PMC4907369_01
Electrocardiograms, CT Scan, and X-Ray Images from the Patient Harboring p.Ile1166Thr-CACNA1C. Electrocardiograms representing 2:1 block at birth (A) and t-wave alternans (B). (C) Non-contrast CT of the head at 3 years 8 months of age showing mild diffuse cerebral parenchymal volume loss. (D) Right hand film at 10 months of age showing 5th finger clinodactyly (arrow). (E) AP tibia-fibula film at 10 months of age demonstrating osteopenia with a permeative appearance of the bone.
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Initial cardiac evaluation revealed 2:1 AV block (Figure 1A) and marked ventricular repolarization delay with QT interval prolongation measurement ranging from 595 to 812 milliseconds (ms). With beta-blockade, 2:1 AV conduction resolved and there was consistent 1:1 AV conduction with significant T-wave alternans (Figure 1B). Brain CT showed progressive cerebral and cerebellar atrophy over the next 18 months (Figure 1C). He had joint hypermobility including the fingers, dimpling at the elbow and shoulder joints, clinodactyly (Figure 1D), short thumbs, bilateral metatarsus adductus, osteopenia (Figure 1E), hypotonia, and unilateral cryptorchidism.
CACNA1C Protein Topology. Topology diagram of the CACNA1C channel alpha-subunit in the membrane. Each repeat is demarcated at the top of the figure. Red circles represent the canonical TS-associated mutations in exon 8 and 8A. The yellow circle represents the p.Ile1166Thr mutation identified within our patient. The blue circle represents the TS mutation identified by Gillis and colleagues.
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A novel missense variant (c. 3497T>C; p.Ile1166Thr) in exon 27 of the CACNA1C gene was identified (Figure 2). A fourth mutation (p.Ala1473Gly; Figure 2) was identified in an individual with features suggestive of TS. One unique observation, as shown in Figure 2, highlights that p.Ile1166Thr, and the four additional
CACNA1C-Ile1166Thr Reduced ICaL Current in Heterologous Cells. (A) Whole cell ICaL current representative tracings from HEK293 cells expressing CACNA1C-WT or CACNA1C-Ile1166Thr determined from a holding potential of -90 mV to testing potential of +70 mV in 10 mV increments with 500 ms duration. (B) Current-voltage relationship for CACNA1C-WT (n=11) and -Ile1166Thr missense mutation (n=11). All values represent mean SEM. *p<0.05 vs. CACNA1C-WT.
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TS. Typical ICaL tracings of voltage-dependent activation for WT and Ile1166Thr are shown in Figure 3A (see inset Figure 3A and figure legend). Analysis of the current-voltage relationship shows that the Ile1166Thr mutant shifted peak current from +30 mV (WT) to +20 mV and dramatically reduced current density at +30 mV by 46.6% from -46.6 +- 8.0 picoamps/picofarad (pA/pF) (WT, n=11) to -24.9 +- 5.6 pA/pF (Ile1166Thr, n=11, P<0.05), indicating a marked loss-of-function electrophysiological phenotype (Figure 3B).
CACNA1C-Ile1166Thr Negatively Shifted ICaL V1/2 in Activation. (A) Activation curves of ICaL CACNA1C-WT (n=11) and -Ile1166Thr mutation (n=11). G/Gmax represents normalized conductance fitted with a Boltzmann function. (B) Inactivation curves of ICaL CACNA1C-WT (n=10) and Ile1166Thr variant (n=4), determined from a holding potential of -90 mV to pre-pulse of 20 mV in 10 mV increments with 10 s duration followed by a test pulse of 30 mV with 500 ms duration. I/Imax represent normalized calcium current fitted with a Boltzmann function. Plot (C) and magnified plot (D) of the activation and inactivation curves shown in panels A and B. (E) Inactivation time constants (tau) for the fast phase of ICaL decay time of CACNA1C-WT (n=10) and Ile1166Thr variant (n=8) as a function of voltage. Time constants for each voltage step were determined by fitting a biexponential function to current decay. (F) Inactivation time constants (tau) for the slow phase of ICaL decay time of CACNA1C-WT (n=10) and Ile1166Thr variant (n=8) as a function of voltage.
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A plot of activation curves shows that the Ile1166Thr mutation shifted V1/2 of activation to less depolarized potential by -8.4 mV from 15.9 +- 1.7 mV (WT, n=11) to 7.5 +- 1.6 mV (Ile1166Thr, n=11; P<0.05; Figure 4A), indicating a significant decrease in the degree of depolarization required for activation of Ile1166Thr-containing channels and gain-of-function electrophysiological phenotype. Steady-state inactivation was assessed by a standard two-pulse voltage-clamp protocol (see inset Figure 4B and figure legend). There was not a significant shift in V1/2 of inactivation when comparing WT (-27.8 +- 1.8 mV; n=10) to the mutant Ile1166Thr (-30.6 +- 2.4 mV; n=4; P>0.05; Figure 4B). In order to better examine the increased window currents, the activation and inactivation curves were plotted together in Figure 4C-D. At 0 mV, Ile1166Thr mutant ICaL revealed a faster inactivation tau in fast component of the decay time (P<0.05; Figure 4E). Slow inactivation tau remained unchanged (Figure 4F).
Simulation of Effects of CACNA1C-Ile1166Thr on the Properties of ICaL, Cardiac Action Potential and Calcium Transients as Compared with WT-CACNA1C. (A) Intrinsic voltage-dependent activation and intrinsic voltage-dependent steady-state inactivation curves for WT ICaL (dashed line) and Ile1166Thr mutant ICaL (solid line). (B) Window ICaL estimated theoretically as a product of the steady-state activation and the steady-state voltage-dependent inactivation curves for WT ICaL (dashed curve) and for mutant ICaL (solid curve). (C) Ventricular action potentials simulated by LR2 model using WT (dashed curve) and heterogeneously (50%/50%) expressed Ile1166Thr mutant (solid curve) ICaL model. The panel shows 100th AP at BCL = 1000 ms. (D) Ca2+ transients, which correspond to the action potentials shown in panel C (solid line - WT; dashed line - 50% of Ile1166Thr mutant). (E) ICaL during corresponding action potentials shown in panel C with WT (dashed line) and 50% of Ile1166Thr mutant (solid line) ICaL; (F) Two components of ICaL (shown by solid line in panel E) with 50% of Ile1166Thr mutant expression (dashed line - WT ICaL component, solid line - Ile1166Thr ICaL component). (G) Effect of a missing beat on action potentials simulated using WT ICaL (dashed line) and heterogeneously expressed mutant ICaL (solid line).
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Theoretical steady-state activation and inactivation curves, which were used to simulate WT and Ile1166Thr mutant ICaL in Luo-Rudy model of the action potential, are shown on Figure 5A. According to this model, there may be a considerable overlap between the steady-state activation and inactivation curves, resulting in a substantial steady-state ("window") calcium current that peaks around -20 mV which is increased by the negative shift of the steady-state activation curve approximately 2.5 times (Figure 5B). Figure 5C shows the effect of the heterozygous expression (50% WT + 50% mutant) of the Ile1166Thr mutation on the shape and duration of simulated APs. The peak of the calcium transient during the action potential with the simulated heterogeneous expression of Ile1166Thr mutation was 97% of that of the WT ICaL(Figure 5D), indicating no significant change in the total calcium influx. Figure 5E illustrates the time course of the calcium current during the action potential for WT and heterozygously expressed Ile1166Thr mutant. The abnormal shape of the ICaL time course due to the mutation is more evident in Figure 5F, which shows the two components (WT and Ile1166Thr mutant) of ICaL comprising of heterogeneously expressed mutant current. Figure 5G shows that the omission of one beat (2000 ms pause) leads to early afterdepolarizations (EADs) evident on AP simulated with 50% of Ile1166Thr-mutant ICaL, which is absent in AP simulated with WT ICaL.