Allelic Complexity in Long QT Syndrome: A Family-Case Study

Abstract

Congenital long QT syndrome (LQTS) is associated with high genetic and allelic heterogeneity. In some cases, more than one genetic variant is identified in the same (compound heterozygosity) or different (digenic heterozygosity) genes, and subjects with multiple pathogenic mutations may have a more severe disease. Standard-of-care clinical genetic testing for this and other arrhythmia susceptibility syndromes improves the identification of complex genotypes. Therefore, it is important to distinguish between pathogenic mutations and benign rare variants. We identified four genetic variants (KCNQ1-p.R583H, KCNH2-p.C108Y, KCNH2-p.K897T, and KCNE1-p.G38S) in an LQTS family. On the basis of in silico analysis, clinical data from our family, and the evidence from previous studies, we analyzed two mutated channels, KCNQ1-p.R583H and KCNH2-p.C108Y, using the whole-cell patch clamp technique. We found that KCNQ1-p.R583H was not associated with a severe functional impairment, whereas KCNH2-p.C108Y, a novel variant, encoded a non-functional channel that exerts dominant-negative effects on the wild-type. Notably, the common variants KCNH2-p.K897T and KCNE1-p.G38S were previously reported to produce more severe phenotypes when combined with disease-causing alleles. Our results indicate that the novel KCNH2-C108Y variant can be a pathogenic LQTS mutation, whereas KCNQ1-p.R583H, KCNH2-p.K897T, and KCNE1-p.G38S could be LQTS modifiers.

Keywords: HERG; KCNH2; KCNQ1; cardiac arrhythmias; electrophysiology; long-QT syndrome; potassium channels.

Figure 1

Pedigree and electrocardiograms. (A) Segregation of the KCNQ1-p.R583H, KCNH2-p.C108Y, KCNH2-p.K897T, and KCNE1-p.G38S variants in the long-QT syndrome (LQTS) family members. Black symbols indicate affected subjects with a mutant genotype; white symbols with a squared or circular black inset indicate unaffected subjects with a positive genotype; the black arrow indicates the proband. The karyotype of subject II-3 is 47, XY, +21; (B) Representative 12-lead electrocardiographic recordings from each member of the family.

Figure 2

Functional characterization of the KCNQ1-p.R583H variant. (A) Time constants of the tail current decay of KCNQ1-WT (solid circles n = 13) and KCNQ1-p.R583H (open circles n = 11). Data were obtained by fitting the tail currents area, delimited by dashed lines shown in (B), with a single exponential function. Data are reported as mean ± standard error of the mean (SEM). * Significant differences between time constants from KCNQ1-WT and KCNQ1-p.R583H (p < 0.05); (B) Stimulation protocol; (C) Representative superimposed tail current traces recorded at −30 mV following +40 to +60 mV activating steps in cells transiently transfected with KCNQ1-WT (black trace) or with KCNQ1-p.R583H (gray trace).

Figure 3

Functional characterization of the KCNQ1-p.R583H+KCNE1 complexes. (A) Representative traces illustrating the potassium currents observed in CHO-K1 cells transiently co-transfected with KCNQ1-WT+KCNE1 or KCNQ1-p.R583H+KCNE1 recorded with the protocol shown in inset (arrows indicate the time points at which currents were compared); (B) The current-voltage relation of potassium current densities (elicited by test pulses to various potentials and normalized to membrane capacitance) from CHO-K1 cells transiently transfected with KCNQ1-WT+KCNE1 (solid circles, n = 7) or KCNQ1-p.R583H+KCNE1 (open circles, n = 8); (C) The current-voltage relation of peak tail current densities after repolarization to −30 mV for KCNQ1-WT+KCNE1 (solid circles, n = 7) and KCNQ1-p.R583H+KCNE1 (open circles, n = 8); (D) Normalized current-voltage relation for peak tail current densities for KCNQ1-WT+KCNE1 (solid circles, n = 7) and KCNQ1-p.R583H+KCNE1 (open circles, n = 8). Data were recorded at test potentials ranging from −80 to +60 mV stepped in 10 mV increments from the holding potential of −80 mV for 2000 ms, followed by repolarization to −30 mV for 1000 ms. Data were fit with a Boltzmann distribution {I = I_max/(1 + exp[(V_1/2 − V)/k])} for KCNQ1-WT+KCNE1 (solid line) and for KCNQ1-p.R583H+KCNE1 (dashed line); (E) Time constants of the tail current from CHO-K1 cells transiently transfected with KCNQ1-WT+KCNE1 (solid line, n = 7) or KCNQ1-p.R583H+KCNE1 (open circles, n = 8) plotted as a function of the activation step voltage. The decay of the potassium current recorded during the test pulse to −30 mV was fit with a single exponential function (area delimited by dotted lines shown in F); (F) Stimulation protocol. Data are shown as mean ± SEM.

Figure 4

Functional properties of the KCNH2-p.C108Y variant. (A) Representative traces illustrating potassium currents recorded from CHO-K1 cells transiently transfected with KCNH2-WT and/or KCNH2-p.C108Y recorded with the protocol shown in the inset (arrows indicate the time points at which currents were compared); (B) Current-voltage relationships of potassium current densities (elicited by test pulses to various potentials and normalized to membrane capacitance) from CHO-K1 cells transiently transfected with KCNH2-WT+empty vector (solid circles, n = 6) or KCNH2-p.C108Y+KCNH2-WT (open circles, n = 9); (C) The current-voltage relation of the amplitude of the peak tail current densities after repolarization to −50 mV for KCNH2-WT+empty vector (solid circles, n = 6) and KCNH2-p.C108Y+KCNH2-WT (open circles, n = 9); (D) Normalized current-voltage relationships for peak tail current densities for KCNH2-WT+empty vector (solid circles, n = 6) and KCNH2-p.C108Y+KCNH2-WT (open circles, n = 9). Data were recorded at test potentials ranging from −80 to +70 mV in 10 mV from the holding potential of −80 mV for 2000 ms, followed by repolarization to −50 mV for 2000 ms. Data were fit with a Boltzmann distribution {I = I_max/(1 + exp[(V_1/2−V)/k])} for KCNH2-WT+empty vector (solid line) and for KCNH2-p.C108Y+KCNH2-WT (dashed line); (E) Time constants of the tail current from CHO-K1 cells transiently transfected with KCNH2-WT+empty vector (solid circles, n = 6) or KCNH2-p.C108Y+KCNH2-WT (open circles, n = 9) plotted as function of the activation step voltage. The decay of potassium current recorded during the test pulse to −50 mV was fit with a single exponential function (area delimited by dotted lines shown in F), representing the time constant for KCNH2 channel deactivation; (F) Stimulation protocol. Data are shown as mean ± SEM. * Significant differences between KCNH2-WT+empty vector and KCNH2-p.C108Y+KCNH2-WT (p < 0.05).

Figure 4

Functional properties of the KCNH2-p.C108Y variant. (A) Representative traces illustrating potassium currents recorded from CHO-K1 cells transiently transfected with KCNH2-WT and/or KCNH2-p.C108Y recorded with the protocol shown in the inset (arrows indicate the time points at which currents were compared); (B) Current-voltage relationships of potassium current densities (elicited by test pulses to various potentials and normalized to membrane capacitance) from CHO-K1 cells transiently transfected with KCNH2-WT+empty vector (solid circles, n = 6) or KCNH2-p.C108Y+KCNH2-WT (open circles, n = 9); (C) The current-voltage relation of the amplitude of the peak tail current densities after repolarization to −50 mV for KCNH2-WT+empty vector (solid circles, n = 6) and KCNH2-p.C108Y+KCNH2-WT (open circles, n = 9); (D) Normalized current-voltage relationships for peak tail current densities for KCNH2-WT+empty vector (solid circles, n = 6) and KCNH2-p.C108Y+KCNH2-WT (open circles, n = 9). Data were recorded at test potentials ranging from −80 to +70 mV in 10 mV from the holding potential of −80 mV for 2000 ms, followed by repolarization to −50 mV for 2000 ms. Data were fit with a Boltzmann distribution {I = I_max/(1 + exp[(V_1/2−V)/k])} for KCNH2-WT+empty vector (solid line) and for KCNH2-p.C108Y+KCNH2-WT (dashed line); (E) Time constants of the tail current from CHO-K1 cells transiently transfected with KCNH2-WT+empty vector (solid circles, n = 6) or KCNH2-p.C108Y+KCNH2-WT (open circles, n = 9) plotted as function of the activation step voltage. The decay of potassium current recorded during the test pulse to −50 mV was fit with a single exponential function (area delimited by dotted lines shown in F), representing the time constant for KCNH2 channel deactivation; (F) Stimulation protocol. Data are shown as mean ± SEM. * Significant differences between KCNH2-WT+empty vector and KCNH2-p.C108Y+KCNH2-WT (p < 0.05).

Figure 5

Immunocytochemical analysis of the KCNH2-p.C108Y variant. (A,B) 100× magnification phase-contrast images of HEK-293 cells transiently expressing KCNH2-p.C108Y or KCNH2-WT channels; (C,D) 100× magnification fluorescence images with cells immunolabeled for KCNH2 (green); (E,F) Merged phase-contrast and fluorescence images showing the localization of KCNH2 in the cells.