A novel mutation in KCNQ1 associated with a potent dominant negative effect as the basis for the LQT1 form of the long QT syndrome

Abstract

Introduction: Long QT Syndrome (LQTS) is an inherited disorder characterized by prolonged QT intervals and life-threatening polymorphic ventricular tachyarrhythmias. LQT1 caused by KCNQ1 mutations is the most common form of LQTS.

Methods and results: Patients diagnosed with LQTS were screened for disease-associated mutations in KCNQ1, KCNH2, KCNE1, KCNE2, KCNJ2, and SCN5A. A novel mutation was identified in KCNQ1 caused by a three-base deletion at the position 824-826, predicting a deletion of phenylalanine at codon 275 in segment 5 of KCNQ1 (DeltaF275). Wild-type (WT) and DeltaF275-KCNQ1 constructs were generated and transiently transfected together with a KCNE1 construct in CHO-K1 cells to characterize the properties of the slowly activating delayed rectifier current (IKs) using conventional whole-cell patch-clamp techniques. Cells transfected with WT-KCNQ1 and KCNE1 (1:1.3 molar ratio) produced slowly activating outward current with the characteristics of IKs. Tail current density measured at -40 mV following a two-second step to +60 mV was 381.3 +/- 62.6 pA/pF (n = 11). Cells transfected with DeltaF275-KCNQ1 and KCNE1 exhibited essentially no current. (Tail current density: 0.8 +/- 2.1 pA/pF, n = 11, P = 0.00001 vs WT). Cotransfection of WT- and DeltaF275- KCNQ1 (50/50), along with KCNE1, produced little to no current (tail current density: 10.3 +/- 3.5 pA/pF, n = 11, P = 0.00001 vs WT alone), suggesting a potent dominant negative effect. Immunohistochemistry showed normal membrane trafficking of DeltaF275-KCNQ1.

Conclusion: Our data suggest that a DeltaF275 mutation in KCNQ1 is associated with a very potent dominant negative effect leading to an almost complete loss of function of IKs and that this defect underlies a LQT1 form of LQTS.

Figure 1. Electrocardiograms of the patient

A. 12-Lead ECG of the patient at rest. Broad-based tall T waves are observed. QTc interval is 515 ms at rest. B. Torsade de Pointes arrhythmias induced following administration of epinephrine (0.3mg/kg/min). Her QTc interval prolonged to 620 ms.

Figure 1. Electrocardiograms of the patient

A. 12-Lead ECG of the patient at rest. Broad-based tall T waves are observed. QTc interval is 515 ms at rest. B. Torsade de Pointes arrhythmias induced following administration of epinephrine (0.3mg/kg/min). Her QTc interval prolonged to 620 ms.

Figure 2. Δ275F mutant at KCNQ

A. Chromatogram showing a heterozygous deletion at the position of 824–826. B. Amino acid sequence alignment showing conservation of Phenylalanine 275 in multiple species C. Schematic topology of KCNQ1 and KCNE1 proteins forming I_Ks. Δ275F mutation was located at the S5 segment of KCNQ1.

Figure 3. Representative current trace of WT- and/or Δ275F-KCNQ1 expressed in CHO-K1 cells

Cells of each panel were transfected as follows: A. 0.75 μg of WT-KCNQ1 and 0.75 μg of KCNE1. B. 0.375 μg of WT-KCNQ1 and 0.75 μg of KCNE1. C. 0.5 μg of WT-KCNQ1, 0.25 μg of Δ275F-KCNQ1 and 0.75μg of KCNE1. D. 0.375μg of WT-KCNQ1, 0.375 μg of Δ275F-KCNQ1 and 0.75μg of KCNE1. E. 0.75μg of Δ275F-KCNQ1 and 0.75μg of KCNE1. Pulse protocol is shown in the inset in the lower right.

Figure 4. Current-voltage relationships of expressed currents

A. Current-Voltage relationship measured at the peak current during the test depolarization pulse. B. Current-Voltage relationship measured of the tail current upon repolarization to −40mV following test depolarization. C. Bar graphs showing current densities of developing (peak) recorded current at +60mV. D. Bar graphs showing current densities of tail current recorded upon repolarization to −40mV from +60mV test depolarization.

Figure 5. Protein localization of WT- and/or mutant-KCNQ1 in CHO-K1 cells visualized by immunohistochemistry

Figure shows 0.15μm optical sections from the center of CHO-K1 cells transfected with WT- KCNQ1 (B), ΔF275-KCNQ1 (C) or ΔF275-KCNQ1 + WT (D). A brighter fluorescence intensity near the plasma membrane reveals positive immunostaining of the KCNQ1 channel. Panel A shows the lack of any fluorescence signal from non transfected cells.