July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Utilising patient-specific retinal organoids to investigate the role of SNRNP200 variants of unknown significance in severe early onset retinitis pigmentosa
Author Affiliations & Notes
  • Carla Bernadette Mellough
    Lions Eye Institute Ltd., Perth, Western Australia, Australia
    Centre for Ophthalmology and Visual Science, University of Western Australia, Perth, Western Australia, Australia
  • Mariska Ackerman
    Lions Eye Institute Ltd., Perth, Western Australia, Australia
    Centre for Ophthalmology and Visual Science, University of Western Australia, Perth, Western Australia, Australia
  • Jennifer A Thompson
    Department of Medical Technology & Physics, Australian Inherited Retinal Disease Registry & DNA Bank, Sir Charles Gairdner Hospital, Perth, Western Australia, Australia
  • John De Roach
    Department of Medical Technology & Physics, Australian Inherited Retinal Disease Registry & DNA Bank, Sir Charles Gairdner Hospital, Perth, Western Australia, Australia
  • Terri McLaren
    Department of Medical Technology & Physics, Australian Inherited Retinal Disease Registry & DNA Bank, Sir Charles Gairdner Hospital, Perth, Western Australia, Australia
  • Tina Lamey
    Department of Medical Technology & Physics, Australian Inherited Retinal Disease Registry & DNA Bank, Sir Charles Gairdner Hospital, Perth, Western Australia, Australia
  • Anthony Akkari
    The Perron Institute for Neurological and Translational Science, Western Australia, Australia
    Industrial Pharmacogenetics, Murdoch University, Western Australia, Australia
  • Ramesh Ram
    Institute for Immunology & Infectious Diseases, Murdoch University, Western Australia, Australia
  • Shay Leary
    Institute for Immunology & Infectious Diseases, Murdoch University, Western Australia, Australia
  • Abha Chopra
    Institute for Immunology & Infectious Diseases, Murdoch University, Western Australia, Australia
  • Shang-Chih Chen
    Lions Eye Institute Ltd., Perth, Western Australia, Australia
  • Dan Zhang
    Lions Eye Institute Ltd., Perth, Western Australia, Australia
  • Samuel McLenachan
    Lions Eye Institute Ltd., Perth, Western Australia, Australia
    Centre for Ophthalmology and Visual Science, University of Western Australia, Perth, Western Australia, Australia
  • Fred Kuanfu Chen
    Lions Eye Institute Ltd., Perth, Western Australia, Australia
    Centre for Ophthalmology and Visual Science, University of Western Australia, Perth, Western Australia, Australia
  • Footnotes
    Commercial Relationships   Carla Mellough, None; Mariska Ackerman, None; Jennifer Thompson, None; John De Roach, None; Terri McLaren, None; Tina Lamey, None; Anthony Akkari, None; Ramesh Ram, None; Shay Leary, None; Abha Chopra, None; Shang-Chih Chen, None; Dan Zhang, None; Samuel McLenachan, None; Fred Chen, None
  • Footnotes
    Support  Retina Australia Grant
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 2868. doi:https://doi.org/
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      Carla Bernadette Mellough, Mariska Ackerman, Jennifer A Thompson, John De Roach, Terri McLaren, Tina Lamey, Anthony Akkari, Ramesh Ram, Shay Leary, Abha Chopra, Shang-Chih Chen, Dan Zhang, Samuel McLenachan, Fred Kuanfu Chen; Utilising patient-specific retinal organoids to investigate the role of SNRNP200 variants of unknown significance in severe early onset retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2868. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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