Multiplexed Assays of Variant Effect and Automated Patch Clamping Improve
KCNH2
-LQTS Variant Classification and Cardiac Event Risk Stratification
Authors: O’Neill Matthew J. , Ng Chai-Ann , Aizawa Takanori , Sala Luca , Bains Sahej , Winbo Annika , Ullah Rizwan , Shen Qianyi , Tan Chek-Ying , Kozek Krystian , Vanags Loren R. , Mitchell Devyn W. , Shen Alex , Wada Yuko , Kashiwa Asami , Crotti Lia , Dagradi Federica , Musu Giulia , Spazzolini Carla , Neves Raquel , Bos J. Martijn , Giudicessi John R. , Bledsoe Xavier , Gamazon Eric R. , Lancaster Megan C. , Glazer Andrew M. , Knollmann Bjorn C. , Roden Dan M. , Weile Jochen , Roth Frederick , Salem Joe-Elie , Earle Nikki , Stiles Rachael , Agee Taylor , Johnson Christopher N. , Horie Minoru , Skinner Jonathan R. , Ackerman Michael J. , Schwartz Peter J. , Ohno Seiko , Vandenberg Jamie I. , Kroncke Brett M. 
Published: September 2024

BACKGROUND:

Long QT syndrome is a lethal arrhythmia syndrome, frequently caused by rare loss-of-function variants in the potassium channel encoded by
KCNH2
. Variant classification is difficult, often because of lack of functional data. Moreover, variant-based risk stratification is also complicated by heterogenous clinical data and incomplete penetrance. Here we sought to test whether variant-specific information, primarily from high-throughput functional assays, could improve both classification and cardiac event risk stratification in a large, harmonized cohort of
KCNH2
missense variant heterozygotes.

METHODS:

We quantified cell-surface trafficking of 18 796 variants in
KCNH2
using a multiplexed assay of variant effect (MAVE). We recorded
KCNH2
current density for 533 variants by automated patch clamping. We calibrated the strength of evidence of MAVE data according to ClinGen guidelines. We deeply phenotyped 1458 patients with
KCNH2
missense variants, including QTc, cardiac event history, and mortality. We correlated variant functional data and Bayesian long QT syndrome penetrance estimates with cohort phenotypes and assessed hazard ratios for cardiac events.

RESULTS:
Variant MAVE trafficking scores and automated patch clamping peak tail currents were highly correlated (Spearman rank-order ρ=0.69; n=433). The MAVE data were found to provide up to pathogenic very strong evidence for severe loss-of-function variants. In the cohort, both functional assays and Bayesian long QT syndrome penetrance estimates were significantly predictive of cardiac events when independently modeled with patient sex and corrected QT interval (QTc); however, MAVE data became nonsignificant when peak tail current and penetrance estimates were also available. The area under the receiver operator characteristic curve for 20-year event outcomes based on patient-specific sex and QTc (area under the curve, 0.80 [0.76–0.83]) was improved with prospectively available penetrance scores conditioned on MAVE (area under the curve, 0.86 [0.83–0.89]) or attainable automated patch clamping peak tail current data (area under the curve, 0.84 [0.81–0.88]).

CONCLUSIONS:

High-throughput
KCNH2
variant MAVE data meaningfully contribute to variant classification at scale, whereas long QT syndrome penetrance estimates and automated patch clamping peak tail current measurements meaningfully contribute to risk stratification of cardiac events in patients with heterozygous
KCNH2
missense variants.