M A Aksit1, A D Bowling1, T A Evans1, A T Joynt1, D Osorio1, S Patel2, N West2, C Merlo2, P R Sosnay2, G R Cutting1, N Sharma3. 1. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States. 2. Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins Hospital, Baltimore, MD, United States. 3. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States. Electronic address: nsharma5@jhmi.edu.
Abstract
BACKGROUND: Cell-based studies have shown that W1282X generates a truncated protein that can be functionally augmented by modulators. However, modulator treatment of primary cells from individuals who carry two copies of W1282X generates no functional CFTR. To understand the lack of response to modulators, we investigated the effect of W1282X on CFTR RNA transcript levels. METHODS: qRT-PCR and RNA-seq were performed on primary nasal epithelial (NE) cells of a previously studied individual who is homozygous for W1282X, her carrier parents and control individuals without nonsense variants in CFTR. RESULTS: CFTR RNA bearing W1282X in NE cells shows a steady-state level of 4.2 ± 0.9% of wild-type (WT) CFTR RNA in the mother and 12.4 ± 1.3% in the father. NMDI14, an inhibitor of nonsense-mediated mRNA decay (NMD), restored W1282X mRNA to almost 50% of WT levels in the parental NE cells. RNA-seq of the NE cells homozygous for W1282X showed that CFTR transcript level was reduced to 1.7% of WT (p-value: 4.6e-3). Negligible truncated CFTR protein was generated by Flp-In 293 cells stably expressing the W1282X EMG even though CFTR transcript was well above levels observed in the parents and proband. Finally, we demonstrated that NMD inhibition improved the stability and response to correctors of W1282X-CFTR protein expressed in the Flp-In-293 cells. CONCLUSION: These results show that W1282X can cause substantial degradation of CFTR mRNA that has to be addressed before efforts aimed at augmenting CFTR protein function can be effective.
BACKGROUND: Cell-based studies have shown that W1282X generates a truncated protein that can be functionally augmented by modulators. However, modulator treatment of primary cells from individuals who carry two copies of W1282X generates no functional CFTR. To understand the lack of response to modulators, we investigated the effect of W1282X on CFTR RNA transcript levels. METHODS: qRT-PCR and RNA-seq were performed on primary nasal epithelial (NE) cells of a previously studied individual who is homozygous for W1282X, her carrier parents and control individuals without nonsense variants in CFTR. RESULTS:CFTR RNA bearing W1282X in NE cells shows a steady-state level of 4.2 ± 0.9% of wild-type (WT) CFTR RNA in the mother and 12.4 ± 1.3% in the father. NMDI14, an inhibitor of nonsense-mediated mRNA decay (NMD), restored W1282X mRNA to almost 50% of WT levels in the parental NE cells. RNA-seq of the NE cells homozygous for W1282X showed that CFTR transcript level was reduced to 1.7% of WT (p-value: 4.6e-3). Negligible truncated CFTR protein was generated by Flp-In 293 cells stably expressing the W1282X EMG even though CFTR transcript was well above levels observed in the parents and proband. Finally, we demonstrated that NMD inhibition improved the stability and response to correctors of W1282X-CFTR protein expressed in the Flp-In-293 cells. CONCLUSION: These results show that W1282X can cause substantial degradation of CFTR mRNA that has to be addressed before efforts aimed at augmenting CFTR protein function can be effective.
Authors: Steven Erwood; Onofrio Laselva; Teija M I Bily; Reid A Brewer; Alexandra H Rutherford; Christine E Bear; Evgueni A Ivakine Journal: Mol Ther Methods Clin Dev Date: 2020-05-12 Impact factor: 6.698
Authors: Puay-Wah Phuan; Joseph-Anthony Tan; Amber A Rivera; Lorna Zlock; Dennis W Nielson; Walter E Finkbeiner; Peter M Haggie; Alan S Verkman Journal: Sci Rep Date: 2019-11-27 Impact factor: 4.379
Authors: Jacelyn E Peabody Lever; Venkateshwar Mutyam; Heather Y Hathorne; Ning Peng; Jyoti Sharma; Lloyd J Edwards; Steven M Rowe Journal: Pediatr Pulmonol Date: 2020-04-13
Authors: Anya T Joynt; Taylor A Evans; Matthew J Pellicore; Emily F Davis-Marcisak; Melis A Aksit; Alice C Eastman; Shivani U Patel; Kathleen C Paul; Derek L Osorio; Alyssa D Bowling; Calvin U Cotton; Karen S Raraigh; Natalie E West; Christian A Merlo; Garry R Cutting; Neeraj Sharma Journal: PLoS Genet Date: 2020-10-21 Impact factor: 5.917