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Joseph C Giacalone, Luke A Wiley, Erin R Burnight, Jeaneen Andorf, Luan M Streb, Louisa M Affatigato, Cathryn Cranston, Dalyz Ochoa, Robert F Mullins, Budd Tucker, Edwin M Stone; Characterization and Treatment of RPGR-associated Photoreceptor Disease. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4515. doi: https://doi.org/.
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Two major transcripts of RPGR exist: 1) a widely expressed form that contains 19 exons and 2) a retinal-predominant transcript that contains a unique purine rich, repetitive 3-prime sequence in exon 15. Interestingly, mutations in RPGR can lead to a predominant dysfunction of rod or cone photoreceptors in different individuals. The purpose of this study was to investigate the frequency, mutation spectrum and phenotypes of RPGR-associated disease and to develop a CRISPR-based genome editing strategy suitable for correcting any RPGR mutation regardless of location.
Over 1000 consecutive families seen by a single retina specialist between January 2010 and June 2016 were reviewed and 500 probands met the clinical criteria for a diagnosis of inherited nonsyndromic photoreceptor disease. Mutations in RPGR were sought in this cohort using a tiered testing strategy consisting of direct Sanger sequencing of PCR products, Sanger sequencing of cloned PCR products, exome sequencing and whole genome sequencing. Using iPSCs generated from patients selected from this cohort, a CRISPR-based genome-editing strategy was developed to achieve genomic correction of mutations anywhere in the gene, including the most commonly mutated exon 15.
Mutations in RPGR were found in 49 of the 500 families (9.8%), making this gene the most common cause of disease in this cohort. The phenotypes ranged from retinitis pigmentosa diagnosed in the first five years of life to a cone selective disease that did not come to medical attention until the fourth decade. Most families exhibited clear X-linked inheritance, but in a few families, the carrier females were as severely affected as the males, mimicking autosomal dominant disease. 37 different mutations were identified, 17 of which were novel. Twenty-three mutations were found in the repetitive portion of exon 15 and required plasmid cloning to identify. Genome-editing strategies were developed to efficiently correct unique mutations in 7 patient lines.
Mutations in RPGR are the most common cause of nonsyndromic photoreceptor disease and 46.9% of these mutations occur in the highly repetitive portion of exon 15 which cannot be easily detected. Technological progress in genome-editing enables efficient correction of patient cells for the purpose of autologous cell replacement.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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