Abstract
Purpose: Nonsense mutation readthrough is used as a gene-specific treatment in some genetic diseases. The response to readthrough treatment is determined by the readthrough efficiency of various nonsense mutations. In this manuscript, we aimed to explore the harmful effects of nonsense mutation suppression.
Methods: HEK293 cells were transfected with two SCN5A (encode cardiac Na+ channel) nonsense mutations, p.R1623X and p.S1812X. We applied two readthrough-enhancing methods (either aminoglycosides or a siRNA-targeting eukaryotic release factor eRF3a (a GTPase that binds eRF1)) to suppress these SCN5A nonsense mutations. When either of readthrough methods was used, the sodium channel proteins were examined by western blot and immunoblotting and recorded by whole cell patch-clamp to observe the functional characterization of the restored channels. Results: Upon readthrough treatment, the sodium currents were restored to the mutant cDNAs. These mutations reduced full-length sodium channel protein levels, and the sodium currents were reduced to 3% of wild-type. The mutant cDNA sodium currents were increased to 30% of wild-type, and the fulllength proteins also increased. However, the functional characterization of these channels from cDNAs carrying p.R1623X and p.S1812X exhibited abnormal biophysical properties, including a negative shift in steady-state sodium channel inactivation, a positive shift in sodium channel activation and robust late sodium currents. The ramp test showed prolonged QT intervals. Conclusion: These results demonstrated that readthrough-enhancing methods effectively suppressed nonsense mutations in SCN5A and restored the expression of full-length channels. However, the restored channels may increase the risk of arrhythmia.Keywords: Eukaryotic release factors, gentamycin, premature termination codon, readthrough, SCN5A, siRNA.
Current Gene Therapy
Title:Readthrough of SCN5A Nonsense Mutations p.R1623X and p.S1812X Questions Gene-therapy in Brugada Syndrome
Volume: 17 Issue: 1
Author(s): Siyong Teng, Jian Huang, Zhan Gao, Jie Hao, Yuejin Yang, Shu Zhang, Jielin Pu, Rutai Hui, Yongjian Wu*Zheng Fan*
Affiliation:
- State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100037, People's,China
- Department of Physiology, University of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163,United States
Keywords: Eukaryotic release factors, gentamycin, premature termination codon, readthrough, SCN5A, siRNA.
Abstract: Purpose: Nonsense mutation readthrough is used as a gene-specific treatment in some genetic diseases. The response to readthrough treatment is determined by the readthrough efficiency of various nonsense mutations. In this manuscript, we aimed to explore the harmful effects of nonsense mutation suppression.
Methods: HEK293 cells were transfected with two SCN5A (encode cardiac Na+ channel) nonsense mutations, p.R1623X and p.S1812X. We applied two readthrough-enhancing methods (either aminoglycosides or a siRNA-targeting eukaryotic release factor eRF3a (a GTPase that binds eRF1)) to suppress these SCN5A nonsense mutations. When either of readthrough methods was used, the sodium channel proteins were examined by western blot and immunoblotting and recorded by whole cell patch-clamp to observe the functional characterization of the restored channels. Results: Upon readthrough treatment, the sodium currents were restored to the mutant cDNAs. These mutations reduced full-length sodium channel protein levels, and the sodium currents were reduced to 3% of wild-type. The mutant cDNA sodium currents were increased to 30% of wild-type, and the fulllength proteins also increased. However, the functional characterization of these channels from cDNAs carrying p.R1623X and p.S1812X exhibited abnormal biophysical properties, including a negative shift in steady-state sodium channel inactivation, a positive shift in sodium channel activation and robust late sodium currents. The ramp test showed prolonged QT intervals. Conclusion: These results demonstrated that readthrough-enhancing methods effectively suppressed nonsense mutations in SCN5A and restored the expression of full-length channels. However, the restored channels may increase the risk of arrhythmia.Export Options
About this article
Cite this article as:
Teng Siyong, Huang Jian, Gao Zhan, Hao Jie, Yang Yuejin, Zhang Shu, Pu Jielin, Hui Rutai, Wu Yongjian*, Fan Zheng*, Readthrough of SCN5A Nonsense Mutations p.R1623X and p.S1812X Questions Gene-therapy in Brugada Syndrome, Current Gene Therapy 2017; 17 (1) . https://dx.doi.org/10.2174/1566523217666170529074758
DOI https://dx.doi.org/10.2174/1566523217666170529074758 |
Print ISSN 1566-5232 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5631 |
Call for Papers in Thematic Issues
Programmed Cell Death Genes in Oncology: Pioneering Therapeutic and Diagnostic Frontiers (BMS-CGT-2024-HT-45)
Programmed Cell Death (PCD) is recognized as a pivotal biological mechanism with far-reaching effects in the realm of cancer therapy. This complex process encompasses a variety of cell death modalities, including apoptosis, autophagic cell death, pyroptosis, and ferroptosis, each of which contributes to the intricate landscape of cancer development and ...read more

- Author Guidelines
- Bentham Author Support Services (BASS)
- Graphical Abstracts
- Fabricating and Stating False Information
- Research Misconduct
- Post Publication Discussions and Corrections
- Publishing Ethics and Rectitude
- Increase Visibility of Your Article
- Archiving Policies
- Peer Review Workflow
- Order Your Article Before Print
- Promote Your Article
- Manuscript Transfer Facility
- Editorial Policies
- Allegations from Whistleblowers
- Announcements
Related Articles
-
Cardiovascular-Active Venom Toxins: An Overview
Current Medicinal Chemistry Cardiovascular Effects of EGFR (Epidermal Growth Factor Receptor) Monoclonal Antibodies
Cardiovascular & Hematological Agents in Medicinal Chemistry Hypomagnesaemia/Hypokalemia Associated with the Use of Esomeprazole
Current Drug Safety Severe Preeclampsia
Current Women`s Health Reviews The Role of Minocycline in Ischemia-Reperfusion Injury: A Comprehensive Review of an Old Drug with New Implications
Recent Patents on Cardiovascular Drug Discovery Genetics and Heart Failure: A Concise Guide for the Clinician
Current Cardiology Reviews Induction of Propranolol Metabolism by Ginkgo biloba Extract EGb 761 in Rats
Current Drug Metabolism Blockade of Renin Angiotensin System in Heart Failure Post-Myocardial Infarction: What is the Best Therapy?
Recent Patents on Cardiovascular Drug Discovery Isoform-Selective PI3K Inhibitors for Various Diseases
Current Topics in Medicinal Chemistry Inhibition of Voltage-Gated Calcium Channels by RGK Proteins
Current Molecular Pharmacology Evolution of the Human Ion Channel Set
Combinatorial Chemistry & High Throughput Screening Benzodiazepine Metabolism: An Analytical Perspective
Current Drug Metabolism Endocrine Orchestration of Cardiovascular, Gastrointestinal and Hypothalamic Control
Current Medicinal Chemistry Chinese Medicinal Herbs as Source of Antioxidant Compounds – Where Tradition Meets the Future
Current Medicinal Chemistry Chronic Latent Magnesium Deficiency in Obesity Decreases Positive Effects of Vitamin D on Cardiometabolic Risk Indicators
Current Vascular Pharmacology Renin Angiotensin System as a Regulator of Cell Volume. Implications to Myocardial Ischemia
Current Cardiology Reviews Heart Failure Models: Traditional and Novel Therapy
Current Vascular Pharmacology CD36 as a Therapeutic Target for Endothelial Dysfunction in Stroke
Current Pharmaceutical Design Functional Properties of Pentacyclic Triterpenes Contained in "Orujo" Olive Oil
Current Nutrition & Food Science Heart Failure in Chronic Myocarditis: A Role for microRNAs?
Current Genomics