title: Using patient-specific induced pluripotent stem cells to interrogate the pathogenicity of a novel retinal pigment epithelium-specific 65 kDa cryptic splice site mutation and confirm eligibility for enrollment into a clinical gene augmentation trial.
abstract: Retinal pigment epithelium-specific 65 kDa (RPE65)-associated Leber congenital amaurosis is an autosomal recessive disease that results in reduced visual acuity and night blindness beginning at birth. It is one of the few retinal degenerative disorders for which promising clinical gene transfer trials are currently underway. However, the ability to enroll patients in a gene augmentation trial is dependent on the identification of 2 bona fide disease-causing mutations, and there are some patients with the phenotype of RPE65-associated disease who might benefit from gene transfer but are ineligible because 2 disease-causing genetic variations have not yet been identified. Some such patients have novel mutations in RPE65 for which pathogenicity is difficult to confirm. The goal of this study was to determine if an intronic mutation identified in a 2-year-old patient with presumed RPE65-associated disease was truly pathogenic and grounds for inclusion in a clinical gene augmentation trial. Sequencing of the RPE65 gene revealed 2 mutations: (1) a previously identified disease-causing exonic leucine-to-proline mutation (L408P) and (2) a novel single point mutation in intron 3 (IVS3-11) resulting in an A>G change. RT-PCR analysis using RNA extracted from control human donor eye-derived primary RPE, control iPSC-RPE cells, and proband iPSC-RPE cells revealed that the identified IVS3-11 variation caused a splicing defect that resulted in a frameshift and insertion of a premature stop codon. In this study, we demonstrate how patient-specific iPSCs can be used to confirm pathogenicity of unknown mutations, which can enable positive clinical outcomes.
authors.affiliation: Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Science, Carver College of Medicine, University of Iowa, Iowa City, Iowa.
authors.forename: Budd A
authors.initials: BA
authors.lastname: Tucker
authors.affiliation: Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Science, Carver College of Medicine, University of Iowa, Iowa City, Iowa.
authors.forename: Cathryn M
authors.initials: CM
authors.lastname: Cranston
authors.affiliation: Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Science, Carver College of Medicine, University of Iowa, Iowa City, Iowa.
authors.forename: Kristin A
authors.initials: KA
authors.lastname: Anfinson
authors.affiliation: Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Science, Carver College of Medicine, University of Iowa, Iowa City, Iowa.
authors.forename: Suruchi
authors.initials: S
authors.lastname: Shrestha
authors.affiliation: Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Science, Carver College of Medicine, University of Iowa, Iowa City, Iowa.
authors.forename: Luan M
authors.initials: LM
authors.lastname: Streb
authors.affiliation: Department of Ophthalmology, Children's Hospital New Orleans, New Orleans, La.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Leon
authors.affiliation: Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Science, Carver College of Medicine, University of Iowa, Iowa City, Iowa.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Mullins
authors.affiliation: Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Science, Carver College of Medicine, University of Iowa, Iowa City, Iowa; Howard Hughes Medical Institute, Department of Ophthalmology and Visual Science, University of Iowa, Iowa City, Iowa. Electronic address: edwin-stone@uiowa.edu.
authors.forename: Edwin M
authors.initials: EM
authors.lastname: Stone
mesh_terms: D002454:Cell Differentiation; D002675:Child, Preschool; D006801:Humans; D009154:Mutation; D039904:Pluripotent Stem Cells / Q000166:cytology*; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D019745:cis-trans-Isomerases / Q000235:genetics*; D000099178:Retinoid Isomerohydrolase
chemical_list: D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase