High-throughput discovery of trafficking-deficient variants in the cardiac potassium channelKCNH2: Deep mutational scan ofKCNH2trafficking

BackgroundKCHN2encodes the KV11.1 potassium channel responsible forIKr, a major repolarization current during the cardiomyocyte action potential. Variants inKCNH2that decreaseIKrcan cause Type 2 Long QT syndrome, usually due to mistrafficking to the cell surface. Accurately discriminating between variants with normal and abnormal trafficking would help clinicians identify and treat individuals at risk of a major cardiac event. The volume of reported non-synonymousKCNH2variants preclude the use of conventional electrophysiologic methods for functional study.ObjectiveTo report a high-throughput, multiplexed screening method forKCNH2genetic variants capable of measuring the cell surface abundance of hundreds of missense variants inKCNH2.MethodsWe develop a method to quantitateKCNH2variant trafficking on a pilot region of 11 residues in the S5 helix, and generate trafficking scores for 220/231 missense variants in this region.ResultsFor 5/5 variants, high-throughput trafficking scores validated when tested in single variant flow cytometry and confocal microscopy experiments. We additionally compare our results with planar patch electrophysiology and find that loss-of-trafficking variants do not produceIKr, but that some variants which traffic normally may still be functionally compromised.ConclusionsHere, we describe a new method for detecting trafficking-deficient variants inKCNH2in a multiplexed assay. This new method accurately generates trafficking data for variants inKCNH2and can be readily extended to all residues in Kv11.1 and to other cell surface proteins.CLINICAL IMPLICATIONSHundreds ofKCNH2variants have been observed to date, and thousands more will be found as clinical and population sequencing efforts become increasingly widespread. The major mechanism of KV11.1 loss of function is misfolding and failure to traffic to the cell surface. Deep mutational scanning ofKCNH2trafficking is a scalable, high-throughput method that can help identify new loss of function variants and decipher the large number ofKCNH2variants being found in the population.