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Transcriptome (RNA-seq) analysis of the retina of crumbs 2a (crb2a m289/m289) zebrafish model of Leber congenital amaurosis and retinitis pigmentosa.

ABSTRACT: The crumbs cell polarity complex plays a crucial role in apical-basal epithelial polarity. When human CRB1 is mutated, it results in autosomal recessive Leber congenital amaurosis and retinitis pigmentosa, with no established genotype-phenotype correlation. Using the oko meduzym289/m289 (crb2a-/-) zebrafish model, we performed integrative transcriptomic and methylomic analysis to identify dysregulated genes and pathways. We reveal delayed retinal cell type specification confirmed in patient-derived retinal organoids, with disruption to cell cycle modulation and epigenetic transcriptional control. Hence, using reduced representation bisulphite sequencing (RRBS) we explored differential DNA methylation, identifying hypermethylated pathways involving biological adhesion, Hippo and transforming growth factor beta (TGFbeta) signalling. Functional epigenetic modules (FEM) were highlighted through the integration of RNA-seq and RRBS, confirming cell cycle involvement and disturbance of TGFbeta, BMP, Hippo and SMAD protein signal transduction. Taken together our work provides insights for epigenetic regulation in early retinal development and considerations for future therapeutic development.

ORGANISM(S): Danio rerio

PROVIDER: GSE178709 | GEO | 2023/01/25

REPOSITORIES: GEO

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Reduced representation bisulfite sequencing (RRBS) of the retina of crumbs 2a (crb2a m289/m289) zebrafish model of Leber congenital amaurosis and retinitis pigmentosa.

Project description:The crumbs cell polarity complex plays a crucial role in apical-basal epithelial polarity. When human CRB1 is mutated, it results in autosomal recessive Leber congenital amaurosis and retinitis pigmentosa, with no established genotype-phenotype correlation. Using the oko meduzym289/m289 (crb2a-/-) zebrafish model, we performed integrative transcriptomic and methylomic analysis to identify dysregulated genes and pathways. We reveal delayed retinal cell type specification confirmed in patient-derived retinal organoids, with disruption to cell cycle modulation and epigenetic transcriptional control. Hence, using reduced representation bisulphite sequencing (RRBS) we explored differential DNA methylation, identifying hypermethylated pathways involving biological adhesion, Hippo and transforming growth factor beta (TGFbeta) signalling. Functional epigenetic modules (FEM) were highlighted through the integration of RNA-seq and RRBS, confirming cell cycle involvement and disturbance of TGFbeta, BMP, Hippo and SMAD protein signal transduction. Taken together our work provides insights for epigenetic regulation in early retinal development and considerations for future therapeutic development.

2023-01-25 | GSE178842 | GEO

AAV-mediated gene augmentation therapy of CRB1-patient derived retinal organoids restores the histological and transcriptional retinal phenotype

Project description:Retinitis pigmentosa (RP) and Leber congenital amaurosis are inherited retinal dystrophies caused by mutations in, among others, the Crumbs homologue 1 (CRB1) gene. CRB1 is required for organizing apical-basal polarity and adhesion between photoreceptors and Müller glial cells. Using human CRB1 patient induced pluripotent stem cells from RP patients we derived CRB1 retinal organoids, with diminished expression of variant CRB1 protein with immunohistochemical analysis. Single cell RNA-sequencing revealed impact on, among others, the endosomal pathway and cell adhesion and migration in CRB1 patient derived retinal organoids compared to isogenic controls. Adeno-associated viral (AAV) vector-mediated hCRB2 or hCRB1 gene augmentation in Müller glial and photoreceptor cells partially restored the histological and differentially expressed genes phenotype. Altogether, we show proof-of-concept that AAV.hCRB1 or AAV.hCRB2 treatment improved the phenotype of CRB1 patient derived retinal organoids, providing essential information for future gene therapy approaches for patients with mutations in the CRB1 gene.

2023-04-20 | GSE212582 | GEO

Histological and single cell transcriptomic analysis of human retinal organoid models of Leber Congenital Amaurosis identifies endoplasmic reticulum stress as a common degenerative pathway

Project description:This study utilized human stem cell derived retinal organoids to characterize two models of Leber Congenital Amaurosis. Using CRISPR/Cas9, we generated CRB1 and RPGRIP1 KO cell lines and differentiated WT and KO lines into retinal organoids. Single cell capture was performed using Dropseq at days 70, 90, 120, 150, 190, and 225 of differentiation. We report upregulation of DDIT3 in both KO organoids relative to WT and confirm DDIT3 induction at the protein level by immunohistochemistry. These results indicate that these genetically different models of disease converge on endoplasmic reticulum stress and DDIT3 induction as a shared degenerative pathway. Therapeutic targeting of ER stress could help alleviate the degenerative phenotype in cases where diseased photorececeptors suffer ER stress.

2021-05-01 | GSE152212 | GEO

Gene therapy of dominant CRX-Leber congenital amaurosis using patient retinal organoids II

Project description:Mutations in the cone-rod homeobox (CRX) transcription factor lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX-I138fs mutation, we established an in vitro model of CRX-LCA in retinal organoids that exhibit defective photoreceptor maturation by histology and gene profiling including diminished expression of visual opsins. Gene therapy by delivery of an additional correct CRX allele using an AAV vector partially restored photoreceptor phenotype and expression of phototransduction-related genes as revealed by single cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX-LCA patient carrying a K88N mutation revealed loss of opsin expression as a common phenotype, which could also be alleviated by AAV-mediated overexpression of CRX. Our studies provide the proof-of-concept for development of gene therapy for dominant CRX-LCA and other CRX-retinopathies.

2021-02-17 | GSE153099 | GEO

Gene therapy of dominant CRX-Leber congenital amaurosis using patient retinal organoids I

2021-02-17 | GSE152939 | GEO

NMNAT1 mutations cause Leber congenital amaurosis.

Project description:Leber congenital amaurosis (LCA) is an infantile-onset form of inherited retinal degeneration characterized by severe vision loss(1,2). Two-thirds of LCA cases are caused by mutations in 17 known disease-associated genes(3) (Retinal Information Network (RetNet)). Using exome sequencing we identified a homozygous missense mutation (c.25G>A, p.Val9Met) in NMNAT1 that is likely to be disease causing in two siblings of a consanguineous Pakistani kindred affected by LCA. This mutation segregated with disease in the kindred, including in three other children with LCA. NMNAT1 resides in the previously identified LCA9 locus and encodes the nuclear isoform of nicotinamide mononucleotide adenylyltransferase, a rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD(+)) biosynthesis(4,5). Functional studies showed that the p.Val9Met alteration decreased NMNAT1 enzyme activity. Sequencing NMNAT1 in 284 unrelated families with LCA identified 14 rare mutations in 13 additional affected individuals. These results are the first to link an NMNAT isoform to disease in humans and indicate that NMNAT1 mutations cause LCA.

| S-EPMC3454532 | biostudies-literature

Peripapillary sparing in RDH12-associated Leber congenital amaurosis.

Project description:BACKGROUND:Peripapillary sparing is a characteristic that is traditionally described as pathognomonic for Stargardt disease. MATERIALS AND METHODS:We present a multimodal assessment of four Leber congenital amaurosis (LCA) cases with congenital macular atrophy and severely attenuated electroretinogram findings caused by bilallelic mutations in RDH12. RESULTS:Fundus autofluorescence imaging revealed a general loss of retinal pigment epithelium across the macula except for the peripapillary region in both eyes of all patients. Spectral domain-optical coherence tomography confirmed relative preservation in this area along with retinal thinning and excavation throughout the rest of the macula. LCA was diagnosed based on clinical exam and retinal imaging, and subsequently confirmed with genetic testing. CONCLUSIONS:Peripapillary sparing is a novel phenotypic feature of RDH12-associated LCA.

| S-EPMC6314194 | biostudies-literature

Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants.

Project description:Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD(+) biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD(+) synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome.

| S-EPMC4498062 | biostudies-literature

Molecular characterization of Leber congenital amaurosis in Koreans.

Project description:PurposeLeber congenital amaurosis (LCA) is the most severe form of inherited retinal dystrophy, and invariably leads to blindness. LCA is a genetically and clinically heterogenous disorder. Although more than nine genes have been found to be associated with LCA, they only account for about half of LCA cases. We performed a comprehensive mutational analysis on nine known genes in 20 unrelated patients to investigate the genetic cause of LCA in Koreans.MethodsAll exons and flanking regions of the nine genes (AIPL1, CRB1, CRX, GUCY2D, RDH12, RPE65, RPGRIP1, LRAT, and TULP1) were analyzed by direct sequencing. We also screened our patients for the common CEP290: c.2991+1655A>G mutation found in Caucasian.ResultsSix different mutations including four novel ones were identified in three patients (15.0%): one frameshift, one nonsense, one splicing, and three missense mutations. These patients were compound heterozygotes and harbored two different mutations in CRB1, RPE65, and RPGRIP1, respectively. We identified three novel unclassified missense variants in RPGRIP1 of the three patients. These patients were heterozygous for each variant and did not have a large deletion or duplication in the same gene.ConclusionsThis comprehensive mutational analysis shows marked genetic heterogeneity in Korean LCA patients and reveals a mutation spectrum that differs from those previously reported. In turn, this suggests that a different strategy should be used for the molecular diagnosis of LCA in Koreans.

| S-EPMC2493025 | biostudies-literature

Treatment Potential for LCA5-Associated Leber Congenital Amaurosis.

Project description:PurposeTo determine the therapeutic window for gene augmentation for Leber congenital amaurosis (LCA) associated with mutations in LCA5.MethodsFive patients (ages 6-31) with LCA and biallelic LCA5 mutations underwent an ophthalmic examination including optical coherence tomography (SD-OCT), full-field stimulus testing (FST), and pupillometry. The time course of photoreceptor degeneration in the Lca5gt/gt mouse model and the efficacy of subretinal gene augmentation therapy with AAV8-hLCA5 delivered at postnatal day 5 (P5) (early, n = 11 eyes), P15 (mid, n = 14), and P30 (late, n = 13) were assessed using SD-OCT, histologic study, electroretinography (ERG), and pupillometry. Comparisons were made with the human disease.ResultsPatients with LCA5-LCA showed a maculopathy with detectable outer nuclear layer (ONL) in the pericentral retina and at least 4 log units of dark-adapted sensitivity loss. The Lca5gt/gt mouse has a similarly severe and rapid photoreceptor degeneration. The ONL became progressively thinner and was undetectable by P60. Rod- and cone-mediated ERGs were severely reduced in amplitudes at P30 and became nondetectable by P60. Subretinal AAV8-hLCA5 administered to Lca5gt/gt mice at P5 and P15, but not at P30, resulted in structural and functional rescue.ConclusionsLCA5-LCA is a particularly severe form of LCA that was recapitulated in the Lca5gt/gt mouse. Gene augmentation resulted in structural and functional rescue in the Lca5gt/gt mouse if delivered before P30. Retained photoreceptors were visible within the central retina in all patients with LCA5-LCA, at a level equivalent to that observed in rescued Lca5gt/gt mice, suggesting a window of opportunity for the treatment of patients with LCA5-LCA.

| S-EPMC7405811 | biostudies-literature

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