Purchase this article with an account.
Tasneem Putliwala Sharma, Joseph C Giacalone, Erin R Burnight, Kristin R. Anfinson, Jill S. Wiley, John Fingert, Robert F Mullins, Edwin M Stone, Budd Tucker; Combining CRISPR based genome editing and patient specific iPSCs to elucidate the pathophysiologic role of WFS1 mutations in optic atrophy.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5585.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Mutations in wolframin ER transmembrane glycoprotein 1 (WFS1) gene have been associated with optic atrophy and Wolfram syndrome. The purpose of this study was to use patient specific iPSCs and CRISPR based genome editing to determine how mutations in WFS1 cause retinal ganglion cell (RGC) death.
Dermal fibroblasts were obtained and expanded from two patients with suspected WFS1 associated optic atrophy and one patient with molecularly confirmed Wolfram syndrome. Fibroblasts were targeted for induced pluripotent stem cell (iPSC) generation using Sendai viruses driving expression of OCT4, SOX2, KLF4 and c-MYC. Pluripotency was confirmed using rt-PCR, immunocytochemistry and the TaqMan Scorecard Assay. Immunofluorescence, quantitative rt-PCR, and Western blot analyses were used to characterize expression of ER stress associated markers. Cells generated from a patient with molecularly confirmed Wolfram syndrome and isogenic CRISPR/Cas9 corrected patient-specific iPSCs were used as controls.
Homozygous Arg558Cys WFS1 variants were identified in two patients with non-syndromic recessive optic atrophy using exome sequencing. As determined by a TaqMan ER stress assay, patient-specific iPSC-derived retinal ganglion cells generated from these two individuals were found to have increased expression of key ER stress genes such as BIP and ERO1LB (P<0.05). Likewise, ER-stress mediated RGC dysfunction, as evident by increased levels of BIP, HSP90AB, CANX, DDIT3, EIF2AK3, CASP3 and BAX were identified (P<0.05). CRISPR/Cas9-mediated homology dependent repair of the WFS1 gene mitigated the mutant phenotype and enabled generation of normal RGCs.
Mutations in WFS1 can cause non-syndromic early onset optic atrophy. By combining patient specific iPSCs and CRISPR based genome editing we have successfully demonstrated that mutations in WFS1 lead to activation of the ER-stress pathway.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only