PMC9453199_01
Electroencephalogram (EEG) tests in patients with developmental and epileptic encephalopathie (DEE) due to the KCNC2 R405G variant. The results showed background (left) generalized, diffuse spike, and wave complexes in both patients. In patient 1 (at the age of 9 years), the EEG showed generalized high amplitude waves at 3-4 Hz and spike-and-slow-wave bursts for 8-18 s in an epileptic attack (upper right). In patient 2, a sleep EEG (lower right) showed slowing of the occipital basic rhythm, multifocal generalized complexes of slow wave, spike and slow wave, sharp slow wave, and polyspike and slow wave, especially in occipital, frontal pole, and frontal regions.
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F1
When the patient was 4 years and 9 months old, upon the last follow-up, the EEG showed the same pattern of slowing of the occipital basic rhythm, multifocal generalized complexes of slow-wave, spike and slow-wave, sharp-slow-wave, and polyspike and slow-wave, especially in occipital, frontal pole, and frontal regions (Figure 1 and Table 1). Seven KCNC2 variants were reported in 11 DEE cases in four published research articles, in which V471L, R351K, and T437N were identified in two unrelated patients, respectively, and a T437A was found in a sporadic DEE case (Figure 2B and Table 1).
PMC9453199_02
Electroencephalogram (EEG) tests in patients with developmental and epileptic encephalopathie (DEE) due to the KCNC2 R405G variant. The results showed background (left) generalized, diffuse spike, and wave complexes in both patients. In patient 1 (at the age of 9 years), the EEG showed generalized high amplitude waves at 3-4 Hz and spike-and-slow-wave bursts for 8-18 s in an epileptic attack (upper right). In patient 2, a sleep EEG (lower right) showed slowing of the occipital basic rhythm, multifocal generalized complexes of slow wave, spike and slow wave, sharp slow wave, and polyspike and slow wave, especially in occipital, frontal pole, and frontal regions.
fnmol-15-950255-g001.jpg
PMC9453199_02_fnmol-15-950255-g001.jpg
F1
When the patient was 4 years and 9 months old, upon the last follow-up, the EEG showed the same pattern of slowing of the occipital basic rhythm, multifocal generalized complexes of slow-wave, spike and slow-wave, sharp-slow-wave, and polyspike and slow-wave, especially in occipital, frontal pole, and frontal regions (Figure 1 and Table 1). Seven KCNC2 variants were reported in 11 DEE cases in four published research articles, in which V471L, R351K, and T437N were identified in two unrelated patients, respectively, and a T437A was found in a sporadic DEE case (Figure 2B and Table 1).
Schematic diagram of known developmental and epileptic encephalopathie (DEE)-linked KCNC2 variants and R405G analysis. (A) Sanger sequencing confirms KCNC2 (NM_139137) c.1213A > G in patient 2 and her parents. (B) Nine well-known DEE-linked variants on eight sites of the KCNC2 amino acid chain. Both previously reported R351K and T437N were found in two unrelated patients with DEE, respectively. (C) Multiple sequence alignment of KCNC1, KCNC2, KCNC3, and KCNC4 subunits across several species is shown by MEGA7 (the R405 residue and KCNC2 human sequence marked in red boxes). Asterisks (*) denote the highly conserved sites. (D) Close view of structural models of the KCNC2 protein (AF-Q96PR1-F1) as ribbons with the p.R405G variant and residues involved in hydrogen-bonding (dotted yellow) network indicated as sticks. (a) Represents wild-type KCNC2 protein with native R405. (b) Represents the mutated KCNC2 with Gly405.
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F2
We then performed Sanger sequencing in patient 2 and her parents, which proved the variant was de novo (Figure 2A). KCNC2 contains cytosolic N- and C-termini and six membrane-spanning segments. KCNC2 R405 is located within the S5-S6 linker, an extracellular loop that forms one of the ion-selective macromolecular protein pores in KCNC2 (Figure 2B). In addition, R405 is strictly conserved in evolution among multiple species and other members of the KV3 subfamily, including KCNC1, KCNC3, and KCNC4 (Figure 2C). AF-P35670-F1) has revealed that glycine substitution has disrupted the hydrogen bonds that bind R405 with Tyr401, Phe421, and Lys422 (Figure 2D). As a result, R405G is predicted to change the pore structure. Seven KCNC2 variants were reported in 11 DEE cases in four published research articles, in which V471L, R351K, and T437N were identified in two unrelated patients, respectively, and a T437A was found in a sporadic DEE case (Figure 2B and Table 1).
Electrophysiological analysis of wild type and mutant KCNC2 channels. (A) Current outward traces from HEK-293 cells transfected with plasmids encoding wild type (WT) or R405G mutant KCNC2 channels in response to the voltage steps from -80 to +80 mV (with an increment of 10 mV). Current scale: 1,000 pA; time scale: 500 ms. (B) Averaged current density-voltage curves of WT (n = 5) and R405G (n = 7) channels. (C) Normalized current densities at + 60 mV from HEK-293 cells expressing WT (n = 5) or R405 (n = 7) channels. (D) Normalized conductance versus voltage relationships of WT (n = 5) and R405G (n = 7) channels. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
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F3
Compared to WT, R405G caused a significantly higher density current at voltages between -40 and +10 mV (Figures 3A,B). At +60 mV, the normalized current densities of the channel (262.2 +- 61.91) revealed that R405G caused about 40% less normalized density current compared to the WT on average (262.2 +- 61.91 vs. 427.9 +- 137.1), although the difference was not statistically significant (Figure 3C). V1/2 = 24.2 mV, k = 21.36) (Figure 3D).