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Seizure
2025 Mar 20;129:14-21. doi: 10.1016/j.seizure.2025.03.014.
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Loss-of-function variant in KCNH3 is associated with global developmental delay, autistic behavior, insomnia, and nocturnal seizures.
Bauer CK
,
Kortüm F
,
Möllring A
,
Grinstein L
,
Denecke J
,
Alawi M
,
Bähring R
,
Harms FL
.
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INTRODUCTION: The KCNH gene family encodes voltage-gated potassium (Kv) channels of the EAG subtype covering three subfamilies (Kv10-12). EAG channels are involved in the control of cardiac and neuronal excitation, and pathogenic variants in KCNH genes encoding Kv10 (eag) and Kv11 (erg) subfamily members cause a broad clinical spectrum ranging from cardiac arrhythmia to neurodevelopmental syndromes. However, no pathogenic variants have been hitherto reported for KCNH genes encoding Kv12 (elk) subfamily members.
METHODS: Clinical, genomic, and functional studies were performed, including voltage-clamp experiments using heterologous channel expression in Xenopus oocytes.
RESULTS: We examined an eight-year-old girl presenting with global developmental delay, intellectual disability, autistic and aggressive behavior, hyperactivity, insomnia, and nocturnal seizures. Focal seizures were successfully treated with sulthiame, which reduced the occurrence of temporo-parietal spike-wave paroxysms. Trio exome sequencing revealed a heterozygous de novo missense variant, NM_012284.3:c.1112C>T; p.(Ala371Val), in KCNH3, which encodes the Kv channel α-subunit Kv12.2. The amino acid substitution associated with the KCNH3 variant identified in the patient is located at a site highly conserved in EAG channels. The analogous variant in KCNH2 causes long-QT-syndrome 2, and has also been associated with epilepsy. Electrophysiological characterization of the KCNH3 p.(Ala371Val) variant demonstrated loss-of-function of the mutant Kv12.2 channels and strongly reduced current amplitudes upon co-expression of wildtype and mutant channel subunits in a dominant-negative manner.
CONCLUSION: Our results propose KCNH3, which is primarily expressed in the nervous system, as a new disease gene associated with a neurodevelopmental phenotype including seizures.
Fig. 1. EAG paralogs and location of the KCNH3 variant-associated amino acid substitution p.Ala371Val in the Kv12.2 channel complex. (A) UniProt single letter amino acid sequence alignment of all human EAG channel subtypes (Kv10, Kv11 and Kv12). Alternative names, which refer to their Drosophila origins eag, erg, and elk (§ also referred to as elk1 [40]), and the corresponding human genes (KCNH1–8) are indicated. The alignment is restricted to transmembrane segment S5 (shaded gray) and flanking intracellular (left) and extracellular regions (right). Numbers on the right specify amino acid residue positions. Residues that are perfectly conserved (*), or exhibit strong similarity (:) or weak similarity (.) are indicated below. Alanine at position 371 in Kv12.2 is conserved throughout the EAG group of Kv channels (indicated in green). (B) Membrane topology scheme of the Kv12.2 α-subunit with six transmembrane segments (S1-S6) flanked by a cytoplasmic amino (NH3+) and carboxy (COO-) terminus (extracellular and cytoplasmic space indicated; see also horizontal dashed lines). Positively charged amino acid residues in S4 constitute the voltage sensor and a re-entrant pore loop (P) between S5 and S6 harbors the selectivity filter sequence. The large extracellular linker between S5 and a pore helix is typical of Kv12.2. The conserved N-terminal structure found in all EAG channel subtypes consists of a PAS (Per-Arnt-Sim) domain and a more distal PAS cap. A cyclic nucleotide-binding homology domain (cNBHD) is located in the cytoplasmic C-terminus. The amino acid substitution A371 V in S5 is indicated by a red dot. (C) Tetrameric Kv12.2 channel structure, obtained with homology modeling based on the Kv11.1 structural coordinates (see Materials and Methods) [25], illustrating transmembrane helices (between dashed lines), extended extracellular loops and large cytoplasmic domains. The depicted Kv12.2 channel complex contains both wildtype (A371, green spheres in S5) and mutant (A371 V, red spheres in S5) α-subunits. (D) Top views of a heteromeric (two WT and two mutant α-subunits co-assembled) Kv12.2 channel. Only membrane spanning and pore helices are shown; intracellular and extracellular domains were removed for clarity. Channel pore loop (P) and pore helix: blue; voltage sensing domains (S1 - S4): orange; S5 and S6: grey; left panel: the pore helix occludes position 371; right panel: removal of pore helices allows a better view of the size and orientation of alanine or valine residues at position 371.
Fig. 2. Electroencephalogram (EEG) recordings of the patient off medication and during sulthiame treatment. Electrode positions follow the 10–20 system, with labels as follows: Fp (prefrontal), F (frontal), C (central), T (temporal), P (parietal), and O (occipital). EEG traces result from source derivation. (A) Interictal EEG at the age of seven years showed high centro-temporo-parietal spike-wave paroxysms. Blue rectangles highlight exemplary EEG abnormalities. (B) EEG recording at the age of eight years showed a decrease of spike-wave paroxysms (blue rectangles) under treatment with 3 mg/kg/day sulthiame. The family reported no further seizures.
Fig. 3. The A371 V amino acid substitution causes loss of channel function and dominant-negative suppression of Kv12.2 currents upon co-expression with WT. (A) Two-electrode voltage clamp recordings from Xenopus oocytes three days after injection with a total of 5 ng Kv12.2 channel cRNA: WT Kv12.2 only, A371 V only, or 2.5 ng of each WT and A371 V cRNA. Current traces were elicited with 1-s depolarizing voltage steps to potentials ranging between −80 and +80 mV to activate the Kv12.2 channels. Capacity transients were removed for clarity. Symbols indicate time points of amplitude measurements for peak current, sustained current and tail current; same color code for all panels. (B) Simplified linear gating scheme with three channel states (C, closed; O, open; I, inactivated). Upon depolarization, channels pass the open state before inactivation, and upon repolarization, channels pass the open state before deactivation. (C) Means ± SEM of maximal current amplitudes (peak currents) during the first 40 ms of the variable test pulses and of current amplitudes just before returning to the holding potential of −80 mV; n = 15 oocytes for each condition. (D) Means ± SEM of the peak current amplitude obtained at +60 mV (left) and of sustained current amplitudes at +10 mV test pulse potential (right). Same experiments as in panel C; Corresponding values from 6 uninjected oocytes from the same donor frog are given for comparison. Co-expression data below 50 % WT indicate a dominant-negative effect of the channel variant. (E) Voltage dependence of activation of WT Kv12.2 channels and WT/A371 V channels. Means ± SEM (n = 15 each) of the tail current amplitude as a function of the preceding test pulse potential. Continuous lines denote Boltzmann functions fitted to the data points. The broken line denotes the WT/A371 V activation curve up-scaled to the amplitude of the WT data. (F) Means ± SEM of the unclamped membrane potential of oocytes injected with WT, A371 V or WT + A371 V cRNA (n = 15 each) and from 6 uninjected oocytes. ** p ≤ 0.01 and *** p ≤ 0.001 versus WT data.
Supplementary Fig. S1. Visualization of the heterozygous de novo KCNH3 variant identified by trio exome
sequencing in the patient and her parents using the Integrative Genomics Viewer (IGV). The KCNH3 variant
NM_012284.3:c.1112C>T; p.(Ala371Val) (NC_000012.12:g.49544305C>T) was heterozygous in leukocyte-derived
DNA of the patient, with a coverage of 250 reads (C vs. T; 52% vs. 48%). The KCNH3 variant was absent in
leukocyte-derived DNA of the patient’s healthy mother and father.