In vivo analysis of trafficking and functional impact of hERG pore-domain missense variants in a CRISPR/Cas9-engineered C. elegans model of long QT syndrome

BACKGROUND: Loss-of-function variants in KCNH2, which encodes hERG, are responsible for long QT syndrome type 2 (LQT2), a major cause of sudden cardiac death. Most hERG pore-domain missense variants are associated with severe LQT2. However, functional heterogeneity exists among these variants, with a small subset associated with milder phenotypes. Only a limited number of these atypical variants have been functionally characterized. Caenorhabditis elegans expresses UNC-103, a voltage-gated K OBJECTIVE: Development of a C. elegans model for in vivo characterization of hERG pore-domain missense variants. METHODS: Using CRISPR/Cas9 genome editing, we generated fluorescent UNC-103 knock-in strains. We established an approach combining fluorescence in vivo imaging to assess channel trafficking efficiency, with behavioral readouts of muscle hyperexcitability to evaluate the integrated functional impact of hERG variants, encompassing defects in trafficking, gating, and potassium permeability. RESULTS: The model faithfully recapitulated the profiles of G628S and G604S, two well-characterized hERG variants associated with severe LQT2. Moreover, it enabled in vivo characterization of G603S, a recently identified atypical pore-domain variant associated with a mild LQT2 phenotype in heterozygous carriers and a severe long QT syndrome in homozygous state. G603S exhibited a trafficking defect responsible for a partial loss-of-function. CONCLUSION: This CRISPR/Cas9-engineered C. elegans model complements in vitro assays and complex transgenic animals, enabling in vivo characterization of hERG pore-domain missense variants.