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De novo mutations in HCN1 cause early infantile epileptic encephalopathy

Nature Genetics volume 46, pages 640–645 (2014)Cite this article

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Abstract

Hyperpolarization-activated, cyclic nucleotide–gated (HCN) channels contribute to cationic Ih current in neurons and regulate the excitability of neuronal networks. Studies in rat models have shown that the Hcn1 gene has a key role in epilepsy, but clinical evidence implicating HCN1 mutations in human epilepsy is lacking. We carried out exome sequencing for parent-offspring trios with fever-sensitive, intractable epileptic encephalopathy, leading to the discovery of two de novo missense HCN1 mutations. Screening of follow-up cohorts comprising 157 cases in total identified 4 additional amino acid substitutions. Patch-clamp recordings of Ih currents in cells expressing wild-type or mutant human HCN1 channels showed that the mutations had striking but divergent effects on homomeric channels. Individuals with mutations had clinical features resembling those of Dravet syndrome with progression toward atypical absences, intellectual disability and autistic traits. These findings provide clear evidence that de novo HCN1 point mutations cause a recognizable early-onset epileptic encephalopathy in humans.

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Figure 1: Identification of HCN1 missense mutations.
Figure 2: Patch-clamp analysis of the functional effects of the de novo HCN1 mutations.
Figure 3: Data analysis of coexpression of wild-type and mutant human HCN1 channels with the patch-clamp technique.

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References

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Acknowledgements

We thank the patients and their families for their participation in this study, J. Stieber (Friedrich Alexander Universität Erlangen-Nürnberg, Germany) for providing a plasmid containing human HCN1 cDNA, the International Parkinson's Disease Genomics Consortium (IPDGC) for granting access to the list of HCN1 variants present in control populations and populations with Parkinson's disease, L. Van de Velde Boermans for genetic tests for SCN1A and PCDH19 and tests on the parents, M. Nizard for case selection, and G. Huguet and T. Bourgeron for helpful discussions. The research generating these results was funded by the University of Würzburg, Biocodex, Fondation de France, ERA-NET NEURON EUHFAUTISM, the “Investissements d'Avenir” program ANR-10-IAIHU-06 (IHU-A-ICM), INSERM, AP-HP, the Eurocores program EuroEPINOMICS of the European Science Foundation, the Fund for Scientific Research Flanders (FWO) and the University of Antwerp. P.S. and F.Z. thank the Genetics Commission of the Italian League Against Epilepsy (LICE) for their support. B.P.C.K. and C.G.F.d.K. were supported by the Netherlands National Epilepsy Fund. A.S. is a postdoctoral fellow of the FWO. C.N., A.B. and C. Depienne are members of the Bio-Psy Labex.

Author information

Author notes

  1. Rudi Balling, Nina Barisic, Stéphanie Baulac, Hande S Caglayan, Dana C Craiu, Peter De Jonghe, Christel Depienne, Padhraig Gormley, Renzo Guerrini, Ingo Helbig, Helle Hjalgrim, Dorota Hoffman-Zacharska, Johanna Jähn, Karl Martin Klein, Bobby P C Koeleman, Vladimir Komarek, Roland Krause, Eric LeGuern, Anna-Elina Lehesjoki, Johannes R Lemke, Holger Lerche, Carla Marini, Patrick May, Rikke S Møller, Hiltrud Muhle, Aarno Palotie, Deb Pal, Felix Rosenow, Kaja Selmer, José M Serratosa, Sanjay Sisodiya, Ulrich Stephani, Katalin Sterbova, Pasquale Striano, Arvid Suls, Tiina Talvik, Sarah von Spiczak, Yvonne Weber, Sarah Weckhuysen and Federico Zara Tiina Talvik and: Full lists of members and affiliations appear at the end of the paper.

  2. Caroline Nava, Carine Dalle and Tiina Talvik: These authors contributed equally to this work

Authors and Affiliations

  1. INSERM UMR 975, Institut du Cerveau et de la Moelle Epinière, Hôpital Pitié-Salpêtrière, Paris, France

    Caroline Nava, Carine Dalle, Agnès Rastetter, Oriane Trouillard, Dahbia Agher, Emmanuel Roze, Suzanne Lesage, Aude Nicolas, Alexis Brice, Michel Baulac, Stéphanie Baulac, Stéphanie Baulac, Christel Depienne, Eric LeGuern, Eric LeGuern & Christel Depienne

  2. CNRS 7225, Hôpital Pitié-Salpêtrière, Paris, France

    Caroline Nava, Emmanuel Roze, Suzanne Lesage, Aude Nicolas, Alexis Brice, Michel Baulac, Stéphanie Baulac, Stéphanie Baulac, Eric LeGuern & Eric LeGuern

  3. Université Pierre et Marie Curie–Paris 6 (UPMC), UMRS 975, Paris, France

    Caroline Nava, Oriane Trouillard, Emmanuel Roze, Suzanne Lesage, Aude Nicolas, Alexis Brice, Michel Baulac, Stéphanie Baulac, Stéphanie Baulac, Christel Depienne, Eric LeGuern, Eric LeGuern & Christel Depienne

  4. Département de Génétique et de Cytogénétique, Assistance Publique–Hôpitaux de Paris (AP-HP), Hôpital Pitié-Salpêtrière, Unité Fonctionnelle de Neurogénétique Moléculaire et Cellulaire, Paris, France

    Caroline Nava, Oriane Trouillard, Alexis Brice, Eric LeGuern & Eric LeGuern

  5. Institut du Cerveau et de la Moelle Epinière, Plateforme d'Electrophysiologie, Paris, France

    Carine Dalle

  6. Department of Neurosciences, Pediatric Neurology and Muscular Diseases Unit, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, 'G Gaslini Institute', Genova, Italy

    Pasquale Striano & Pasquale Striano

  7. Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands

    Carolien G F de Kovel, Eva H Brilstra, Bobby P C Koeleman & Bobby P C Koeleman

  8. Department of Pediatric Neurology, Centre de Référence Epilepsies Rares, Hôpital Necker–Enfants Malades, AP-HP, Paris, France

    Rima Nabbout

  9. INSERM U663, Université Paris Descartes, Sorbonne Paris Cité, Hôpital Necker–Enfants Malades, Paris, France

    Rima Nabbout

  10. Service de Neurologie Pédiatrique, Hôpital des Enfants, Centre Hospitalier Universitaire de Toulouse, Toulouse, France

    Claude Cancès

  11. Service de Neurologie Pédiatrique, Hôpital Femme Mère Enfant, Centre Hospitalier Universitaire de Lyon, Bron, France

    Dorothée Ville

  12. Child Neurology Unit, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Institute of Neurological Sciences of Bologna, Bologna, Italy

    Giuseppe Gobbi

  13. Service de Neuropédiatrie, Hôpital d'Enfants de Brabois, Centre Hospitalier Universitaire de Nancy, Vandoeuvre Les Nancy, France

    Emmanuel Raffo

  14. Institut du Cerveau et de la Moelle Epinière, Plateforme de Génotypage et Séquençage, Paris, France

    Delphine Bouteiller & Yannick Marie

  15. Department of Neurosciences, Laboratory of Neurogenetics, Gaslini Institute, Genova, Italy

    Angela Robbiano, Federico Zara & Federico Zara

  16. Département de Génétique et de Cytogénétique, AP-HP, Hôpital Pitié-Salpêtrière, Unité Fonctionnelle de Cytogénétique, Paris, France

    Boris Keren

  17. Institüt für Humangenetik, Universität Würzburg, Würzburg, Germany

    Cornelia Vogt, Nady El Hajj, Eberhard Schneider, Christel Depienne, Thomas Haaf & Christel Depienne

  18. Department of Molecular Genetics, Neurogenetics Group, VIB, Antwerp, Belgium

    Arvid Suls, Sarah Weckhuysen, Peter De Jonghe, Peter De Jonghe, Arvid Suls & Sarah Weckhuysen

  19. Laboratory of Neurogenetics, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium

    Arvid Suls, Sarah Weckhuysen, Peter De Jonghe, Peter De Jonghe, Arvid Suls & Sarah Weckhuysen

  20. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, UK

    Padhraig Gormley, Padhraig Gormley & Aarno Palotie

  21. Folkhälsan Institute of Genetics, Helsinki, Finland

    Anna-Elina Lehesjoki & Anna-Elina Lehesjoki

  22. Research Programs Unit, Molecular Neurology and Neuroscience Center, University of Helsinki, Helsinki, Finland

    Anna-Elina Lehesjoki & Anna-Elina Lehesjoki

  23. Department of Neuropediatrics, University Medical Center Schleswig-Holstein, Christian Albrechts University, Kiel, Germany

    Ingo Helbig, Ingo Helbig, Johanna Jähn, Hiltrud Muhle, Ulrich Stephani & Sarah von Spiczak

  24. Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Esch-sur-Alzette, Luxembourg

    Rudi Balling, Roland Krause & Patrick May

  25. Department of Paediatrics, University of Zagreb, Medical School, University Hospital Centre Zagreb, Zagreb, Croatia

    Nina Barisic

  26. Department of Molecular Biology and Genetics, Boğaziçi University, Istanbul, Turkey

    Hande S Caglayan

  27. Department of Neurology, Pediatric Neurology Clinic II, Pediatric Neurology, Psychiatry, Neurosurgery, “Carol Davila” University of Medicine, Bucharest, Romania

    Dana C Craiu

  28. Pediatric Neurology Clinic, “Professor Doctor Alexandru Obregia” Clinical Hospital, Bucharest, Romania

    Dana C Craiu

  29. Pediatric Neurology Unit and Laboratories, Children's Hospital A. Meyer, University of Florence, Florence, Italy

    Renzo Guerrini & Carla Marini

  30. Danish Epilepsy Centre, Dianalund, Denmark

    Helle Hjalgrim & Rikke S Møller

  31. Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland

    Dorota Hoffman-Zacharska

  32. Department of Neurology, Epilepsy Center Hessen, University Hospitals Marburg and Philipps, University Marburg, Marburg, Germany

    Karl Martin Klein & Felix Rosenow

  33. Child Neurology Department, University Hospital Motol, Prague, Czech Republic

    Vladimir Komarek & Katalin Sterbova

  34. Division of Human Genetics, University Children's Hospital Inselspital, Bern, Switzerland

    Johannes R Lemke

  35. Department of Neurology and Epileptology, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany

    Holger Lerche & Yvonne Weber

  36. Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland

    Aarno Palotie

  37. Department of Clinical Neuroscience, Institute of Psychiatry, King's College London, London, UK

    Deb Pal

  38. Department of Medical Genetics, Oslo University Hospital, Oslo, Norway

    Kaja Selmer

  39. Institute of Medical Genetics, University of Oslo, Oslo, Norway

    Kaja Selmer

  40. Epilepsy Unit, Neurology Service, Hospital Universitario Fundación Jiménez Díaz and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain

    José M Serratosa

  41. Department of Clinical and Experimental Epilepsy, University College London Institute of Neurology, Queen Square, London, UK

    Sanjay Sisodiya

  42. Department of Pediatrics, University of Tartu, Tartu, Estonia

    Tiina Talvik

  43. Department of Neurology and Neurorehabilitation, Children's Clinic, Tartu University Hospital, Tartu, Estonia

    Tiina Talvik

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  1. Caroline Nava
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Consortia

EuroEPINOMICS RES Consortium

  • Rudi Balling
  • , Nina Barisic
  • , Stéphanie Baulac
  • , Hande S Caglayan
  • , Dana C Craiu
  • , Peter De Jonghe
  • , Christel Depienne
  • , Padhraig Gormley
  • , Renzo Guerrini
  • , Ingo Helbig
  • , Helle Hjalgrim
  • , Dorota Hoffman-Zacharska
  • , Johanna Jähn
  • , Karl Martin Klein
  • , Bobby P C Koeleman
  • , Vladimir Komarek
  • , Roland Krause
  • , Eric LeGuern
  • , Anna-Elina Lehesjoki
  • , Johannes R Lemke
  • , Holger Lerche
  • , Carla Marini
  • , Patrick May
  • , Rikke S Møller
  • , Hiltrud Muhle
  • , Aarno Palotie
  • , Deb Pal
  • , Felix Rosenow
  • , Kaja Selmer
  • , José M Serratosa
  • , Sanjay Sisodiya
  • , Ulrich Stephani
  • , Katalin Sterbova
  • , Pasquale Striano
  • , Arvid Suls
  • , Tiina Talvik
  • , Sarah von Spiczak
  • , Yvonne Weber
  • , Sarah Weckhuysen
  •  & Federico Zara

Contributions

Clinical and genetic data. French cohort. C. Depienne and C.N. analyzed whole-exome sequencing data. C.N., A. Rastetter, D.A., D.B., Y.M., C.V., N.E.H. and E.S. contributed to pyrosequencing and/or Sanger sequencing and sequence analysis. B.K. and C.N. contributed to CNV analysis. O.T. performed quantitative PCR. C.C., D.V., R.N. and E. Raffo phenotyped and sampled the cases and provided clinical information. E. Roze was involved in case selection. A.B., A.N. and S.L. contributed to variant analysis in populations from IPDGC. S.B., M.B. and A.B. contributed to study design and discussions. C. Depienne and E.L. supervised the projects related to EIEE, including cohort collection. C. Depienne, E.L. and T.H. designed this study. Dutch cohort. B.P.C.K. and C.G.F.d.K. designed the study and analyzed sequencing data. B.P.C.K. supervised the study. E.H.B. phenotyped and sampled the cases. Italian case. This case was included in the EuroEPINOMICS RES Consortium. A.S., I.H. and P.G. analyzed whole-exome sequencing data. P.S., A. Robbiano and F.Z. contributed to validation of exome sequencing data and supervision of the study. G.G. phenotyped the case and provided clinical information. P.D.J., I.H., A.S., S.W. and A.-E.L. designed the study and/or coordinated projects in the EuroEPINOMICS RES Consortium. Functional studies. C.N. and A. Rastetter performed site-directed mutagenesis, cell transfection, immunohistochemistry and plasma membrane enrichment experiments. C. Dalle performed electrophysiology analysis and wrote the sections relating to electrophysiology. C. Depienne supervised the collaborative study and drafted the manuscript. All authors critically revised the manuscript.

Corresponding authors

Correspondence to Eric LeGuern or Christel Depienne.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Comparison of the mean number of rare variants per base and per individual between HCN1 and selected genes expressed in the brain.

The numbers of truncating variants (nonsense and splice site), rare missense variants (with MAF < 1‰), very rare missense variants (present in fewer than ten individuals) and very rare missense variants predicted to be possibly or probably damaging by Polyphen-2 per base and per individual have been calculated for selected genes expressed in the brain, associated with autosomal dominant epilepsies or epileptic encephalopathies (SCN1A, SCN2A, SCN8A, SCN1B, CHD2, STXBP1, KCNT1, GRIN2A, GRIN2B, SYNGAP1), autosomal recessive various phenotypes (CLCN2, EPM2A, NHLRC1, POLG, RELN) and X-linked epileptic encephalopathy (PCDH19) and for genes associated with complex phenotypes (DRD4) or no known disorder in humans (HTR3B), from data of the European ESP population (Exome Variant Server, http://evs.gs.washington.edu/EVS/).

Supplementary Figure 2 Identification of a heterozygous deletion encompassing exon 4 of HCN1 in a female subject with ID and ASD, inherited from her asymptomatic father.

(a) Cyto-12 SNP array (Illumina) profiles of the patient with HCN1 exon 4 deletion and details of the coding sequences included the deletion: the y axis indicates the B allele frequency (above) and the log R ratio (below), and the x axis indicates the position on chromosome 5. The deletion spans ~33 kb and theoretically leads to an in-frame deletion of 73 amino acids (p.Asn338_Lys410del) in the extracellular loop between the S5 and S6 domains containing the pore region. (b) Confirmation of the presence of the HCN1 heterozygous deletion in the proband and her father by quantitative PCR using primers located on exon 4 (forward: 5'-CCACCTGCTATGCCATGTTT-3'; reverse: 5'-ATACTGCCGCCTCGAAGsAAT-3'). RT-PCR experiments were performed using 10 ng of genomic DNA, 0.8 μM of each primer and 12.5 μl of SYBR Green PCR master mix (Applied Biosystems) in a total volume of 25 μl. RNase P (RNAse P Control Assay, Applied Biosystems) was used as the reference amplicon. Each sample was run in triplicate on an ABI PRISM 7700 Detection System (Applied Biosystems), and three different experiments were used for final quantification. Relative ratios were calculated using the formula r = 2 − Δ Δ C T with ΔΔCt = (Ct mutation – Ct RNaseP) ind tested – (Ct mutation – Ct RNaseP) ind ref.

Supplementary Figure 3 Alignment of the regions flanking the six missense variants in orthologous proteins, showing the conservation of the altered amino acids.

Multiple pairwise alignments were performed using ClustalW 2.1 (http://www.genome.jp/tools/clustalw/). The amino acids altered by the mutations are highlighted. Full alignments including all species are provided as a Supplementary Note.

Supplementary Figure 4 Reversal potential (Erev) of gain-of-function mutations affecting the hHCN1 channel.

(a) Representative whole-cell current trace recorded from a CHO-K1 cell transfected with construct for wild-type (WT) hHCN1 channel. For determination of Erev, currents were fully activated by a prepulse to –130 mV for 1 s from a holding potential of –20 mV, and test pulses were then applied from –100 mV to +40 mV in 10-mV increments, with tail current measured immediately after the prepulse. (b) Plot of mean tail current density as a function of test voltage for WT, S100F, H279Y and D401H hHCN1 channels. The number of cells for each condition is indicated in parentheses. The red line represents a linear regression through the mean values for the tail current density and is used to obtain Erev. (c) Bar graph of Erev for WT hHCN1 and the three mutant channels (one-way ANONA followed by a Dunnett's test, ***P < 0.001). Data are represented as mean values ± s.e.m. with numbers of experiments given in parentheses.

Supplementary Figure 5 Data analysis of loss-of-function mutations affecting the hHCN1 channel identified with the patch-clamp technique.

Plot of mean current density as a function of test voltage for control cells (cells transfected only with pEGFP plasmid, reflecting the endogenous current) and cells expressing S272P and R297T mutant channels. The number of cells for each condition is indicated in parentheses. Currents were elicited by test pulses ranging from –140 mV to +40 mV in 10-mV increments from a holding potential of –20 mV and were normalized to cell capacitance (representative traces in Fig. 2a). Current densities of both mutant channels were not significantly different compared with endogenous currents from control cells (one-way ANOVA followed by a Dunnett's test, P > 0.05). Data are presented as means ± s.e.m. with numbers of experiments given in parentheses.

Supplementary Figure 6 Impact of de novo HCN1 mutations on protein expression and localization at the plasma membrane.

(a) Semiquantitative analysis of WT and mutant (S100F, S272P, R297T, H279Y or D401H) HCN1 protein expression in whole-cell lysates and at the plasma membrane by protein blot. CHO-K1 cells were transiently cotransfected with 12 μg of WT or mutant HCN1 expression plasmid using the Neon electroporation system (Invitrogen); 24 h after transfection, proteins present at the plasma membrane were isolated using the Cell Surface Protein Isolation kit (Pierce), following the manufacturer's recommendations. Whole-cell lysates were kept before isolation of membrane protein from the same experiments. Proteins present in both fractions were resolved by SDS-PAGE on 4–12% gradient gels (Invitrogen) and electrotransferred onto nitrocellulose membranes. HCN1 was probed using a monoclonal mouse anti-HCN1 antibody (ab84816, Abcam; 1:10,000 dilution), and the signal was visualized with enhanced chemiluminescence (Pierce). Membranes were probed with anti–Flotillin-1 antibody (610820, BD Biosciences; 1:1,000 dilution) for normalization. The image shows the result of a representative experiment. (b) Semiquantitative measurement of WT and mutant HCN1 proteins present in whole-cell lysates (blue) and at the plasma membrane (red) from three independent experiments using the ImageJ program (http://rsb.info.nih.gov/ij/). The values obtained for WT and mutant HCN1 proteins were corrected for the intensity of the corresponding flotillin bands. Data are presented as means ± s.e.m. Note that, although the expression of S100F, S272P and R297T HCN1 channels was similarly decreased, an Ih current was recorded for S100F but nor for S272P and R297T. In addition, the S272P and R297T mutants showed a dominant-negative effect on the WT-mutant heteromeric channels, suggesting that mutant homomeric channels are less stable than WT-mutant heteromeric channels. (c) Effect of proteasome inhibition on mutant (S100F, S272P, R297T, H279Y or D401H) HCN1 protein expression in whole-cell lysates. CHO-K1 cells were transiently cotransfected with WT or mutant HCN1 expression plasmids; 24 h after transfection, half of the cells was treated with 1 μM epoxomicin (E3652, Sigma) for 16 h. Proteins were then resolved by SDS-PAGE on 4–12% gradient gels (Invitrogen) and electrotransferred onto nitrocellulose membranes. HCN1 was probed using the monoclonal mouse anti-HCN1 antibody, and the signal was detected by enhanced direct near-infrared fluorescence detection system (LI-COR Biosciences). Membranes were probed with anti-actin antibody (ab3280, Abcam; 1:4,000 dilution) for normalization. The image shows the result of a representative experiment.

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Nava, C., Dalle, C., Rastetter, A. et al. De novo mutations in HCN1 cause early infantile epileptic encephalopathy. Nat Genet 46, 640–645 (2014). https://doi.org/10.1038/ng.2952

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