Molecular autopsy: using the discovery of a novel de novo pathogenic variant in the KCNH2 gene to inform healthcare of surviving family

Figures

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  Fig. 1. Key Findings to Establish Cause of Death in the Decedent and Results of Family Study (A). Ante-mortem EKG showed normal sinus rhythm with a heart rate of 85 bpm and prolonged QT interval QTc = 518 ms. (B) molecular testing results of the index; (C) family study results. Index is shown as blue circle. (+) or (-) above the pedigree symbols denotes presence or absence of the pathogenic variant in KCNH2; Status of EKG and targeted variant testing results of the CACNA2D1 VUS is shown below the symbols (D) DNA sequences surrounding the de novo variant in KCNH2 (underscore in red).

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  Fig. 2. Activation Properties and Fully-activated Current-voltage Relationships. COS-7 cells were transfected with wild-type hERG or hERG-ΔQ1068 cDNAs and subjected to whole-cell patch clamping. (A) Channels were activated by 4 s conditioning steps, applied at 0.05 Hz, to voltages between -70 mV to +70 mV. Tail currents were recorded during a 5 s test pulse at -50 mV. (B) Currents at the end of the conditioning pulses were plotted as a function of the conditioning voltage. (C) Tail current amplitude density recorded during the test pulse to -50 mV. (D) Normalized tail currents, plotted as a function of the conditioning voltage. Data points were subjected to curve fitting to a Boltzmann equation, 1/[1 + exp[(Vm - V½)/k]], where Vm is the membrane potential, V½ is the voltage of half-maximal activation, and k represents a slope factor. (E) Representative recording of the fully-activated hERG currents. Membrane currents were elicited by a 1 s depolarizing pulse to +60 mV repeated at 0.05 Hz, followed by variable 4 s test pulses between -100 mV and +40 mV, with 10 mV increments, to a final step to -80 mV. (F) Maximal tail current amplitudes during the variable test pulses were plotted as a…

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  Fig. 3. Reduced surface expression of hERG-ΔQ1068 channels. (A) HEK-293 cells were transfected with 0.5 μg of wild-type hERG or hERG-ΔQ1068 cDNAs. Cell lysates were treated with Endo H, PNGase F or no treatment before subjecting to immunoblotting with an anti-hERG antibody. The 150 kDa band representing the mature, fully-glycosylated form is illustrated with an arrow. (B) HEK-293 cells were transfected with 2 μg of wild-type hERG or hERG-ΔQ1068 cDNAs. Live cells were surface biotinylated, cell lysates were prepared, and purified with streptavidin-agarose beads. The cell lysates, or biotinylated samples, were subjected to Western blotting with anti-hERG antibodies, or anti-GAPDH antibodies as a loading control. GAPDH was not observed in the surface biotinylated protein pool, verifying the identity of the sample. (C) Total hERG protein of wild-type hERG (n = 3) and hERG-ΔQ1068 (n = 3) in cell lysates was calculated by expressing the band intensities as a function of that of GAPDH. The two hERG-ΔQ1068 bands were summed to calculate expression of the variant. Surface expression was calculated by expressing their band intensities relative to total protein respectively for wild-type hERG (n =…

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  Fig. 4. Effects of hERG-ΔQ1068 on activation kinetics. (A) Representative trace demonstrating the measurement of the time course of activation. From the holding potential of -80 mV, the membrane was clamped to +20 mV for various durations before repolarizing to -100 mV for 2000 ms. (B) The tail current amplitudes at -100 mV were normalized to maximal tail current amplitude and plotted as a function of the preceding depolarization duration to produce the “envelope of tail currents”. Data points were subjected to curve fitting to a single exponential function, normalized current = 1-e-t/τ, where t is time and τ is the activation time constant. (C) Representative trace to demonstrate the recording of deactivation kinetics. From a holding potential of -80 mV, the membrane was clamped to 20 mV for 1000 ms, followed by repolarization for 14 ms to test voltages between -40 mV and -100 mV. Tail current data points were subjected to curve fitting to a two exponential function, f(t) = [Af * exp (tf/τf) ]+ [As * exp (ts/τs) ]+C, with A the amplitude, τ the deactivation time constant and c the offset parameter. (D and E) Summary data respectively of the slow and fast deactivation time constants.…

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  Fig. 5. Voltage dependence of steady-state inactivation. (A) A typical recording demonstrating the method. From a holding potential of -80 mV, the membrane was clamped to 20 mV for 500 ms, followed by a conditioning pulse of 20 ms between -110 and +20 mV, before measuring tail current amplitudes during a test pulse of 800 ms at 20 mV. The protocol was repeated every 15 s. (B) Tail current amplitudes were normalized to the maximal tail amplitude and plotted against the conditioning voltage to obtain the steady-state inactivation curve. Data points were subjected to curve fitting to a modified Boltzmann equation, y = 1/[1+exp[(Vm-V1/2)/k]], where V½ is the voltage of half-maximal inactivation, and k represents the slope factor. Data points represent mean ± SEM. *p < 0.05 with Student's t-test.

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  Fig. 6. hERG currents recorded using rapid repetitive pulses as the command waveform. (A) Slow time scale recording of wild-type hERG or hERG-ΔQ1068 during 250 ms voltage clamp steps from -88 mV to 0 mV, repeated at 4 Hz. (B) The total current during the depolarization step was integrated and corrected for cell size, and plotted as a function of the pulse number. Data points represent mean ± SEM. *p < 0.05 with Student's t-test.

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  Fig. 7. Simulated heterozygosity of the ΔQ1068 variant. COS-7 cells were transfected with 1 μg wild-type hERG (WT), 1 μg hERG-ΔQ1068, or 0.5 μg each of wild-type or hERG-ΔQ1068 cDNAs and subjected to whole-cell patch clamping. (A) The fully-activated hERG currents were recorded as described in Fig. 2. Currents at -40 mV (B) or -100 mV (C) were corrected for cell surface area by dividing by the capacitance, averaged and displayed as bar graphs. (D) Western blotting with an anti-hERG antibody of cell lysates of the three experimental groups. The error bars represent SEM. *p < 0.05 with Student's t-test.

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  Fig. 8. Comparison of the Healthcare for Surviving Family Members. Cost, time and quality of healthcare for surviving family members with (A) or without molecular autopsy (B) using a three-generation family of parents, five siblings and a grandchild as an example where one of the siblings died suddenly and unexpectedly in an undetermined cause of natural death.