Hyperpolarization activated Cyclic Nucleotide (HCN) gated channels are crucial for various neurophysiological functions, including learning and sensory functions, and their dysfunction are responsible for brain disorders, such as epilepsy. To date, HCN2 variants have only been associated with mild epilepsy and recently, one monoallelic missense variant has been linked to developmental and epileptic encephalopathy. Here, we expand the phenotypic spectrum of HCN2-related disorders by describing twenty-one additional individuals from fifteen unrelated families carrying <HCN2> variants. Seventeen individuals had developmental delay/intellectual disability (DD/ID), two had borderline DD/ID, and one had borderline DD. Ten individuals had epilepsy with DD/ID, with median age of onset of 10 months, and one had epilepsy with normal development. Molecular diagnosis identified thirteen different pathogenic HCN2 variants, including eleven missense variants affecting highly conserved amino acids, one frameshift variant, and one in-frame deletion. Seven variants were monoallelic of which five occurred de novo, one was not maternally inherited, one was inherited from a father with mild learning disabilities, and one was of unknown inheritance. The remaining six variants were biallelic, with four homozygous and two compound heterozygous variants. Functional studies using two-electrode voltage-clamp recordings in <Xenopus laevis> oocytes were performed on three monoallelic variants, p.(Arg324His), p.(Ala363Val), and p.(Met374Leu), and three biallelic variants, p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp). The p.(Arg324His) variant induced a strong increase of HCN2 conductance, while p.(Ala363Val) and p.(Met374Leu) displayed dominant negative effects, leading to a partial loss of HCN2 channel function. By confocal imaging, we found that the p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp) pathogenic variants impaired membrane trafficking, resulting in a complete loss of HCN2 elicited currents in <Xenopus> oocytes. Structural 3D-analysis in depolarized and hyperpolarized states of HCN2 channels, revealed that the pathogenic variants p.(His205Gln), p.(Ser409Leu), p.(Arg324Cys), p.(Asn369Ser) and p.(Gly460Asp) modify molecular interactions altering HCN2 function. Taken together, our data broadens the clinical spectrum associated with HCN2 variants, and disclose that HCN2 is involved in developmental encephalopathy with or without epilepsy.

The authors have declared no competing interest.

This work was supported by a grant from the Groupama Foundation Vaincre les maladies rares. For individual 1, sequencing and analysis were provided by the Broad Institute of MIT and Harvard Center for Mendelian Genomics (Broad CMG) and were funded by the National Human Genome Research Institute grants UM1 HG008900 (with additional support from the National Eye Institute, and the National Heart, Lung and Blood Institute), U01HG011755, and R01 HG009141, and in part by grant number 2020-224274 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation. For individual 8, SS received funding by the Dietmar Hopp Stiftung [1DH1813319]. Dr Howell was supported by the Melbourne Children's Clinician Scientist Fellowship scheme, and grants from the National Health and Medical Research Council and the Medical Research Futures Fund. Dr Howell has received project funding (for unrelated work) from Praxis Precision Medicines, RogCon, Inc, and UCB Australia. The Murdoch Children's Research Institute is supported by the Victorian State Government Operational Infrastructure Program.

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