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.
Abbreviations:
Best1 (Bestrophin1), bFGF (basic fibroblast growth factor), DMEM (Dulbecco's Modified Eagle Medium), DNMT (DNA methyltransferases), ERG (Electroretinography), KLF4 (Kruppel-like factor 4), LB (Lysogeny broth), LCA (Leber congenital amaurosis), MEM (minimum essential medium), NEAA (non-essential amino acids), MITF (Microphthalmia-Associated Transcription Factor), MOI (multiplicity of infection), RPE (Retinal pigment epithelium), RPE65 (Retinal pigment epithelium–specific 65 kDa), SOX2 (SRY (sex determining region Y)-box 2), SSEA4 (Stage-Specific Embryonic Antigen)To read this article in full you will need to make a payment
Purchase one-time access:
Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online accessOne-time access price info
- For academic or personal research use, select 'Academic and Personal'
- For corporate R&D use, select 'Corporate R&D Professionals'
Subscribe:
Subscribe to Translational ResearchAlready a print subscriber? Claim online access
Already an online subscriber? Sign in
Register: Create an account
Institutional Access: Sign in to ScienceDirect
References
- Review and update on the molecular basis of Leber congenital amaurosis.World J Clin Cases. 2015; 3: 112-124
- Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle.Nat Genet. 1998; 20: 344-351
- Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle.Proc Natl Acad Sci U S A. 2005; 102: 13658-13663
- Activation, deactivation, and adaptation in vertebrate photoreceptor cells.Annu Rev Neurosci. 2001; 24: 779-805
- Focus on Molecules: RPE65, the visual cycle retinol isomerase.Exp Eye Res. 2009; 88: 846-847
- Nature of the visual loss in observers with Leber's congenital amaurosis caused by specific mutations in RPE65.Invest Ophthalmol Vis Sci. 2014; 55: 6817-6828
- Effect of gene therapy on visual function in Leber's congenital amaurosis.N Engl J Med. 2008; 358: 2231-2239
- Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial.Lancet. 2009; 374: 1597-1605
- Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial.Hum Gene Ther. 2008; 19: 979-990
- Gene therapy for leber congenital amaurosis caused by RPE65 mutations: safety and efficacy in 15 children and adults followed up to 3 years.Arch Ophthalmol. 2012; 130: 9-24
- CEP290 gene transfer rescues Leber congenital amaurosis cellular phenotype.Gene Ther. 2014; 21: 662-672
- Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676
- Use of a synthetic xeno-free culture substrate for induced pluripotent stem cell induction and retinal differentiation.Stem Cells Transl Med. 2013; 2: 16-24
- Exome sequencing and analysis of induced pluripotent stem cells identify the cilia-related gene male germ cell-associated kinase (MAK) as a cause of retinitis pigmentosa.Proc Natl Acad Sci U S A. 2011; 108: E569-E576
- Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa.Elife. 2013; 2: e00824
- Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease.Hum Mol Genet. 2013; 22: 5136-5145
- Duplication of TBK1 stimulates autophagy in iPSC-derived retinal cells from a patient with normal tension glaucoma.J Stem Cell Res Ther. 2014; 3: 161
- The phenotype of Severe Early Childhood Onset Retinal Dystrophy (SECORD) from mutation of RPE65 and differentiation from Leber congenital amaurosis.Invest Ophthalmol Vis Sci. 2011; 52: 292-302
- Rescue of enzymatic function for disease-associated RPE65 proteins containing various missense mutations in non-active sites.J Biol Chem. 2014; 289: 18943-18956
- Leber congenital amaurosis—a model for efficient genetic testing of heterogeneous disorders: LXIV Edward Jackson Memorial Lecture.Am J Ophthalmol. 2007; 144: 791-811
- Predicting the pathogenicity of RPE65 mutations.Hum Mutat. 2009; 30: 1183-1188
- Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs.Nat Commun. 2014; 5: 4047
- Blood-derived human iPS cells generate optic vesicle-like structures with the capacity to form retinal laminae and develop synapses.Invest Ophthalmol Vis Sci. 2012; 53: 2007-2019
- Generation, purification and transplantation of photoreceptors derived from human induced pluripotent stem cells.PLoS One. 2010; 5: e8763
- Differentiation of human pluripotent stem cells to retinal pigmented epithelium in defined conditions using purified extracellular matrix proteins.J Tissue Eng Regen Med. 2013; 7: 642-653
- Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application.Stem Cell Reports. 2014; 2: 205-218
- Self-formation of optic cups and storable stratified neural retina from human ESCs.Cell Stem Cell. 2012; 10: 771-785
Article info
Publication history
Published online: August 27, 2015
Accepted:
August 24,
2015
Received in revised form:
August 18,
2015
Received:
March 16,
2015
Identification
Copyright
© 2015 Elsevier Inc. Published by Elsevier Inc. All rights reserved.
