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>_h</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 _nano 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.