Decreased mRNA and protein stability of W1282X limits response to modulator therapy

M. A. Aksit, A. D. Bowling, T. A. Evans, A. T. Joynt, D. Osorio, S. Patel, Natalie E West, Christian Merlo, Patrick Ryan Sosnay, Garry R Cutting, Neeraj Sharma

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
JournalJournal of Cystic Fibrosis
DOIs
StatePublished - Jan 1 2019

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Protein Stability
RNA Stability
Nose
Cystic Fibrosis Transmembrane Conductance Regulator
RNA
Epithelial Cells
Nonsense Mediated mRNA Decay
Parents
Therapeutics
Fathers
Mothers
Polymerase Chain Reaction
Messenger RNA
Proteins

ASJC Scopus subject areas

  • Pediatrics, Perinatology, and Child Health
  • Pulmonary and Respiratory Medicine

Cite this

Decreased mRNA and protein stability of W1282X limits response to modulator therapy. / Aksit, M. A.; Bowling, A. D.; Evans, T. A.; Joynt, A. T.; Osorio, D.; Patel, S.; West, Natalie E; Merlo, Christian; Sosnay, Patrick Ryan; Cutting, Garry R; Sharma, Neeraj.

In: Journal of Cystic Fibrosis, 01.01.2019.

Research output: Contribution to journalArticle

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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.",
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T1 - Decreased mRNA and protein stability of W1282X limits response to modulator therapy

AU - Aksit, M. A.

AU - Bowling, A. D.

AU - Evans, T. A.

AU - Joynt, A. T.

AU - Osorio, D.

AU - Patel, S.

AU - West, Natalie E

AU - Merlo, Christian

AU - Sosnay, Patrick Ryan

AU - Cutting, Garry R

AU - Sharma, Neeraj

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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.

AB - 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.

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DO - 10.1016/j.jcf.2019.02.009

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JO - Journal of Cystic Fibrosis

JF - Journal of Cystic Fibrosis

SN - 1569-1993

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