Human iPSC-Derived Retinal Organoids and Retinal Pigment Epithelium for Novel Intronic <i>RPGR</i> Variant Assessment for Therapy Suitability
The <i>RPGR</i> gene encodes Retinitis Pigmentosa GTPase Regulator, a known interactor with ciliary proteins, which is involved in maintaining healthy photoreceptor cells. Variants in <i>RPGR</i> are the main contributor to X-linked rod-cone dystrophy (RCD), and <i>RPGR...
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MDPI AG
2022-03-01
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| Series: | Journal of Personalized Medicine |
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| Online Access: | https://www.mdpi.com/2075-4426/12/3/502 |
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| author | Fidelle Chahine Karam To Ha Loi Alan Ma Benjamin M. Nash John R. Grigg Darshan Parekh Lisa G. Riley Elizabeth Farnsworth Bruce Bennetts Anai Gonzalez-Cordero Robyn V. Jamieson |
| author_facet | Fidelle Chahine Karam To Ha Loi Alan Ma Benjamin M. Nash John R. Grigg Darshan Parekh Lisa G. Riley Elizabeth Farnsworth Bruce Bennetts Anai Gonzalez-Cordero Robyn V. Jamieson |
| author_sort | Fidelle Chahine Karam |
| collection | DOAJ |
| description | The <i>RPGR</i> gene encodes Retinitis Pigmentosa GTPase Regulator, a known interactor with ciliary proteins, which is involved in maintaining healthy photoreceptor cells. Variants in <i>RPGR</i> are the main contributor to X-linked rod-cone dystrophy (RCD), and <i>RPGR</i> gene therapy approaches are in clinical trials. Hence, elucidation of the pathogenicity of novel <i>RPGR</i> variants is important for a patient therapy opportunity. Here, we describe a novel intronic <i>RPGR</i> variant, c.1415 − 9A>G, in a patient with RCD, which was classified as a variant of uncertain significance according to current clinical diagnostic criteria. The variant lay several base pairs intronic to the canonical splice acceptor site, raising suspicion of an <i>RPGR</i> RNA splicing abnormality and consequent protein dysfunction. To investigate disease causation in an appropriate disease model, induced pluripotent stem cells were generated from patient fibroblasts and differentiated to retinal pigment epithelium (iPSC-RPE) and retinal organoids (iPSC-RO). Abnormal RNA splicing of <i>RPGR</i> was demonstrated in patient fibroblasts, iPSC-RPE and iPSC-ROs, leading to a predicted frameshift and premature stop codon. Decreased RPGR expression was demonstrated in these cell types, with a striking loss of RPGR localization at the ciliary transitional zone, critically in the photoreceptor cilium of the patient iPSC-ROs. Mislocalisation of rhodopsin staining was present in the patient’s iPSC-RO rod photoreceptor cells, along with an abnormality of L/M opsin staining affecting cone photoreceptor cells and increased photoreceptor apoptosis. Additionally, patient iPSC-ROs displayed an increase in F-actin expression that was consistent with an abnormal actin regulation phenotype. Collectively, these studies indicate that the splicing abnormality caused by the c.1415 − 9A>G variant has an impact on RPGR function. This work has enabled the reclassification of this variant to pathogenic, allowing the consideration of patients with this variant having access to gene therapy clinical trials. In addition, we have identified biomarkers of disease suitable for the interrogation of other <i>RPGR</i> variants of uncertain significance. |
| first_indexed | 2024-03-09T13:38:26Z |
| format | Article |
| id | doaj.art-bd10374474d04ff3ad319d610201f24e |
| institution | Directory Open Access Journal |
| issn | 2075-4426 |
| language | English |
| last_indexed | 2024-03-09T13:38:26Z |
| publishDate | 2022-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Personalized Medicine |
| spelling | doaj.art-bd10374474d04ff3ad319d610201f24e2023-11-30T21:09:22ZengMDPI AGJournal of Personalized Medicine2075-44262022-03-0112350210.3390/jpm12030502Human iPSC-Derived Retinal Organoids and Retinal Pigment Epithelium for Novel Intronic <i>RPGR</i> Variant Assessment for Therapy SuitabilityFidelle Chahine Karam0To Ha Loi1Alan Ma2Benjamin M. Nash3John R. Grigg4Darshan Parekh5Lisa G. Riley6Elizabeth Farnsworth7Bruce Bennetts8Anai Gonzalez-Cordero9Robyn V. Jamieson10Eye Genetics Research Unit, Children’s Medical Research Institute, Sydney Children’s Hospitals Network, Save Sight Institute, University of Sydney, Westmead, Sydney 2145, AustraliaEye Genetics Research Unit, Children’s Medical Research Institute, Sydney Children’s Hospitals Network, Save Sight Institute, University of Sydney, Westmead, Sydney 2145, AustraliaEye Genetics Research Unit, Children’s Medical Research Institute, Sydney Children’s Hospitals Network, Save Sight Institute, University of Sydney, Westmead, Sydney 2145, AustraliaEye Genetics Research Unit, Children’s Medical Research Institute, Sydney Children’s Hospitals Network, Save Sight Institute, University of Sydney, Westmead, Sydney 2145, AustraliaEye Genetics Research Unit, Children’s Medical Research Institute, Sydney Children’s Hospitals Network, Save Sight Institute, University of Sydney, Westmead, Sydney 2145, AustraliaRare Diseases Functional Genomics Laboratory, Sydney Children’s Hospitals Network and Children’s Medical Research Institute, Westmead, Sydney 2145, AustraliaRare Diseases Functional Genomics Laboratory, Sydney Children’s Hospitals Network and Children’s Medical Research Institute, Westmead, Sydney 2145, AustraliaSpecialty of Genomic Medicine, Faculty of Medicine and Health, University of Sydney, Westmead, Sydney 2145, AustraliaSpecialty of Genomic Medicine, Faculty of Medicine and Health, University of Sydney, Westmead, Sydney 2145, AustraliaStem Cell Medicine Group, Children’s Medical Research Institute, University of Sydney, Westmead, Sydney 2145, AustraliaEye Genetics Research Unit, Children’s Medical Research Institute, Sydney Children’s Hospitals Network, Save Sight Institute, University of Sydney, Westmead, Sydney 2145, AustraliaThe <i>RPGR</i> gene encodes Retinitis Pigmentosa GTPase Regulator, a known interactor with ciliary proteins, which is involved in maintaining healthy photoreceptor cells. Variants in <i>RPGR</i> are the main contributor to X-linked rod-cone dystrophy (RCD), and <i>RPGR</i> gene therapy approaches are in clinical trials. Hence, elucidation of the pathogenicity of novel <i>RPGR</i> variants is important for a patient therapy opportunity. Here, we describe a novel intronic <i>RPGR</i> variant, c.1415 − 9A>G, in a patient with RCD, which was classified as a variant of uncertain significance according to current clinical diagnostic criteria. The variant lay several base pairs intronic to the canonical splice acceptor site, raising suspicion of an <i>RPGR</i> RNA splicing abnormality and consequent protein dysfunction. To investigate disease causation in an appropriate disease model, induced pluripotent stem cells were generated from patient fibroblasts and differentiated to retinal pigment epithelium (iPSC-RPE) and retinal organoids (iPSC-RO). Abnormal RNA splicing of <i>RPGR</i> was demonstrated in patient fibroblasts, iPSC-RPE and iPSC-ROs, leading to a predicted frameshift and premature stop codon. Decreased RPGR expression was demonstrated in these cell types, with a striking loss of RPGR localization at the ciliary transitional zone, critically in the photoreceptor cilium of the patient iPSC-ROs. Mislocalisation of rhodopsin staining was present in the patient’s iPSC-RO rod photoreceptor cells, along with an abnormality of L/M opsin staining affecting cone photoreceptor cells and increased photoreceptor apoptosis. Additionally, patient iPSC-ROs displayed an increase in F-actin expression that was consistent with an abnormal actin regulation phenotype. Collectively, these studies indicate that the splicing abnormality caused by the c.1415 − 9A>G variant has an impact on RPGR function. This work has enabled the reclassification of this variant to pathogenic, allowing the consideration of patients with this variant having access to gene therapy clinical trials. In addition, we have identified biomarkers of disease suitable for the interrogation of other <i>RPGR</i> variants of uncertain significance.https://www.mdpi.com/2075-4426/12/3/502<i>RPGR</i>inherited retinal diseaserod-cone dystrophyciliopathyiPSCretinal organoids |
| spellingShingle | Fidelle Chahine Karam To Ha Loi Alan Ma Benjamin M. Nash John R. Grigg Darshan Parekh Lisa G. Riley Elizabeth Farnsworth Bruce Bennetts Anai Gonzalez-Cordero Robyn V. Jamieson Human iPSC-Derived Retinal Organoids and Retinal Pigment Epithelium for Novel Intronic <i>RPGR</i> Variant Assessment for Therapy Suitability Journal of Personalized Medicine <i>RPGR</i> inherited retinal disease rod-cone dystrophy ciliopathy iPSC retinal organoids |
| title | Human iPSC-Derived Retinal Organoids and Retinal Pigment Epithelium for Novel Intronic <i>RPGR</i> Variant Assessment for Therapy Suitability |
| title_full | Human iPSC-Derived Retinal Organoids and Retinal Pigment Epithelium for Novel Intronic <i>RPGR</i> Variant Assessment for Therapy Suitability |
| title_fullStr | Human iPSC-Derived Retinal Organoids and Retinal Pigment Epithelium for Novel Intronic <i>RPGR</i> Variant Assessment for Therapy Suitability |
| title_full_unstemmed | Human iPSC-Derived Retinal Organoids and Retinal Pigment Epithelium for Novel Intronic <i>RPGR</i> Variant Assessment for Therapy Suitability |
| title_short | Human iPSC-Derived Retinal Organoids and Retinal Pigment Epithelium for Novel Intronic <i>RPGR</i> Variant Assessment for Therapy Suitability |
| title_sort | human ipsc derived retinal organoids and retinal pigment epithelium for novel intronic i rpgr i variant assessment for therapy suitability |
| topic | <i>RPGR</i> inherited retinal disease rod-cone dystrophy ciliopathy iPSC retinal organoids |
| url | https://www.mdpi.com/2075-4426/12/3/502 |
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