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

Figures

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  Figure 1. Pedigrees of index patients depicting genotype and phenotype. The KCNQ1 variant is pathogenic (class 5), the others are of unknown significance (VUS). Phenotype is based on QTc, ECG morphology, symptoms and, if available, Holter measurements and exercise test. A clinical overview can be found in Supplementary Material, Table S1. AF: atrial fibrillation, AKAP9: A-kinase anchoring protein 9, AV: aortic valve, BAV: bicuspid aortic valve, bp: blood pressure, bpm: beats per minute, BrS: Brugada syndrome, CACNA1C: calcium voltage-gated channel subunit alpha1 C, HF: heart failure, ICD: implantable cardioverter defibrillator, KCNH2: potassium voltage-gated channel subfamily h member 2, KCNQ1: potassium voltage-gated channel subfamily Q member 1, LVH: left ventricular hypertrophy, OHCA: out-of-hospital cardiac arrest, PKP2: plakophilin-2, QTc: QT interval corrected for heart rate, RYR2: ryanodine receptor 2, SCD: sudden cardiac death, TdP: Torsade de Pointes.

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  Figure 2. Representative ECGs of index patients carrying the KCNH2-p. (Ser906Leu) variant. (A) 12-Lead ECG from the index of family C, showing a flattened ST-segment in lead I, V4 and V5, and QT prolongation (QTc 470). (B) Holter recording of index E during a TdP episode. (C) Holter recording of index E showing QT prolongation (QTc 534).

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  Figure 3. Location of amino acid substitution resulting from the genetic variant in KCNH2. (A) Conservation of the serine amino acid located at position 906 (bold) in KCNH2 among animal species. (_:non-identical amino acids). (B) Localization of amino acid substitute in the KCNH2 protein. The variant is located at the distal end of the c-terminus, distal from the cyclic nucleotide binding domain (CNBD).

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  Figure 4. Current density and activation gating. (A) Representative traces of HEK293A cells expressing wild-type (WT) KCNH2, KCNH2-p.S906L heterozygous (S906L Hz) and homozygous (S906L Hm). Inset, double-pulse protocol used. (B) Average density of steady-state current. (C) Average density of peak tail current. (D) Voltage dependency of activation. (E) Speed of activation. (F and H) Half maximal activation (V1/2), and slope factor (k), determined by Boltzmann fit through activation curves. Statistics by two-way ANOVA-RM, or one-way ANOVA, and Bonferroni’s correction for multiple testing. *; P ≤ 0.05, **; P ≤ 0.01. WT; wild type, Hz; heterozygous, Hm; homozygous.

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  Figure 5. Variant interpretation using calibrated automated patch clamp assay: (A) The -50 and -120mV tail current traces corresponding to the highlighted region within the voltage protocol for WT and heterozygous p.Ser906Leu. Peak tail current and current amplitude 500ms after peak were indicated by * and #, respectively. (B) Data distribution of normalized current density (sqrt) by WT from the same plate for all the benign controls (n=1027) from Jiang et al., (2022), p.Ser906Leu (n=73 from 4 replicates) and negative control (n=66). (C) Data distribution of normalized deactivation ratio for all the benign controls (n=919) and p.Ser906Leu (n=61 from 4 replicates). (D) Data distribution of normalized inactivation ratio for all the benign controls (n=859) and p.Ser906Leu (n=50 from 4 replicates). (E) Data distribution of shift in the V0.5 relative from WT for all the benign controls (n=996) and p.Ser906Leu (n=67 from 4 replicates). (F) p.Ser906Leu is indeterminate for the current density as the confidence interval (CI) is spreading between functionally normal (blue region) and abnormal range. (G) p.Ser906Leu is indeterminate for shift in activation as the CI spreads between functionally…