Comprehensive classification of HCN1 variants linked to neurodevelopmental disorders with and without epilepsy

Hyperpolarization-activated cyclic nucleotide-gated 1 channels (HCN1) mediate the I <i><sub>h</sub></i> cationic current and play a central role in regulating neuronal excitability and synaptic integration. HCN1 is predominantly expressed in the neocortex and hippocampus. Pathogenic variants in <i>HCN1</i> have been increasingly identified in individuals presenting with a broad spectrum of epileptic disorders, ranging from severe developmental and epileptic encephalopathy (DEE) to milder epilepsies. Here, we used patch-clamp electrophysiology in combination with confocal imaging in HEK293 cells to functionally characterize 43 <i>HCN1</i> variants found in patients presenting with neurodevelopmental disorders, with or without epilepsy. Based on their biophysical properties, we defined four functional classes: (I) low or no current, (II) hyperpolarizing (i.e. left) shift in voltage dependence, (III) depolarizing (i.e. right) shift in voltage dependence, and (IV) generation of an instantaneous current. Integration of this functional classification with detailed clinical data from a cohort of 49 patients revealed a striking genotype-phenotype correlation. Loss-of-function variants were strongly enriched among individuals without epilepsy or with milder generalized phenotypes, whereas gain-of-function and mixed variants were predominantly associated with epilepsy, including all cases of DEE. Notably, non-epileptic cases clustered within a subgroup of loss-of-function variants affecting the selectivity filter. We further show that allosteric modulators, including the peptides NB6 and TRIP8b <sub>nano</sub> and the small molecule J&J12e, normalize the functional properties of mutant HCN1 channels in three classes. These findings establish a clinically relevant framework for interpreting <i>HCN1</i> gain- and loss-of-function variants suggesting that the direction of channel dysfunction is a major determinant of epilepsy risk and severity.

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Journal
bioRxiv (Cold Spring Harbor Laboratory)
Published
2026-03-20
DOI
https://doi.org/10.64898/2026.03.18.712601
Primary Topic
Epilepsy research and treatment
Type
article
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article

Comprehensive classification of HCN1 variants linked to neurodevelopmental disorders with and without epilepsy

Andrea Saponaro, Lauren E. Bleakley, Susanna Rizzi, Elena Pavlidis et al.
bioRxiv (Cold Spring Harbor Laboratory)
Epilepsy research and treatment
article

Comprehensive classification of HCN1 variants linked to neurodevelopmental disorders with and without epilepsy

Andrea Saponaro, Lauren E. Bleakley, Susanna Rizzi, Elena Pavlidis, Christel Depienne, Roberta Castelli, Vishnu Anand Cuddapah, Carlo Fusco, Gianpiero L. Cavalleri, Shimriet Zeidler, Dario DiFrancesco, Richard Caswell, Dario Pruna, Bina Santoro, Yline Capri, Charlotte Sherlaw, Ghayda Mirzaa, Nathalie Couque, Gerhard Thiel, A. Moroni, A Guariglia F Porro, Frederic Tran Mau Them, Anna Castellini, Amitav Parida, Ingrid Scheffer, Fabio Sirchia, Krzystof Szczaluba, Carla Marini, Chris Read, Christian M Korff, Laurence Faivre, Deb Pal, Katerine B Howell, Greta Fontana
article en

Abstract

Hyperpolarization-activated cyclic nucleotide-gated 1 channels (HCN1) mediate the I <i><sub>h</sub></i> cationic current and play a central role in regulating neuronal excitability and synaptic integration. HCN1 is predominantly expressed in the neocortex and hippocampus. Pathogenic variants in <i>HCN1</i> have been increasingly identified in individuals presenting with a broad spectrum of epileptic disorders, ranging from severe developmental and epileptic encephalopathy (DEE) to milder epilepsies. Here, we used patch-clamp electrophysiology in combination with confocal imaging in HEK293 cells to functionally characterize 43 <i>HCN1</i> variants found in patients presenting with neurodevelopmental disorders, with or without epilepsy. Based on their biophysical properties, we defined four functional classes: (I) low or no current, (II) hyperpolarizing (i.e. left) shift in voltage dependence, (III) depolarizing (i.e. right) shift in voltage dependence, and (IV) generation of an instantaneous current. Integration of this functional classification with detailed clinical data from a cohort of 49 patients revealed a striking genotype-phenotype correlation. Loss-of-function variants were strongly enriched among individuals without epilepsy or with milder generalized phenotypes, whereas gain-of-function and mixed variants were predominantly associated with epilepsy, including all cases of DEE. Notably, non-epileptic cases clustered within a subgroup of loss-of-function variants affecting the selectivity filter. We further show that allosteric modulators, including the peptides NB6 and TRIP8b <sub>nano</sub> and the small molecule J&J12e, normalize the functional properties of mutant HCN1 channels in three classes. These findings establish a clinically relevant framework for interpreting <i>HCN1</i> gain- and loss-of-function variants suggesting that the direction of channel dysfunction is a major determinant of epilepsy risk and severity.

bioRxiv (Cold Spring Harbor Laboratory)
Marche Polytechnic University (IT), Royal Children's Hospital (AU), Royal College of Surgeons in Ireland (IE), Inserm (FR), The University of Melbourne (AU), Science Foundation Ireland (IE), Université de Bourgogne (FR), Baylor College of Medicine (US), King's College London (GB), University of Milan (IT), University of Washington (US), Université Paris Cité (FR), Florey Institute of Neuroscience and Mental Health (AU), University of Pavia (IT), Medical University of Warsaw (PL), Royal Devon & Exeter NHS Foundation Trust (GB), Erasmus MC (NL), Technische Universität Darmstadt (DE), Norcliffe Foundation (US), Ospedale di Bolzano (IT), Assistance Publique – Hôpitaux de Paris (FR), Birmingham Children's Hospital (GB), University Hospital of Geneva (CH), Ospedale Santa Maria (IT), Neurological Research Institute (US), Hôpital Robert-Debré (FR), Texas Children's Hospital (US), Murdoch Children's Research Institute (AU), Istituti di Ricovero e Cura a Carattere Scientifico (IT), Birmingham Women’s and Children’s NHS Foundation Trust (GB), Azienda Ospedaliera G. Brotzu (IT), University of Duisburg-Essen (DE), Geneva College (US), Columbia University (US), University of Birmingham (GB)
Good health and well-being
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Epilepsy research and treatment
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