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Epilepsia Open
2017 Aug 05;23:334-342. doi: 10.1002/epi4.12068.
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Functional variants in HCN4 and CACNA1H may contribute to genetic generalized epilepsy.
Becker F
,
Reid CA
,
Hallmann K
,
Tae HS
,
Phillips AM
,
Teodorescu G
,
Weber YG
,
Kleefuss-Lie A
,
Elger C
,
Perez-Reyes E
,
Petrou S
,
Kunz WS
,
Lerche H
,
Maljevic S
.
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Objective: Genetic generalized epilepsy (GGE) encompasses seizure disorders characterized by spike-and-wave discharges (SWD) originating within thalamo-cortical circuits. Hyperpolarization-activated (HCN) and T-type Ca2+ channels are key modulators of rhythmic activity in these brain regions. Here, we screened HCN4 and CACNA1H genes for potentially contributory variants and provide their functional analysis.
Methods: Targeted gene sequencing was performed in 20 unrelated familial cases with different subtypes of GGE, and the results confirmed in 230 ethnically matching controls. Selected variants in CACNA1H and HCN4 were functionally assessed in tsA201 cells and Xenopus laevis oocytes, respectively.
Results: We discovered a novel CACNA1H (p.G1158S) variant in two affected members of a single family. One of them also carried an HCN4 (p.P1117L) variant inherited from the unaffected mother. In a separate family, an HCN4 variant (p.E153G) was identified in one of several affected members. Voltage-clamp analysis of CACNA1H (p.G1158S) revealed a small but significant gain-of-function, including increased current density and a depolarizing shift of steady-state inactivation. HCN4 p.P1117L and p.G153E both caused a hyperpolarizing shift in activation and reduced current amplitudes, resulting in a loss-of-function.
Significance: Our results are consistent with a model suggesting cumulative contributions of subtle functional variations in ion channels to seizure susceptibility and GGE.
Figure 1. Pedigrees of the two GGE families. (A) Pedigree of Family 1 showing a complete cosegregation of the CACNA1H variant (orange) with the GGE phenotype and the inheritance pattern of the HCN4 variant (blue). (B) Pedigree of Family 2 in which the second HCN4 mutation p.E153G was identified in a single patient; EGMA, epilepsy with grand mal seizures on awakening; TLE, temporal lobe epilepsy; FS, febrile seizures; CAE, childhood absence epilepsy; EGTCS, epilepsy with generalized tonic‐clonic seizures.
Figure 2. Conservation and localization of affected amino acids. (A) Alignment of protein sequences showing the conservation of affected amino acids in Cav3.2 (encoded by CACNA1H) and HCN4 proteins among different species. (B) Predicted localization of detected variants within the Cav3.2 and HCN4 channel proteins.
Figure 3. Functional analysis of the CACNA1H variant in tsA201 cells. (A) Whole cell currents of WT Cav3.2 and G1158S channels elicited by depolarizing the membrane between −70 mV and 70 mV in 5‐mV steps from a holding potential of −90 mV. (B) Current density for the Cav3.2 WT and G1158S mutant channels at different potentials. (C) Steady‐state activation and inactivation for Cav3.2 WT and G1158S mutant channels obtained by standard protocols using a holding potential of −90 mV. Recordings were performed with Ca2+ as the charge carrier. Parameters for activation were as follows: WT – V0.5 = −41.4 ± 0.8 mV, k = −6.7 ± 0.3 mV (n = 13); G1158S – V0.5 = −40.8 ± 0.8 mV, k −1.3 ± 0.3 (n = 10). For the inactivation (inset), parameters were: WT – V0.5 = −69.2 ± 1.1 mV, k = 4.3 ± 0.2 mV (n = 11); G1158S – V0.5 = −64.1 ± 1.1 mV, k = −4.4 ± 0.1 (n = 7), **p < 0.01, unpaired t test. (D) Recovery from inactivation for Cav3.2 WT and G1158S mutant channels. Curves represent monoexponential fits to the averaged data (n = 4–12). Inset: mean time constant for recovery from inactivation for Cav3.2 WT and G1158S mutant, obtained from monoexponential fits to the normalized current recovery curve (n = 4–12, n.s. = not significant).
Figure 4. Electrophysiological characterization of HCN4 epilepsy variants. (A) Steady‐state (left) and tail (right) currents from oocytes expressing HCN4 WT and E153G and P1117L epilepsy variants. Vertical scale bar represents 1 μA. (B) Average tail current–voltage (I–V) relationship of WT (n = 21), E153G (n = 19), and P1117L (n = 30) channels. (C) Activation curve constructed from average normalized tail currents and fit with the Boltzmann equation for HCN4 WT and E153G and P1117L variants. (D) Bar graph of the average half‐activation voltage (V0.5) for each situation.*p < 0.05, one‐way ANOVA.
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