Utilising patient-specific retinal organoids to investigate the role of SNRNP200 variants of unknown significance in severe early onset retinitis pigmentosa

Abstract

Purpose : The consequence of variants in many genes implicated in retinitis pigmentosa (RP) remain unknown. This applies to SNRNP200, which encodes for a component of the spliceosome complex. To investigate the effect of SNRNP200 variants on the human retina, we generated retinal organoids (ROs) from a patient with severe early-onset RP found to harbour compound heterozygous missense variants in SNRNP200 that segregated with the disease. Methods : Computer-assisted pathogenicity programs and other tools were used to predict the pathogenicity of candidate variants detected by a 537-gene next-generation sequencing panel (MVL, Oregon, US). Induced pluripotent stem cells (iPSCs) derived from patient and control dermal fibroblasts were differentiated into ROs and maintained for up to 350 days. Western Blotting and immunohistochemical (IHC) analysis was performed using antibodies directed against SNRNP200 and retinal-specific proteins. Gene expression was analysed by qRT-PCR and retinal ultrastructure examined by transmission electron microscopy. Single cell sorting of control and patient iPSCs and ROs was performed to capture individual cells which were then subjected to RNA-Seq and bioinformatic analysis. Results : A patient-specific in vitro model of RP was generated. The emergence of retinal-specific phenotypes in ROs was confirmed by IHC and qRT-PCR. Several morphological differences in patient ROs were identified including (1) reduced neuroepithelial integrity, (2) a high number of vacuole-like inclusions within photoreceptor soma, and (3) abnormal mitochondrial morphology at later stages of differentiation. Single cell transcriptomic analysis revealed significantly significant differential gene expression between control and patient cells. Notably, multiple genes associated with immunologic signatures were upregulated, and genes associated with cell coenzyme biosynthesis, an important process for normal vision, were differentially expressed. Conclusions : Using a human RO-based model of RP we have identified patient-specific differences in gene, protein and transcriptomic expression, neuroepithelial integrity, photoreceptor morphology and ultrastructure. These data show the potential of using ROs to assess functional consequences in patients affected by variants of unknown significance