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De novo mutations in HCN1 cause early infantile epileptic encephalopathy


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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  1. Depienne, C., Gourfinkel-An, I., Baulac, S. & LeGuern, E. in Jasper's Basic Mechanisms of the Epilepsies 4th edn. 62 (eds. Noebels, J.L., Avoli, M., Rogawski, M.A., Olsen, R.W. & Delgado-Escueta, A.V.) 62 797–812 (Oxford University Press, Oxford, New York, 2012).

  2. O'Brien, J.E. & Meisler, M.H. Sodium channel SCN8A (Nav1.6): properties and de novo mutations in epileptic encephalopathy and intellectual disability. Front. Genet. 4, 213 (2013).

    Article  Google Scholar 

  3. Dravet, C. The core Dravet syndrome phenotype. Epilepsia 52 (suppl. 2), 3–9 (2011).

    Article  Google Scholar 

  4. Claes, L. et al. De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy. Am. J. Hum. Genet. 68, 1327–1332 (2001).

    CAS  Article  Google Scholar 

  5. Depienne, C. et al. Spectrum of SCN1A gene mutations associated with Dravet syndrome: analysis of 333 patients. J. Med. Genet. 46, 183–191 (2009).

    CAS  Article  Google Scholar 

  6. Depienne, C. et al. Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females. PLoS Genet. 5, e1000381 (2009).

    Article  Google Scholar 

  7. Dibbens, L.M. et al. X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment. Nat. Genet. 40, 776–781 (2008).

    CAS  Article  Google Scholar 

  8. Pinto, D. et al. Comprehensive assessment of array-based platforms and calling algorithms for detection of copy number variants. Nat. Biotechnol. 29, 512–520 (2011).

    CAS  Article  Google Scholar 

  9. Itsara, A. et al. Population analysis of large copy number variants and hotspots of human genetic disease. Am. J. Hum. Genet. 84, 148–161 (2009).

    CAS  Article  Google Scholar 

  10. Cooper, G.M. et al. A copy number variation morbidity map of developmental delay. Nat. Genet. 43, 838–846 (2011).

    CAS  Article  Google Scholar 

  11. Nava, C. et al. Prospective diagnostic analysis of copy number variants using SNP microarrays in individuals with autism spectrum disorders. Eur. J. Hum. Genet. 22, 71–78 (2014).

    CAS  Article  Google Scholar 

  12. Santoro, B. et al. Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 93, 717–729 (1998).

    CAS  Article  Google Scholar 

  13. Benarroch, E.E. HCN channels: function and clinical implications. Neurology 80, 304–310 (2013).

    Article  Google Scholar 

  14. Biel, M., Wahl-Schott, C., Michalakis, S. & Zong, X. Hyperpolarization-activated cation channels: from genes to function. Physiol. Rev. 89, 847–885 (2009).

    CAS  Article  Google Scholar 

  15. Lörincz, A., Notomi, T., Tamas, G., Shigemoto, R. & Nusser, Z. Polarized and compartment-dependent distribution of HCN1 in pyramidal cell dendrites. Nat. Neurosci. 5, 1185–1193 (2002).

    Article  Google Scholar 

  16. Poolos, N.P. in Jasper's Basic Mechanisms of the Epilepsies 4th edn. 7 (eds. Noebels, J.L., Avoli, M., Rogawski, M.A., Olsen, R.W. & Delgado-Escueta, A.V.) 7 85–96 (Oxford University Press, Oxford, New York, 2012).

  17. Kase, D. & Imoto, K. The role of HCN channels on membrane excitability in the nervous system. J. Signal Transduct. 2012, 619747 (2012).

    Article  Google Scholar 

  18. Nolan, M.F. et al. A behavioral role for dendritic integration: HCN1 channels constrain spatial memory and plasticity at inputs to distal dendrites of CA1 pyramidal neurons. Cell 119, 719–732 (2004).

    CAS  PubMed  Google Scholar 

  19. Santoro, B. et al. Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS. J. Neurosci. 20, 5264–5275 (2000).

    CAS  Article  Google Scholar 

  20. Bender, R.A. et al. Differential and age-dependent expression of hyperpolarization-activated, cyclic nucleotide–gated cation channel isoforms 1–4 suggests evolving roles in the developing rat hippocampus. Neuroscience 106, 689–698 (2001).

    CAS  Article  Google Scholar 

  21. Chen, K. et al. Persistently modified h-channels after complex febrile seizures convert the seizure-induced enhancement of inhibition to hyperexcitability. Nat. Med. 7, 331–337 (2001).

    CAS  Article  Google Scholar 

  22. Noam, Y., Bernard, C. & Baram, T.Z. Towards an integrated view of HCN channel role in epilepsy. Curr. Opin. Neurobiol. 21, 873–879 (2011).

    CAS  Article  Google Scholar 

  23. Dibbens, L.M. et al. Augmented currents of an HCN2 variant in patients with febrile seizure syndromes. Ann. Neurol. 67, 542–546 (2010).

    CAS  Article  Google Scholar 

  24. Tang, B., Sander, T., Craven, K.B., Hempelmann, A. & Escayg, A. Mutation analysis of the hyperpolarization-activated cyclic nucleotide–gated channels HCN1 and HCN2 in idiopathic generalized epilepsy. Neurobiol. Dis. 29, 59–70 (2008).

    CAS  Article  Google Scholar 

  25. Brewster, A. et al. Developmental febrile seizures modulate hippocampal gene expression of hyperpolarization-activated channels in an isoform- and cell-specific manner. J. Neurosci. 22, 4591–4599 (2002).

    CAS  Article  Google Scholar 

  26. Brãuer, A.U. et al. Molecular and functional analysis of hyperpolarization-activated pacemaker channels in the hippocampus after entorhinal cortex lesion. FASEB J. 15, 2689–2701 (2001).

    Article  Google Scholar 

  27. Jung, S. et al. Progressive dendritic HCN channelopathy during epileptogenesis in the rat pilocarpine model of epilepsy. J. Neurosci. 27, 13012–13021 (2007).

    CAS  Article  Google Scholar 

  28. Powell, K.L. et al. Decreases in HCN mRNA expression in the hippocampus after kindling and status epilepticus in adult rats. Epilepsia 49, 1686–1695 (2008).

    CAS  Article  Google Scholar 

  29. Kole, M.H., Brauer, A.U. & Stuart, G.J. Inherited cortical HCN1 channel loss amplifies dendritic calcium electrogenesis and burst firing in a rat absence epilepsy model. J. Physiol. (Lond.) 578, 507–525 (2007).

    CAS  Article  Google Scholar 

  30. Surges, R., Freiman, T.M. & Feuerstein, T.J. Gabapentin increases the hyperpolarization-activated cation current Ih in rat CA1 pyramidal cells. Epilepsia 44, 150–156 (2003).

    CAS  Article  Google Scholar 

  31. Munsch, T. & Pape, H.C. Upregulation of the hyperpolarization-activated cation current in rat thalamic relay neurones by acetazolamide. J. Physiol. (Lond.) 519, 505–514 (1999).

    CAS  Article  Google Scholar 

  32. Nolan, M.F. et al. The hyperpolarization-activated HCN1 channel is important for motor learning and neuronal integration by cerebellar Purkinje cells. Cell 115, 551–564 (2003).

    CAS  Article  Google Scholar 

  33. Santoro, B. et al. Increased seizure severity and seizure-related death in mice lacking HCN1 channels. Epilepsia 51, 1624–1627 (2010).

    Article  Google Scholar 

  34. Huang, Z., Walker, M.C. & Shah, M.M. Loss of dendritic HCN1 subunits enhances cortical excitability and epileptogenesis. J. Neurosci. 29, 10979–10988 (2009).

    CAS  Article  Google Scholar 

  35. Ludwig, A. et al. Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2. EMBO J. 22, 216–224 (2003).

    CAS  Article  Google Scholar 

  36. Chung, W.K. et al. Absence epilepsy in apathetic, a spontaneous mutant mouse lacking the h channel subunit, HCN2. Neurobiol. Dis. 33, 499–508 (2009).

    CAS  Article  Google Scholar 

  37. DiFrancesco, J.C. et al. Recessive loss-of-function mutation in the pacemaker HCN2 channel causing increased neuronal excitability in a patient with idiopathic generalized epilepsy. J. Neurosci. 31, 17327–17337 (2011).

    CAS  Article  Google Scholar 

  38. Cestèle, S., Schiavon, E., Rusconi, R., Franceschetti, S. & Mantegazza, M. Nonfunctional Nav1.1 familial hemiplegic migraine mutant transformed into gain of function by partial rescue of folding defects. Proc. Natl. Acad. Sci. USA 110, 17546–17551 (2013).

    Article  Google Scholar 

  39. Rush, A.M. et al. A single sodium channel mutation produces hyper- or hypoexcitability in different types of neurons. Proc. Natl. Acad. Sci. USA 103, 8245–8250 (2006).

    CAS  Article  Google Scholar 

  40. Ishida, S. et al. Mutations of DEPDC5 cause autosomal dominant focal epilepsies. Nat. Genet. 45, 552–555 (2013).

    CAS  Article  Google Scholar 

  41. Suls, A. et al. De novo loss-of-function mutations in CHD2 cause a fever-sensitive myoclonic epileptic encephalopathy sharing features with Dravet syndrome. Am. J. Hum. Genet. 93, 967–975 (2013).

    CAS  Article  Google Scholar 

  42. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).

    Article  Google Scholar 

  43. McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).

    CAS  Article  Google Scholar 

  44. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  45. Albers, C.A. et al. Dindel: accurate indel calls from short-read data. Genome Res. 21, 961–973 (2011).

    CAS  Article  Google Scholar 

  46. Reumers, J. et al. Optimized filtering reduces the error rate in detecting genomic variants by short-read sequencing. Nat. Biotechnol. 30, 61–68 (2012).

    CAS  Article  Google Scholar 

  47. Conrad, D.F. et al. Variation in genome-wide mutation rates within and between human families. Nat. Genet. 43, 712–714 (2011).

    CAS  Article  Google Scholar 

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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.

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Authors and Affiliations




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,

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 mutationCt RNaseP) ind tested – (Ct mutationCt 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 ( 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 ( 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).

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