RESUMEN
Mutations in CEP290 are the most common cause of Leber congenital amaurosis (LCA), a severe inherited retinal degenerative disease for which there is currently no cure. Autosomal recessive CEP290-associated LCA is a good candidate for gene replacement therapy, and cells derived from affected individuals give researchers the ability to study human disease and therapeutic gene correction in vitro. Here we report the development of lentiviral vectors carrying full-length CEP290 for the purpose of correcting the CEP290 disease-specific phenotype in human cells. A lentiviral vector containing CMV-driven human full-length CEP290 was constructed. Following transduction of patient-specific, iPSC-derived, photoreceptor precursor cells, reverse transcriptase-PCR analysis and western blotting revealed vector-derived expression. As CEP290 is important in ciliogenesis, the ability of fibroblast cultures from CEP290-associated LCA patients to form cilia was investigated. In cultures derived from these patients, fewer cells formed cilia compared with unaffected controls. Cilia that were formed were shorter in patient-derived cells than in cells from unaffected individuals. Importantly, lentiviral delivery of CEP290 rescued the ciliogenesis defect. The successful construction and viral transfer of full-length CEP290 brings us closer to the goal of providing gene- and cell-based therapies for patients affected with this common form of LCA.
Asunto(s)
Antígenos de Neoplasias/genética , Células Madre Pluripotentes Inducidas/trasplante , Amaurosis Congénita de Leber/terapia , Lentivirus/genética , Proteínas de Neoplasias/genética , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Animales , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Cilios/metabolismo , Cilios/patología , Proteínas del Citoesqueleto , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibroblastos/patología , Vectores Genéticos/farmacología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/patología , Ratones , Proteínas de Neoplasias/metabolismo , Retina/patología , Transducción GenéticaRESUMEN
PURPOSE: To determine the disease expression in heterozygotes for mutations in the RP1 gene, a newly identified cause of autosomal dominant retinitis pigmentosa (adRP). METHODS: Screening strategies were used to detect disease-causing mutations in the RP1 gene, and detailed studies of phenotype were performed in a subset of the detected RP1 heterozygotes using electroretinography (ERG), psychophysics, and optical coherence tomography (OCT). RESULTS: Seventeen adRP families had heterozygous RP1 changes. Thirteen families had the Arg677ter mutation, whereas four others had one of the following: Pro658 (1-bp del), Ser747 (1-bp del), Leu762-763 (5-bp del), and Tyr1053 (1-bp del). In Arg677ter RP1 heterozygotes, there was regional retinal variation in disease, with the far peripheral inferonasal retina being most vulnerable; central and superior temporal retinal regions were better preserved. The earliest manifestation of disease was rod dysfunction, detectable as reduced rod ERG photoresponse maximum amplitude, even in heterozygotes with otherwise normal clinical, functional, and OCT cross-sectional retinal imaging results. At disease stages when cone abnormalities were present, there was greater rod than cone dysfunction. Patients with the RP1 frameshift mutations showed similarities in phenotype to those with the Arg677ter mutation. CONCLUSIONS: Earliest disease expression of RP1 gene mutations causing adRP involves primarily rod photoreceptors, and there is a gradient of vulnerability of retinopathy with more pronounced effects in the inferonasal peripheral retina. At other disease stages, cone function is also affected, and severe retina-wide degeneration can occur. The nonpenetrance or minimal disease expression in some Arg677ter mutation-positive heterozygotes suggests important roles for modifier genes or environmental factors in RP1-related disease.
Asunto(s)
Proteínas del Ojo/genética , Mutación , Retinitis Pigmentosa/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Adaptación a la Oscuridad/fisiología , Electrorretinografía , Femenino , Humanos , Masculino , Proteínas Asociadas a Microtúbulos , Persona de Mediana Edad , Fenotipo , Células Fotorreceptoras de Vertebrados/fisiología , Retinitis Pigmentosa/fisiopatología , Tomografía/métodos , Campos Visuales/fisiologíaRESUMEN
PURPOSE: To assess the allelic variation of the VMD2 gene in patients with Best disease and age-related macular degeneration (AMD). METHODS: Three hundred twenty-one AMD patients, 192 ethnically similar control subjects, 39 unrelated probands with familial Best disease, and 57 unrelated probands with the ophthalmoscopic findings of Best disease but no family history were screened for sequence variations in the VMD2 gene by single-strand conformation polymorphism (SSCP) analysis. Amplimers showing a bandshift were reamplified and sequenced bidirectionally. In addition, the coding regions of the VMD2 gene were completely sequenced in six probands with familial Best disease who showed no SSCP shift. RESULTS: Forty different probable or possible disease-causing mutations were found in one or more Best disease or AMD patients. Twenty-nine of these variations are novel. Of the 39 probands with familial Best disease, mutations were detected in all 39 (33 by SSCP and 6 by DNA sequencing). SSCP screening of the 57 probands with a clinical diagnosis of Best disease but no family history revealed 16 with mutations. Mutations were found in 5 of 321 AMD patients (1.5%), a fraction that was not significantly greater than in control individuals (0/192, 0%). CONCLUSIONS: Patients with the clinical diagnosis of Best disease are significantly more likely to have a mutation in the VMD2 gene if they also have a positive family history. These findings suggest that a small fraction of patients with the clinical diagnosis of AMD may actually have a late-onset variant of Best disease, whereas at the same time, a considerable fraction of isolated patients with the ophthalmoscopic features of Best disease are probably affected with some other macular disease.
Asunto(s)
Alelos , Proteínas del Ojo/genética , Variación Genética/genética , Degeneración Macular/genética , Mutación Puntual , Adulto , Anciano , Bestrofinas , Niño , Canales de Cloruro , Análisis Mutacional de ADN , Cartilla de ADN/química , Femenino , Humanos , Degeneración Macular/diagnóstico , Masculino , Linaje , Polimorfismo Conformacional Retorcido-Simple , Degeneración Retiniana/diagnóstico , Degeneración Retiniana/genética , Análisis de Secuencia de ADNRESUMEN
PURPOSE: To define the phenotypes of retinal degenerations associated with mutations in the gene encoding CRX (cone-rod homeobox), a photoreceptor-specific transcription factor. METHODS: Heterozygotes with the E168 [delta1 bp], E168 [delta2 bp], or G217 [delta1 bp] CRXgene mutation were studied clinically, with visual function tests, including rod and cone perimetry and electroretinography (ERG), and with optical coherence tomography (OCT). RESULTS: Clinical diagnoses included autosomal dominant cone-rod dystrophy in one family (E168 [delta1 bp] mutation) and simplex Leber congenital amaurosis in two families (E168 [delta2 bp], G217 [delta1 bp] mutations). In the family with the E168 [delta1 bp] mutation, two siblings had relatively mild disease expression in the third decade of life. The central retinas of these two patients had profound loss of rod and short wavelength cone function; long/middle wavelength cone thresholds were elevated at fixation, but there were greater paracentral than central abnormalities. Peripheral retinal dysfunction was evident by psychophysics and by maximum amplitude loss for rod- and cone-isolated ERG photoreceptor responses. OCT cross-sectional reflectance images showed decreased central retinal thickness consistent with photoreceptor loss. An additional member of this family (E168 [delta1 bp] mutation) and two other patients (representing E168 [delta2 bp] and G217 [delta1 bp] mutations) had a severe phenotype with retina-wide loss of function and islands of function remaining only in the temporal periphery. CONCLUSIONS: Truncation mutations in CRX are associated with retinopathies that share phenotypic features but vary in disease severity. The disease mechanism could involve abnormal photoreceptor development compounded by a disturbance in the maintenance of photoreceptors in the mature retina.
Asunto(s)
Proteínas de Homeodominio/genética , Mutación , Células Fotorreceptoras de Vertebrados/fisiología , Degeneración Retiniana/genética , Transactivadores/genética , Adulto , Niño , Electrorretinografía , Femenino , Humanos , Persona de Mediana Edad , Linaje , Fenotipo , Psicofísica , Degeneración Retiniana/fisiopatología , Tomografía , Agudeza Visual , Pruebas del Campo Visual , Campos VisualesRESUMEN
Crx is a novel paired-like homeodomain protein that is expressed predominantly in retinal photoreceptors and pinealocytes. Its gene has been mapped to chromosome 19q13.3, the site of a disease locus for autosomal dominant cone-rod dystrophy (CORDII). Analysis of the proband from a family with autosomal dominant CORD revealed an Arg41Trp substitution in the third residue of the CRX homeodomain. The sequence change cosegregated with the disease phenotype and was not detected in 247 normal controls. Recombinant CRX homeodomain containing the Arg41Trp substitution showed decreased DNA binding activity. Analysis of another 169 CORD probands identified three additional CRX sequence variations (Arg41Gln, Val242Met, and a 4 bp deletion in codons 196/7) that were not found among the controls. This data suggests that mutations in the CRX gene are associated with photoreceptor degeneration and that the Crx protein is necessary for the maintenance of normal cone and rod function.