RESUMO
Retinoblastoma (RB) is a common intraocular malignancy mostly caused by variation of the tumour suppressor gene RB1. In this study, we successfully generated two induced pluripotent stem cell (iPSC) lines from an infant with non-heritable RB. Both cell clones exhibited typical iPSC characteristics with normal karyotypes, consistent pluripotency markers expression and the capability of trilineage differentiation.
Assuntos
Células-Tronco Pluripotentes Induzidas , Neoplasias da Retina , Retinoblastoma , Lactente , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Retinoblastoma/genética , Retinoblastoma/metabolismo , Diferenciação Celular/genética , Neoplasias da Retina/genética , Neoplasias da Retina/metabolismo , Neoplasias da Retina/patologia , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/patologiaRESUMO
CLCN2 encodes a two-pore homodimeric chloride channel protein (CLC-2) that is widely expressed in human tissues. The association between Clcn2 and the retina is well-established in mice, as loss-of-function of CLC-2 can cause retinopathy in mice; however, the ocular phenotypes caused by CLCN2 mutations in humans and the underlying mechanisms remain unclear. The present study aimed to define the ocular features and reveal the pathogenic mechanisms of CLCN2 variants associated with retinal degeneration in humans using an in vitro overexpression system, as well as patient-induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) cells and retinal organoids (ROs). A patient carrying the homozygous c.2257C > T (p.R753X) nonsense CLCN2 mutation was followed up for > 6 years. Ocular features were comprehensively characterized with multimodality imaging and functional examination. The patient presented with severe bilateral retinal degeneration with loss of photoreceptor and RPE. In vitro, mutant CLC-2 maintained the correct subcellular localization, but with reduced channel function compared to wild-type CLC-2 in HEK293T cells. Additionally, patient iPSC-derived RPE cells carrying the CLCN2 mutation exhibited dysfunctional ClC-2 chloride channels and outer segment phagocytosis. Notably, these functions were rescued following the repair of the CLCN2 mutation using the CRISPR-Cas9 system. However, this variant did not cause significant photoreceptor degeneration in patient-derived ROs, indicating that dysfunctional RPE is likely the primary cause of biallelic CLCN2 variant-mediated retinopathy. This study is the first to establish the confirmatory ocular features of human CLCN2-related retinal degeneration, and reveal a pathogenic mechanism associated with biallelic CLCN2 variants, providing new insights into the cause of inherited retinal dystrophies.
Assuntos
Células-Tronco Pluripotentes Induzidas , Distrofias Retinianas , Animais , Humanos , Camundongos , Canais de Cloreto/genética , Códon sem Sentido , Células HEK293 , Mutação , Fagocitose/genética , Espécies Reativas de Oxigênio/metabolismo , Distrofias Retinianas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologiaRESUMO
PROM1-related retinal dystrophy (PROM1-RD) is a group of hereditary retinal disorder characterized by the progressive damage of the photoreceptors. We generated and identified two induced pluripotent stem cell (iPSC) lines carrying homozygous or heterozygous nonsense mutation c.619G > T (p.E207X) in PROM1 gene from a patient with PROM1-RD and his healthy mother, respectively. Both iPSC lines maintained the typical stem cell morphology, genomic stability and pluripotency. These iPSC lines have great potential to elucidate the disease mechanisms and develop the feasible treatments of PROM1-RD.
