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
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.