Functional characterization and allele-specific RNA interference-based rescue of KCNH2 p.F68C variant associated with long QT syndrome

Long QT syndrome (LQTS) is an inherited life-threatening cardiac disorder characterized by delayed ventricular repolarization and increased risk of malignant arrhythmias. Among its subtypes, long QT syndrome type 2 (LQT2) is primarily caused by pathogenic variants in KCNH2 , which encodes the human ether-à-go-go–related gene ( hERG ) potassium channel responsible for the rapid delayed rectifier current ( I Kr ). However, the substantial functional heterogeneity among KCNH2 variants poses a major challenge for clinical interpretation and precision intervention. In this study, we sought to functionally characterize KCNH2 p.F68C variant (c.203T > G) identified in a Chinese LQT2 patient and to evaluate the feasibility of RNA interference–based modulation of its functional impact on the hERG channel. Using biochemical and electrophysiological analyses in HEK293T cells, we show that variant p.F68C causes a severe trafficking defect and exerts a dominant-negative effect on wild-type hERG channels, leading to markedly reduced rapid delayed rectifier potassium current ( I Kr ). In contrast to several previously reported Per–Arnt–Sim (PAS) domain variants, the trafficking defect of p.F68C was resistant to reduced culture temperature, chemical chaperones, and pharmacological chaperones. Notably, allele-specific RNA interference selectively suppressed mutant hERG expression, alleviated dominant-negative interference, and partially restored hERG current density without detectable cytotoxicity. Together, these findings establish p.F68C as a loss-of-function KCNH2 variant and highlight allele-specific RNA interference as a variant-directed strategy that may serve as an alternative to suppression–replacement approaches, providing a basis for functional interpretation and precision therapeutic exploration of individual KCNH2 variants.