High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel KCNH2: Deep mutational scan of KCNH2 trafficking

Background KCHN2 encodes the K_V11.1 potassium channel responsible for I_Kr, a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that decrease I_Kr can 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-synonymous KCNH2 variants preclude the use of conventional electrophysiologic methods for functional study.

Objective To report a high-throughput, multiplexed screening method for KCNH2 genetic variants capable of measuring the cell surface abundance of hundreds of missense variants in KCNH2.

Methods We develop a method to quantitate KCNH2 variant trafficking on a pilot region of 11 residues in the S5 helix, and generate trafficking scores for 220/231 missense variants in this region.

Results For 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 produce I_Kr, but that some variants which traffic normally may still be functionally compromised.

Conclusions Here, we describe a new method for detecting trafficking-deficient variants in KCNH2 in a multiplexed assay. This new method accurately generates trafficking data for variants in KCNH2 and can be readily extended to all residues in Kv11.1 and to other cell surface proteins.

CLINICAL IMPLICATIONS Hundreds of KCNH2 variants have been observed to date, and thousands more will be found as clinical and population sequencing efforts become increasingly widespread. The major mechanism of K_V11.1 loss of function is misfolding and failure to traffic to the cell surface. Deep mutational scanning of KCNH2 trafficking is a scalable, high-throughput method that can help identify new loss of function variants and decipher the large number of KCNH2 variants being found in the population.