De novo loss- or gain-of-function mutations in KCNA2 cause epileptic encephalopathy

Abstract

Epileptic encephalopathies are a phenotypically and genetically heterogeneous group of severe epilepsies accompanied by intellectual disability and other neurodevelopmental features. Using next-generation sequencing, we identified four different de novo mutations in KCNA2, encoding the potassium channel KV1.2, in six isolated patients with epileptic encephalopathy (one mutation recurred three times independently). Four individuals presented with febrile and multiple afebrile, often focal seizure types, multifocal epileptiform discharges strongly activated by sleep, mild to moderate intellectual disability, delayed speech development and sometimes ataxia. Functional studies of the two mutations associated with this phenotype showed almost complete loss of function with a dominant-negative effect. Two further individuals presented with a different and more severe epileptic encephalopathy phenotype. They carried mutations inducing a drastic gain-of-function effect leading to permanently open channels. These results establish KCNA2 as a new gene involved in human neurodevelopmental disorders through two different mechanisms, predicting either hyperexcitability or electrical silencing of KV1.2-expressing neurons.

Figure 1

Mutations in the K_V1.2 channel. (A) Structure of the voltage-gated potassium channel K_V1.2 with transmembrane segments S1–S4 forming the voltage sensor domain (light gray) and the pore region S5-S6 (in dark gray) with its pore-forming loop. Mutations are localized in highly-conserved regions in the S3 segment (I263T, light blue), the S4 segment constituting the voltage sensor (R297Q, red; L298F, orange) and the S6 segment (P405L, dark blue). (B) I263, R297, L298 and P405 and the respective surrounding amino acids show evolutionary conservation. (C) Pedigrees of patients #1, #2 and #4–7.

Figure 2

Functional effects of the KCNA2 mutations P405L and I263T. (A) Representative current traces of K_V1.2 wildtype (WT) channels recorded in a Xenopus laevis oocyte during voltage steps (from −80 mV to +70 mV). (B) Effect of increasing amounts of injected WT-KCNA2 cRNA on current amplitude (0.25: n=13; 0.5: n=18; 1: n=22; 2: n=17; 4: n=20; 8: n=19). Shown are means ± SEM. (C) Current traces derived from K_V1.2-P405L (top) and K_V1.2-I263T (bottom) channels recorded as described in (A). (D) K⁺-currents were reduced for mutants P405L (top) and I263T (bottom) compared to WT-cRNA (top: P405L: n=10; WT: n=44; bottom: I263T: n=10; WT: n=34). A dominant-negative effect of P405L and I263T mutants on K_V1.2-WT channels was shown when a constant amount of WT cRNA (amount 1 in (B)) was injected with either H₂O or increasing amounts of mutant cRNA (top: P405L: ratio 1:1: n=47; ratio 1:2: n=40; ratio 1:4: n=36; bottom: I263T: ratio 1:1: n=34; ratio 1:2: n=42; ratio 1:4: n=38). Co-expression of P405L or I263T and the WT led to a significant reduction of the current amplitude compared to the WT alone. Groups were statistically different (One-way ANOVA (p<0.001), posthoc Dunn’s method (p<0.05)). Shown are means ± SEM. (E) Western blot analysis from lysates of Xenopus laevis oocytes injected with equal amounts of K_V1.2-WT or mutant cRNA (P405L: top; I263T: bottom) or from lysates of CHO cells transiently transfected with K_V1.2-WT and P405L cDNAs (middle). For P405L-mutant channels there was a shift from 57 kDa to ~58.5 kDa (n=3). K_V1.2-WT or I263T (n=3) mutant channels revealed similar bands (57 kDa).

Figure 3

Functional effects of the K_V1.2 mutations R297Q and L298F. (A) Representative current traces derived from K_V1.2-WT (top), R297Q (middle) or L298F mutant channels (bottom) recorded as described in Fig. 2A. (B) Mean current amplitudes of top: K_V1.2-WT (1.0, n=23), WT + R297Q (0.5:0.5, n=37), R297Q (1.0, n=35) and H₂O injection (n=25); bottom: K_V1.2-WT (1.0, n=13), WT + L298F (0.5:0.5, n=26), L298F (1.0, n=14), and H₂O injection (n=10). Shown are means±SEM. There was a statistical significant difference between WT and tested groups (ANOVA on ranks; p<0.001) with posthoc Dunn’s Method (p<0.05)). (C) Mean voltage dependence of K_V1.2 channel activation for WT, R297Q (red, top) or L298F channels (orange, bottom). Shown are means ± SEM. Lines represent Boltzmann functions fit to data points. Activation curves of mutant channels were significantly shifted to more hyperpolarized potentials (p<0.05). For details see Supplementary notes. (D) Resting membrane potentials of oocytes injected with: top: WT (1.0, n=44), WT+R297Q (0.5:0.5, n=42), R297Q (1.0; n=38) or H₂O (n=24); bottom: WT (1.0, n=30), WT+L298F (0.5:0.5, n=34), L298F (1.0; n=28) or H₂O (n=13). Shown are means ± SEM. Statistically significant differences between WT and tested groups was verified by ANOVA on ranks (p<0.001) with posthoc Dunn’s Method (p<0.05). (E) Western blot analysis from lysates of Xenopus oocytes injected with K_V1.2-WT (1.0), K_V1.2-WT (0.5) + R297Q (0.5, top), mutant R297Q (1.0, top), K_V1.2-WT (0.5) + L298F (0.5, bottom) or mutant L298F (1.0, bottom) cRNA (n=3). All channels revealed similar bands (57 kDa).