (92_97) insC (p.W33fs) mutation was identified for another patient, indicating the variant is possibly damaging in silico analysis. The third case was identified with a pathogenic mutation c.422C>G (p.R141H), HGMD CM981753. These are the first cases of XLRS in the Lithuanian population confirmed by molecular genotyping. Presented patients had a different genotype but similar phenotypic traits.

| S-EPMC6232335 | biostudies-literature

Retinal detachment repair with perfluoro-N-octane endotamponade in an infant with juvenile X-linked retinoschisis.

Project description:PurposeWe present a case of successful surgical management of an infant with X-linked retinoschisis with a giant retinal tear and retinal detachment of the right eye.ObservationsA 10-month-old male presented with retinoschisis of both eyes and a retinal detachment of the right eye. The patient underwent two-stage pars plana vitrectomy utilizing perfluoro-N-octane to stabilize the detached retina and facilitate posterior hyaloid separation. Retained perfluoro-N-octane tamponade was later exchanged with silicone oil. The retina remained attached at last follow up.Conclusions and importanceRetinal detachment repair in infants presents unique challenges. This is a safe and effective strategy for complex retinal detachment repair in the infant population.

| S-EPMC7670199 | biostudies-literature

Retinal morphological changes of patients with X-linked retinoschisis evaluated by Fourier-domain optical coherence tomography.

Project description:ObjectiveTo investigate the retinal microstructure and lamination of patients affected with X-linked retinoschisis (XLRS) using high-resolution imaging modalities.MethodsPatients diagnosed as having XLRS underwent assessment. Visual function testing included visual acuity, color vision, and full-field electroretinography. We used a high-resolution Fourier-domain optical coherence tomography (FD-OCT) system (4.5-mum axial resolution; 9 frames/s; 1000 A-scans per frame) combined with a handheld scanner. Macular image evaluation included schisis localization and retinal layer integrity.ResultsSix patients with XLRS and identified mutations in the XLRS1 gene underwent testing. Visual acuity ranged from 0.2 to 1.6 logMAR (logarithm of the minimum angle of resolution). Results of FD-OCT revealed foveal schisis extending from the outer to the inner plexiform layer in 4 of 6 patients. Bullous foveal schisis was associated with younger age. All patients showed extrafoveal schisis within the outer and inner nuclear and ganglion cell layer, alone or in combination. Photoreceptor outer and inner segment layers were disrupted and irregular in all patients.ConclusionsRetinal dystrophy in XLRS is reflected by morphological changes within the inner and outer retinal layers. Disturbed foveal photoreceptor integrity was identified in all patients. Retinal layer abnormalities correlated with age but did not appear to correlate with visual acuity or genotypic variation.

| S-EPMC2612690 | biostudies-literature

Novel RS1 mutations associated with X-linked juvenile retinoschisis.

Project description:To identify mutations in the retinoschisin (RS1) gene in families with X-linked retinoschisis (XLRS). Twenty families with XLRS were enrolled in this study. All six coding exons and adjacent intronic regions of RS1 were amplified by polymerase chain reaction (PCR). The nucleotide sequences of the amplicons were determined by Sanger sequencing. Ten hemizygous mutations in RS1 were detected in patients from 14 of the 20 families. Four of the ten mutations were novel, including c:176G>A (p:Cys59Tyr) in exon 3, c:531T>G (p:Tyr177X), c:607C>G (p:Pro203Ala) and c:668G>A (p:Cys223Tyr) in exon 6. These four novel mutations were not present in 176 normal individuals. The remaining six were recurrent mutations, including c:214G>A (p:Glu72Lys), c:304C>T (p:Arg102Trp), c:436G>A (p:Glu146Lys), c:544C>T (p:Arg182Cys), c:599G>A (p:Arg200His) and c:644A>T (p:Glu215Val). Our study expanded the mutation spectrum of RS1 and enriches our understanding of the molecular basis of XLRS.

| S-EPMC3573736 | biostudies-literature

Outcome measures in juvenile X-linked retinoschisis: A systematic review.

Project description:X-linked retinoschisis (XLRS) is a leading cause of hereditary juvenile macular degeneration in males resulting in significant vision impairment. Outcome measures to monitor disease progression or therapeutic interventions have evolved with technology. A systematic review was undertaken to evaluate outcome measures for XLRS. Inclusion criteria were all publications examining outcome measures for natural history studies or following an interventional approach for patients with XLRS. Studies which did not present follow-up data were excluded. We searched medical databases including CENTRAL, Ovid Medline, pre-Medline and ahead of Print up to February 2019. Two authors independently assessed the risk of bias. Twelve studies meet the inclusion criteria with four prospective and eight retrospective case series. Five series were natural history observational studies and seven were interventional series using either topical or systemic carbonic anhydrase inhibitors. Visual acuity (VA) declined very slowly in the natural history studies equivalent to 0.22-0.5 letters per year. Five of the six interventional studies showed an improvement in VA and four a reduction in spectral domain optical coherence tomography (SD-OCT) parameters for central macular thickness (CMT). The full-field electroretinogram identified the 30-Hz latency as a further parameter to monitor function. VA was the measure most likely to show a statistically significant outcome. How functionally meaningful this is, requires further evaluation. CMT SD-OCT outcomes are variable depending on cystic changes. More refined measures are required to better correlate structure with function.

| S-EPMC7608480 | biostudies-literature

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