Abstract
Retinitis pigmentosa (RP) represents a group of inherited retinopathies with early-onset nyctalopia followed by progressive photoreceptor degeneration causing irreversible vision loss. Mutations in USH2A are the most common cause of non-syndromic RP. Here, we reprogrammed induced pluripotent stem cells (iPSCs) from a RP patient with a mutation in USH2A (c.8559-2A > G/c.9127_9129delTCC). Then, multilayer retinal organoids including neural retina (NR) and retinal pigment epithelium (RPE) were generated by three-step “induction-reversal culture.” The early retinal organoids derived from the RP patient with the USH2A mutation exhibited significant defects in terms of morphology, immunofluorescence staining and transcriptional profiling. To the best of our knowledge, the pathogenic mutation (c.9127_9129delTCC) in USH2A has not been reported previously among RP patients. Notably, the expression of laminin in the USH2A mutation organoids was significantly lower than in the iPSCs derived from healthy, age- and sex-matched controls during the retinal organogenesis. We also observed that abnormal retinal neuroepithelium differentiation and polarization caused defective retinal progenitor cell development and retinal layer formation, disordered organization of NRs in the presence of the USH2A mutation. Furthermore, the USH2A mutation bearing RPE cells presented abnormal morphology, lacking pigmented foci and showing an apoptotic trend and reduced expression of specific makers, such as MITF, PEDF, and RPE65. In addition, the USH2A mutation organoids had lower expression of cilium-associated (especially CFAP43, PIFO) and dopaminergic synapse-related genes (including DLGAP1, GRIK1, SLC17A7, and SLC17A8), while there was higher expression of neuron apoptotic process-related genes (especially HIF1A, ADARB1, and CASP3). This study may provide essential assistance in the molecular diagnosis and screening of RP. This work recapitulates the pathogenesis of USH2A using patient-specific organoids and demonstrated that alterations in USH2A function due to mutations may lead to cellular and molecular abnormalities.
Highlights
Retinitis pigmentosa (RP) is a group of inherited and progressive eye diseases that cause vision loss (Hartong et al, 2006)
These results collectively suggest that the USH2A mutation in induced pluripotent stem cells (iPSCs) is associated with abnormal neural retina (NR) organoids induction, which is accompanied by decreased proliferation
By combining iPSCs and 3D retinal organoids technologies with panel sequencing, we were able to demonstrate the two disease-causing mutations in a patient with non-syndromic USH2A-associated RP were associated with abnormal NR and retinal pigment epithelium (RPE) development
Summary
Retinitis pigmentosa (RP) is a group of inherited and progressive eye diseases that cause vision loss (Hartong et al, 2006). The patient visual field (VF) gradually becomes only tunnel vision, eventually leading to dysfunction and blindness (Hartong et al, 2006). More than 95 RP pathogenic genes have been mapped and identified, among which 65 are implicated in the autosomal recessive form. More than 95 RP pathogenic genes have been mapped and identified, among which 65 are implicated in the autosomal recessive form1 These known genes account for only approximately 60–70% of all RP cases and the other 40% have not been identified (Chen et al, 2018). Over 600 different mutations in USH2A have been identified and mutations in this gene are by far the most frequent cause of autosomal recessive non-syndromic RP (12–25%) (Slijkerman et al, 2018)
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