Abstract 15875: Zebrafish as a High-Throughput System for Long QT Syndrome
Introduction: Since the advent of commercial Long QT Syndrome (LQTS) screening, majority of novel hERG (KCNH2) mutations are not functionally characterized. A high-throughput system for functional characterization of LQTS mutations is needed to distinguish between disease-causing and normal variants. Previously, we validated the zebrafish as model of human LQT2. Here, we test the validity of an in vivo embryonic zebrafish cardiac assay to determine functional consequences of human LQT2 mutations.
Methods: Kcnh2 is the sole repolarizing ion channel in embryonic zebrafish ventricle. Injection of kcnh2 morpholino (MO) into fertilized embryos at 1-2 cell stage results in abnormal ventricular repolarization in ~90% of embryos. Normal repolarization is restored when MO is co-injected with WT hERG cRNA. Functional consequences of LQT2 mutations were defined by restoration of normal repolarization similar to WT hERG. Fertilized embryos were injected with MO, MO+WT hERG or MO+mutant hERG. Cardiac repolarization in embryonic heart was assessed in embryonic heart as normal (1:1 AV conduction) or abnormal (2:1 AV block or ventricular asystole).
Results: A variant was classified as “disease-causing” if the ability to restore normal repolarization was below 95% confidence interval (CI) for WT (dotted blue lines). Of 8 hERG variants, 6 resulted in a spectrum of rescued- repolarization below CI, consistent with previous designations as disease-causing in HEK heterologous system. R56Q and P347S (a common polymorphism) rescued repolarization similar to WT. There was a linear fit between fraction of WT normal phenotype (zebrafish assay) and fraction of WT current (HEK assay), slope 0.9, R 0.92.
Conclusion: This study supports the utility of a zebrafish cardiac assay to assess functional consequences of LQT2 mutations. Functional characterization of novel LQTS mutations will allow clinician to discern between disease-causing variant vs. “variant of unknown significance.”
- © 2011 by American Heart Association, Inc.