RESUMEN
Inherited retinal diseases (IRDs) are a heterogeneous group of blinding genetic disorders caused by pathogenic variants in genes expressed in the retina. In this study, we sought to develop a method for rapid evaluation of IRD gene variant pathogenicity by inducing expression of retinal genes in patient-derived fibroblasts using CRISPR-activation (CRISPRa). We demonstrate CRISPRa of CRB1 expression in fibroblasts derived from patients with retinitis pigmentosa, enabling investigation of pathogenic mechanisms associated with specific variants. We show the CRB1 c.4005 + 1G>A variant caused exon 11 skipping in CRISPR-activated fibroblasts and retinal organoids (ROs) derived from the same RP12 patient. The c.652 + 5G>C variant was shown to enhance exon 2 skipping in CRISPR-activated fibroblasts and differentially affected CRB1 isoform expression in fibroblasts and ROs. Our study demonstrates an accessible platform for transcript screening of IRD gene variants in patient-derived fibroblasts, which can potentially be applied for rapid pathogenicity assessments of any gene variant.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Especies Reactivas de Oxígeno/metabolismo , Virulencia , Edición Génica , Expresión Génica , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Retinitis pigmentosa 11 (RP11) is caused by dominant mutations in PRPF31, however a significant proportion of mutation carriers do not develop retinopathy. Here, we investigated the relationship between CNOT3 polymorphism, MSR1 repeat copy number and disease penetrance in RP11 patients and non-penetrant carriers (NPCs). We further characterized PRPF31 and CNOT3 expression in fibroblasts from eight RP11 patients and one NPC from a family carrying the c.1205C>T variant. Retinal organoids (ROs) and retinal pigment epithelium (RPE) were differentiated from induced pluripotent stem cells derived from RP11 patients, an NPC and a control subject. All RP11 patients were homozygous for the 3-copy MSR1 repeat in the PRPF31 promoter, while 3/5 NPCs carried a 4-copy MSR1 repeat. The CNOT3 rs4806718 genotype did not correlate with disease penetrance. PRFP31 expression declined with age in adult cadaveric retina. PRPF31 and CNOT3 expression was reduced in RP11 fibroblasts, RO and RPE compared with controls. Both RP11 and NPC RPE displayed shortened primary cilia compared with controls, however a subpopulation of cells with normal cilia lengths was present in NPC RPE monolayers. Our results indicate that RP11 non-penetrance is associated with the inheritance of a 4-copy MSR1 repeat, but not with CNOT3 polymorphisms.
Asunto(s)
Proteínas del Ojo/genética , Penetrancia , Retinitis Pigmentosa/genética , Adolescente , Adulto , Anciano , Células Cultivadas , Niño , Proteínas del Ojo/metabolismo , Femenino , Genes Modificadores , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Retina/metabolismo , Retina/patología , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/patología , Receptores Depuradores de Clase A/genética , Receptores Depuradores de Clase A/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Two human iPSC lines were generated from dermal fibroblasts derived from a patient with retinitis pigmentosa caused by CRB1 mutation using episomal plasmids containing OCT4, SOX2, LIN28, KLF4, L-MYC and mp53DD. These clonal iPSC lines carry compound heterozygous mutations in CRB1 (c.2555 T > C and c.3014A > T). Both lines expressed pluripotency markers, displayed a normal karyotype and demonstrated the ability to differentiate into the three primary germ layers, as well as retinal organoids.
Asunto(s)
Células Madre Pluripotentes Inducidas , Retinitis Pigmentosa , Diferenciación Celular , Línea Celular , Proteínas del Ojo/genética , Fibroblastos , Humanos , Factor 4 Similar a Kruppel , Proteínas de la Membrana , Mutación , Proteínas del Tejido Nervioso , Retinitis Pigmentosa/genéticaRESUMEN
Autosomal recessive Stargardt disease is the most common cause of inherited retinal disease. In this report, we describe the generation and characterization of two human induced pluripotent stem cell (iPSC) lines from a patient with compound heterozygous mutations in the ABCA4 gene (c.[768G>T];[6079C>T]). Patient dermal fibroblasts were reprogrammed using episomal plasmids encoding OCT4, SOX2, KLF4, L-MYC, LIN28, mir302/367 microRNA and shRNA for P53. The clonal iPSC lines LEIi012-A and LEIi012-B were established. Both lines had a normal karyotype, displayed iPSC morphology, expressed pluripotency genes at similar levels to control iPSC and displayed trilineage differentiation potential during embryoid body differentiation.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Células Madre Pluripotentes Inducidas , Enfermedad de Stargardt , Transportadoras de Casetes de Unión a ATP/genética , Diferenciación Celular , Línea Celular , Humanos , Factor 4 Similar a Kruppel , Mutación , Enfermedad de Stargardt/genéticaRESUMEN
We report the generation of the iPSC line LEIi005-B from a patient with retinitis pigmentosa caused by a dominant nonsense mutation in the RP1 gene (c.2098G>T p.E700X). Reprogramming of dermal fibroblasts was performed using episomal plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, mir302/367 microRNA and shRNA for p53 to establish the clonal iPSC line LEIi005-B. LEIi005-B expressed pluripotent stem cell markers, had a normal karyotype and differentiated into endoderm, mesoderm and ectoderm.
Asunto(s)
Diferenciación Celular , Reprogramación Celular , Fibroblastos/patología , Células Madre Pluripotentes Inducidas/patología , Proteínas Asociadas a Microtúbulos/genética , Mutación , Retinitis Pigmentosa/genética , Anciano , Células Cultivadas , Femenino , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Fenotipo , Retinitis Pigmentosa/patologíaRESUMEN
The human iPSC lines LEIi010-A and LEIi010-B were generated from the dermal fibroblasts of a patient with Usher syndrome using episomal plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, mir302/367 microRNA and shRNA for p53. These iPSC lines carry compound heterozygous mutations (c.949Câ¯>â¯A and c.1256Gâ¯>â¯T) in USH2A. LEIi010-A and LEIi010-B expressed pluripotent stem cell markers, had a normal karyotype and could be differentiated into endoderm, mesoderm and ectodermal lineages.
Asunto(s)
Línea Celular , Proteínas de la Matriz Extracelular/genética , Células Madre Pluripotentes Inducidas , Síndromes de Usher/genética , Diferenciación Celular , Técnicas de Reprogramación Celular , Fibroblastos , Heterocigoto , Humanos , Cariotipo , Factor 4 Similar a Kruppel , PielRESUMEN
We report the generation of the human iPSC line LEIi007-A from a patient with autosomal recessive Stargardt disease caused by compound heterozygous mutations in the ABCA4 gene (c.[5461-10â¯Tâ¯>â¯C];[4139Câ¯>â¯T]). Reprogramming of patient dermal fibroblasts was performed using episomal plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, shRNA for p53 and mir302/367 microRNA to establish the clonal iPSC line LEIl007-A. LEIl007-A displayed normal pluripotent stem cell colony morphology, expressed pluripotent stem cell markers, displayed a normal karyotype and differentiated into ectodermal, mesodermal and endodermal germ layer lineages.