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 potassium channel orthologous to hERG, with high conservation in sequence and channel biogenesis regulators, supporting its use as an in vivo model to investigate hERG variants.

OBJECTIVE

This study aimed to develop 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 knockin 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, 2 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 the 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.