Abstract
Epileptic encephalopathies are a phenotypically and genetically heterogeneous group of severe epilepsies accompanied by intellectual disability and other neurodevelopmental features1,2,3,4,5,6. 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.
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Acknowledgements
We thank all patients and family members for their participation in this study, S. Grissmer (Ulm University) for providing the human cDNA clone of KCNA2, and F. Lang and his colleagues (University of Tübingen) for providing Xenopus oocytes. J.R.L. (32EP30_136042/1), J.M.S. (EUI-EURC2011-4325), H. Lerche (DFG Le1030/11-1), P.D.J. (G.A.136.11.N, FWO/ESF-ECRP), H.S.C. (TUBITAK 110S518) and I.H. (DFG HE 5415 3-1) received financial support within the EuroEPINOMICS RES and EuroEPINOMICS CoGIE networks (http://www.euroepinomics.org/), a Eurocores project of the European Science Foundation. R.S. received funding from the European Union (E-Rare JTC grants 01GM1408B and PIOF-GA-2012-326681). J.M.S. received further support from the Ministerio de Economía y Competitividad (SAF2010-18586). H. Lerche, S.B. and S. Maljevic received further support from the Federal Ministry for Education and Research (BMBF, program on rare diseases, IonNeurONet: 01GM1105A). S.Z. received support from the US National Institutes of Health (R01NS072248). S.M.S. received support from the Wellcome Trust (084730), National Institute for Health Research (NIHR) University College London Hospital Biomedical Research Centre and Epilepsy Society, UK. M. Synofzik received support from the Interdisciplinary Center for Clinical Research (IZKF) Tübingen (2191-0-0). A.S. received funding for a postdoctoral fellowship from the Fonds Wetenschappelijk Onderzoek. T. Djémié is a PhD fellow of the Institute of Science and Technology (IWT).
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Study design: S.S., U.B.S.H., S.W., H. Lerche and J.R.L. Subject ascertainment and phenotyping: S.S., E.R., R.S.M., B.M., L.H.-H., H.S.C., M.A., J.M.S., T. Dorn, H.V., G.K., M. Synofzik, L.S., P.E.M., T.L., A.-E.L., K.S., D.C.C., D.H.-Z., C.M.K., Y.G.W., M. Steinlin, S.G., A.B., M.K.B., A.M., W.K., A.P., A.S., P.D.J., I.H., S.B., M.W., S.M.S., S.W., H. Lerche, J.R.L. and the EuroEPINOMICS RES Consortium. Mutation and CNV analysis: E.R., T. Djémié, B.M., L.H.-H., R.S., M.G., S.Z., A.S., P.D.J., S.B., S.M.S., S.W., H. Lerche and J.R.L. Statistical analysis: M.N., P.M., R.K., H. Lerche and J.R.L. Functional analysis: U.B.S.H., S. Müller, H. Löffler, K.D., S. Maljevic and H. Lerche. Interpretation of data: S.S., U.B.S.H., M.W., S. Maljevic, S.M.S., S.W., H. Lerche and J.R.L. Writing of the manuscript: S.S., U.B.S.H., T. Djémié, M.N., P.M., S. Maljevic, S.M.S., S.W., H. Lerche and J.R.L. Revision of the manuscript: all authors.
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Integrated supplementary information
Supplementary Figure 1 Overview of the patient cohorts used in this study.
Screening of patients with different epilepsy phenotypes by panel sequencing (dark gray) or parent-offspring trio WES (light gray) revealed several KCNA2 de novo mutations resulting in loss of function (red) or gain of function (blue) as well as one variant of unknown inheritance with no detectable effect on channel function (white). Sanger sequencing of two follow-up cohorts revealed no additional mutations in KCNA2. MAQ screening in most WES cohorts as well as the two follow-up cohorts did not reveal CNVs affecting KCNA2.
Supplementary Figure 2 Electropherograms of identified mutations.
Electropherograms of patients 1, 2 and 4–7 (top) and their parents (father in the middle, mother at bottom) with patients 1, 4 and 5 showing the same de novo mutation.
Supplementary Figure 3 Functional analysis of the KCNA2 mutation R147K.
(a) Structure of the voltage-gated potassium channel KV1.2 with transmembrane segments S1–S4 forming the voltage sensor domain (light gray) and the pore region S5–S6 (dark gray) with its pore-forming loop. Mutation R147K is in the N terminus. (b) R147 and the respective surrounding amino acids show evolutionary conservation. (c) Pedigree of patients 3 and electropherogram of the mutation. (d) Representative current traces derived from KV1.2 wild-type (WT, top) and mutant KV1.2 R147K (bottom) channels recorded in a Xenopus oocyte 2 d after cRNA injection during voltage steps ranging from –80 mV to +70 mV. (e) Mean current amplitudes of KV1.2-WT (1.0; n = 20), R147K (1.0; n = 20) and coexpressed WT + R147K (0.5:0.5) (n = 24) did not show significant differences between each other. All currents were normalized to the mean current amplitude of 1.0 WT with H2O recorded on the same day. (f) Western blot analysis from lysates of Xenopus oocytes injected with equal amounts of KV1.2-WT or mutant (R147K) cRNA were performed using a mouse anti-KV1.2 antibody. KV1.2-WT and R147K mutant (n = 4) channels showed similar bands (57 kDa) in total cell lysates. Water-injected oocytes were used as controls.
Supplementary Figure 4 Titrating expression of KV1.2 channels in Xenopus oocytes.
Shown is the effect of an increasing amount of injected WT-KV1.2 cRNA on the absolute, i.e., non-normalized current amplitudes (in contrast to Fig. 2b showing the relative amplitudes normalized to the amount of 1.0 WT-KV1.2 cRNA). The differences with Figure 2b are small, reflecting the relative stability of our experiments on different experimental days and different batches of oocytes: 0.25, n = 13; 0.5, n = 18; 1, n = 22; 2, n = 17; 4, n = 20; 8, n = 19. There was an overall difference between the groups (one-way ANOVA, P < 0.001); pairwise multiple comparisons between the single groups (Dunn’s method) showed a statistically significant difference between all tested groups of mutants (*P < 0.05).
Supplementary Figure 5 Gating parameters of WT and mutated KV1.2 channels.
(a) Mean voltage dependence of KV1.2 channel activation for P405L (left, dark blue), I263T (middle, light blue) and R147K (right, green) mutant and WT channels. Shown are means ± s.e.m. with lines showing Boltzmann functions fit to data points. For P405L (left): V1/2 = –12.5 ± 1.5 mV for WT (n = 16) versus V1/2 = –13.5 ± 2.0 mV for P405L (n = 7); P < 0.05, Student’s t test. kV = –6.9 ± 0.2 for WT versus kV = –6.9 ± 0.3 for P405L; n.s., Student’s t test. For I263T (middle): V1/2 = –14.8 ± 1.0 mV for WT (n = 16) versus V1/2 = –5.8 ± 1.5 mV for I263T (n = 22); P < 0.001, Mann-Whitney rank-sum test; kV = –6.8 ± 1.8 for WT versus kV = –11.5 ± 1.3 for I263T; P < 0.001, Mann-Whitney rank-sum test. For R147K (right): V1/2 = –12.1 ± 1.1 mV for WT (n = 20) versus V1/2 = –12.5 ± 0.8 mV for R147K (n = 12); n.s., Student’s t test. kV = –6.8 ± 0.3 for WT versus kV = –6.7 ± 0.2 for WT; n.s., Student’s t test. (b) Mean voltage dependence of KV1.2 channel inactivation for P405L (left, dark blue), I263T (middle, light blue) and R147K (right, green) mutants. Shown are means ± s.e.m. fitted to a standard Boltzmann function. For P405L (left): V1/2 = –24.4 ± 0.5 mV for WT (n = 11) versus V1/2 = –28.9 ± 0.9 mV for P405L (n = 7); P < 0.05, Student’s t test. kV = 3.8 ± 0.8 for WT versus kV = 3.6 ± 0.6 for P405L; n.s., Student’s t test. For I263T (middle): V1/2 = –23.3 ± 1.1 mV for WT (n = 13) versus V1/2 = –23.5 ± 1.1 mV for I263T (n = 7); n.s., Student’s t test. kV = 3.9 ± 0.4 for WT versus kV = 5.2 ± 0.6 for I263T; n.s., Student’s t test. For R147K (right): V1/2 = –24.8 ± 0.1 mV for WT (n = 18) versus V1/2 = –23.4 ± 0.5 mV for R147K (n = 16); n.s., Student’s t test. kV = 3.4 ± 0.1 for WT versus kV = 3.6 ± 0.1 for WT; n.s., Student’s t test.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–5 and Supplementary Note. (PDF 674 kb)
Supplementary Table 1
List of all variants detected in patients 1, 3 and 5 by panel analysis. (XLS 41 kb)
Supplementary Table 2
Prediction of the pathogenicity of all KCNA2 variants detected within this study as well as of all nonsynonymous KCNA2 variants reported in the ExAC database. (XLS 95 kb)
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Syrbe, S., Hedrich, U., Riesch, E. et al. De novo loss- or gain-of-function mutations in KCNA2 cause epileptic encephalopathy. Nat Genet 47, 393–399 (2015). https://doi.org/10.1038/ng.3239
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DOI: https://doi.org/10.1038/ng.3239
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