Molecular mechanisms of arrhythmogenesis in a loss of function KCND3-mutation linked to clinical diagnosis of long-QT-syndrome and aborted SCD

A.-K. Rahm (Heidelberg)¹, M. E. Müller (Heidelberg)¹, E. Kayvanpour (Heidelberg)¹, F. Sedaghat-Hamedani (Heidelberg)¹, J. Haas (Heidelberg)¹, K. Streckfuß-Bömeke (Würzburg)², X. Zhou (Mannheim)³, I. Akin (Mannheim)³, J. Heijman (Maastricht)⁴, X. Wan (Columbus)⁵, I. Deschenes (Columbus)⁵, M. P. Penazola Amnion (Karlsruhe)⁶, W. Wenzel (Karlsruhe)⁶, N. Ullrich (Bern)⁷, C. Afting (Heidelberg)⁸, J. Wittbrodt (Heidelberg)⁸, D. Gramlich (Stuttgart)⁹, P. Schweizer (Heidelberg)¹, P. Lugenbiel (Heidelberg)¹, N. Frey (Heidelberg)¹, B. Meder (Heidelberg)¹, D. Thomas (Heidelberg)^1
1Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland; ²Universitätsklinikum Würzburg Institut für Pharmakologie und Toxikologie Würzburg, Deutschland; ³Universitätsklinikum Mannheim GmbH I. Medizinische Klinik Mannheim, Deutschland; ⁴Maastricht University Cardiovascular Research Institute Maastricht, Niederlande; ⁵The Ohio State University, Frick Center for Heart Failure and Arrhythmias, Davis Heart and Lung Research Institute Department of Physiology and Cell Biology Columbus, USA; ⁶Karlsruher Institut für Technologie (KIT) Institut für Biomedizinische Technik Karlsruhe, Deutschland; ⁷Universität Bern Institut für Physiologie Bern, Schweiz; ⁸Heidelberg University Centre for Organismal Studies (COS) Heidelberg, Deutschland; ⁹Robert-Bosch-Krankenhaus Kardiologie und Angiologie Stuttgart, Deutschland

Background
Sixteen genes encoding for cardiac ion channels and associated proteins have been linked to long-QT-syndrome (LQTS) in humans. So far, no pathogenic variant in the KCND3 gene coding for K_v4.3, the main component of the transient-outward potassium current (I_to), has been described in LQTS. 

Purpose
This study presents a novel KCND3 variant in a patient with aborted sudden cardiac death with LQTS as the most likely clinical diagnosis and aimed to identify the underlying genetic and biophysical basis for arrhythmogenesis.

Methods
The index-patient and index-family were examined by electrocardiography, echocardiography and cardiac magnetic resonance imaging. Whole-genome sequencing identified a novel  KCND3-C1469G variant, which was generated by in-vitro mutagenesis and heterologously expressed in Xenopus laevis oocytes and CHO cells for studies using dual-electrode voltage clamp, patch-clamp, western blot and immunofluorescence staining. Current measurements in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) of the index-patient were compared to a genotype-negative family member. 

Results
QTc prolongation and T wave alterans led to the clinical diagnosis of LQTS in the index-patient. Brugada syndrome and cardiac structural abnormalities were ruled out. The KCND3-C1469G (rs1256867747) variant resulted in a serine-to-cysteine substitution at position 490 in the K_v4.3 C-terminus. Heterologous expression of K_v4.3-S490C revealed reduced I_to amplitude and a change in K_v4.3 channel inactivation kinetics. A comparison of protein expression levels between wildtype and mutant samples isolated from injected Xenopus laevis oocytes showed no significant difference. We propose the formation of a new disulfide bond as potential biophysical mechanism, as in-vitro mutagenesis of serine-457 partially restored current amplitudes. Reduced I_to amplitudes, identified as a specific electrophysiological alteration underlying LQTS, were confirmed in hiPSC-CM derived from the index-patient compared to a genotype-negative family member.

Conclusion
The novel KCND3-C1469G (rs1256867747) variant leads to reduced I_to amplitude and is linked to LQTS and aborted sudden cardiac death. Further studies are needed to identify the arrhythmogenic mechanism in more detail.