Reclassification of a likely pathogenic Dutch founder variant in KCNH2; implications of reduced penetrance

Abstract

Background Variants in KCNH2, encoding the hERG channel which is responsible for the rapid component of the cardiac delayed rectifier K+ current (IKr), are causal to Long QT Syndrome type 2 (LQTS2). We identified eight index patients with a new variant of unknown significance (VUS), KCNH2:c.2717C > T:p.(Ser906Leu). We aimed to elucidate the biophysiological effect of this variant, to enable reclassification and consequent clinical decision-making. Methods A genotype–phenotype overview of the patients and relatives was created. The biophysiological effects were assessed independently by manual and automated calibrated patch-clamp. HEK293a cells expressing: (I) wild type (WT) KCNH2, (II) KCNH2-p.S906L alone (homozygous, Hm) or (III) KCNH2-p.S906L in combination with WT (1:1) (heterozygous, Hz), were used for manual patching. Automated patch-clamp measured the variants function against known benign and pathogenic variants, using Flp-In T-rex HEK293 KCNH2-variant cell lines. Results Incomplete penetrance of LQTS2 in KCNH2:p.(Ser906Leu) carriers was observed. In addition, some patients were heterozygous for other VUSs in CACNA1C, PKP2, RYR2, or AKAP9. The phenotype of carriers of KCNH2:p.(Ser906Leu) ranged from asymptomatic to life-threatening arrhythmic events. Manual patch-clamp showed a reduced current density by 69.8%, and 60.4% in KCNH2-p.S906L-Hm and KCNH2-p.S906L-Hz, respectively. The time constant of activation was significantly increased with 80.1% in KCNH2-p.S906L-Hm compared to KCNH2-WT. Assessment of KCNH2-p.S906L-Hz, by calibrated automatic patch-clamp assay showed a reduction in current density by 35.6%. Conclusion The reduced current density in the KCNH2-p.S906L-Hz indicates a moderate loss-of-function. Combined with the reduced penetrance and variable phenotype, we conclude that KCNH2:p.(Ser906Leu) is a low penetrant likely pathogenic variant for LQTS2.

The Action Potential Clamp Technique as a Tool for Risk Stratification of Sinus Bradycardia Due to Loss-of-Function Mutations in HCN4: An In Silico Exploration Based on In Vitro and In Vivo Data

“…However, mammalian cell lines in which the wild-type and the variant cDNA of a given ion channel can be expressed are still widely used to study the functional properties of ion currents [ 37 , 38 ]. Initially, patch clamp experiments were performed manually, but in recent years, the pathogenicity of SCN5A , KCNQ1 , and KCNH2 variants involved in the inherited cardiac channelopathies Brugada syndrome and long QT syndrome types 1 and 2, respectively, have also been determined using high-throughput reclassification assays based on automated patch clamp devices [ 25 , 39 , 40 , 41 , 42 , 43 , 44 ]. Various biophysical parameters of the membrane currents have been used to classify the risk of genetic variants, but current density is the most reliable marker for predicting the risk of SCN5A [ 45 ], KCNQ1 [ 39 ], and KCNH2 [ 46 ] ion channel variants, followed by their voltage dependence of activation (through their half-activation voltage, V ½ ).…”