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Budd Tucker, Robert Mullins, Kristin Anfinson, Louisa Affatigato, Jennifer Halder, Peter Brzeskiewicz, Edwin Stone; Investigating the pathophysiology of USH2A associated retinal degeneration with patient specific iPSCs. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4054.
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Mutations in the USH2A gene are the most common cause of both Usher syndrome type II and non-syndromic USH-associated RP. As USH2A is a large highly polymorphic gene, our ability to functionally confirm that newly discovered mutations are disease causing is a critical step prior to development of gene and/or cell based therapies. This is especially true for non-exomic mutations with difficult to confirm pathogenicity at the DNA level. By combining next generation sequencing and iPSC technologies, this study was designed to determine whether mutations identified in the USH2A gene were reflected in the retinal transcript and as such were truly disease causing.
iPSCs were generated via transduction of human keratinocytes, obtained from patients with USH2A-associated retinitis pigmentosa, using the transcription factors Oct4, Sox2, C-Myc and KLF4. Capacity for retinal differentiation was determined via ICC, rt-PCR, and WB analysis targeted at detecting the markers recoverin, rhodopsin, ROM-1, R/G opsin, B-opsin, and tubulin. Pathogenicity of USH2A mutations was determined via rt-PCR and WB targeted against USH2A.
iPSCs generated from a patient with suspected non-syndromic USH2A associated RP were generated. Cell lines were expanded in feeder free conditions and determined to be pluripotent based on immunocytochemical and rt-PCR pluripotency marker expression (Nanog, SSEA3, SSEA4, Tra-1-60, and Tra-1-81) and ability to generate tissues specific to all three germ layers (determined via teratoma and embryoid body formation). Following differentiation, eyecups containing both RPE and neural retina developed in vitro. Isolation of the neural retina and subsequent analysis of the USH2A transcript revealed that a suspected splice site mutation within IVS40, caused exonification of the intron and insertion of a premature stop codon. In addition, a novel mutation identified via next generation whole exome sequencing was confirmed within the iPSC derived neural retina of the patient.
By combining next generation sequencing and induced pluripotent stem cell (iPSC) technologies we have been able to definitively demonstrate the pathogenicity of two disease-causing mutations in a patient with non-syndromic USH2A associated RP. These findings have enabled us to proceed with patient specific studies focused on USH2A gene correction and photoreceptor cell replacement.
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