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

Budd A Tucker; Cathryn M Cranston; Kristin A Anfinson; Suruchi Shrestha; Luan M Streb; Alejandro Leon; Robert F Mullins; Edwin M Stone

doi:10.1016/j.trsl.2015.08.007

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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

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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

Budd A Tucker, Cathryn M Cranston, Kristin A Anfinson, Suruchi Shrestha, Luan M Streb, Alejandro Leon, Robert F Mullins and Edwin M Stone

Translational research : the journal of laboratory and clinical medicine, Vol.166(6), pp.740-749.e1

12/2015

DOI: 10.1016/j.trsl.2015.08.007

PMCID: PMC4702513

PMID: 26364624

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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.

Retinal Pigment Epithelium - metabolism

Pluripotent Stem Cells - cytology

cis-trans-Isomerases - genetics

Humans

Child, Preschool

Cell Differentiation

Mutation

Details

Title: Subtitle: 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
Creators: Budd A Tucker - Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Science, Carver College of Medicine, University of Iowa, Iowa City, Iowa
Cathryn M Cranston - Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Science, Carver College of Medicine, University of Iowa, Iowa City, Iowa
Kristin A Anfinson - Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Science, Carver College of Medicine, University of Iowa, Iowa City, Iowa
Suruchi Shrestha - Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Science, Carver College of Medicine, University of Iowa, Iowa City, Iowa
Luan M Streb - Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Science, Carver College of Medicine, University of Iowa, Iowa City, Iowa
Alejandro Leon - Department of Ophthalmology, Children's Hospital New Orleans, New Orleans, La
Robert F Mullins - Stephen A Wynn Institute for Vision Research, Department of Ophthalmology and Visual Science, Carver College of Medicine, University of Iowa, Iowa City, Iowa
Edwin M Stone - 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
Resource Type: Journal article
Publication Details: Translational research : the journal of laboratory and clinical medicine, Vol.166(6), pp.740-749.e1
DOI: 10.1016/j.trsl.2015.08.007
PMID: 26364624
PMCID: PMC4702513
NLM abbreviation: Transl Res
ISSN: 1931-5244
eISSN: 1878-1810
Publisher: United States
Grant note: DP2 OD007483 / NIH HHS DP2OD007483-01 / NIH HHS
Language: English
Date published: 12/2015
Academic Unit: The University of Iowa Institute for Vision Research; Iowa Neuroscience Institute; Ophthalmology and Visual Sciences
Record Identifier: 9983980056802771

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