ABSTRACT _HCN1_ is one of four genes encoding hyperpolarization-activated cyclic nucleotide-gated channels. The phenotypic spectrum associated with _HCN1_ variants ranges from neonatal

developmental and epileptic encephalopathy to idiopathic generalized epilepsy. We report a Japanese patient with repetitive focal seizures and super-refractory status epilepticus since early

infancy caused by a _de novo HCN1_ variant, NM_021072.4, c.1195T>C, p.(Ser399Pro). This variant might have a dominant-negative effect on channel function, leading to severe epileptic

encephalopathy. SIMILAR CONTENT BEING VIEWED BY OTHERS DE NOVO Y1460C MISSENSE VARIANT IN NAV1.1 IMPEDES THE PORE REGION AND RESULTS IN EPILEPTIC ENCEPHALOPATHY Article Open access 13

cyclic nucleotide-gated (HCN) channels mediate a cationic current that stabilizes the neuronal membrane potential against excitatory or inhibitory input and regulates neuronal network

excitability1,2. _HCN1_ (NM_021072), one of the four genes that encode HCN channels1, is highly expressed in the neocortex, hippocampus, and brainstem of the central nervous system3. It was

therefore presumed that pathogenic _HCN1_ variants could produce pharmacoresponsive epilepsy or developmental and epileptic encephalopathy (DEE). In 2014, Nava _et al_. identified five

different _de novo HCN1_ variants in patients with Dravet-like syndrome without _SCN1A_ and _PCDH19_ variants or fever-sensitive epileptic encephalopathy4. Additionally, recent studies have

revealed that the phenotypic spectrum associated with _HCN1_ variants ranges from genetic epilepsy with febrile seizures plus (OMIM #618482) or genetic generalized epilepsy to neonatal- or

infantile-onset DEE5,6,7,8,9,10 (OMIM #615871). Herein, we report a Japanese patient with a de novo heterozygous _HCN1_ variant who had presented since early infancy with repetitive focal

seizures and recurrent super-refractory status epilepticus, which were associated with profound developmental delay. This report provides detailed clinical findings that expand the known

phenotypic spectrum of _HCN1_-related DEE. The male patient, the second child of unrelated parents, was born at 39 weeks gestational age after a normal pregnancy and delivery. His birth

weight, length, and occipitofrontal circumference were 3380 g (+0.8 standard deviation score [SDS]), 50.8 cm (+0.9 SDS), and 33.8 cm (+0.3 SDS), respectively. His father, elder brother, and

maternal uncle had a history of febrile seizures. At 2 months of age, the patient developed convulsive seizures that gradually increased in frequency, occurring in clusters despite

administration of phenobarbital and sodium valproate. He was first admitted to our hospital at 4 months of age. His anthropometric measurements were as follows: body weight 7.6 kg (+0.6

SDS), height 60.5 cm (−1.6 SDS), and occipitofrontal circumference 42.4 cm (+1.3 SDS). Psychomotor development was normal. After admission, right or left hemi-clonic seizures and focal to

bilateral tonic–clonic seizures were observed. Shortly afterward, a respiratory infection elicited a high fever and provoked the onset of convulsive status epilepticus. Intravenous

anti-seizure medications, including diazepam, midazolam, phenytoin, and lidocaine, were ineffective. Ultimately, thiopental was effective in terminating seizure activity. An interictal

electroencephalogram (EEG) showed spike discharges in the left frontal area. An ictal EEG revealed spike-wave discharge with onset in the left fronto-centro-parietal region (Fig. 1A) or

right fronto-central region. Laboratory examinations and brain magnetic resonance imaging (MRI) findings showed no abnormalities at that time (Fig. 1B). At 6 months of age, during another

respiratory tract infection, the patient developed a prolonged generalized tonic convulsion lasting 30 min, which evolved into repetitive generalized convulsions with prolonged hypoxia. This

prolonged seizure was refractory to anticonvulsants, including thiopental, and lasted 36 h. Pentobarbital was given intravenously, and mechanical ventilation was required for 2 weeks. After

extubation, seizures were controlled by high-dose phenobarbital treatment combined with potassium bromide and zonisamide. However, the patient’s motor and cognitive development were

severely impaired. Brain MRI at 8 months of age showed severe cortical and white matter atrophy (largely in the frontal lobe) and ventricular enlargement (Fig. 1C). At 10 months of age,

brief daily seizures involving grinning or a pale face were noted. At 13 years of age, the patient had generalized hypotonia and spastic quadriplegia with profound intellectual and motor

impairment. He could not control his head, spoke no meaningful words and was almost entirely bedridden. An increase in brief seizures with tonic movements elicited the addition of

levetiracetam and topiramate to existing medications (phenobarbital, potassium bromide, and zonisamide). However, these medications were only partially effective. At 18 years of age, the

patient continued to have brief focal seizures, often in clusters requiring intravenous phenobarbital for seizure control. Whole exome sequencing was performed using a SureSelectXT Human All

Exon v5 (Agilent Technologies, Santa Clara, CA), and captured libraries were sequenced using an Illumina HiSeq 2500 (Illumina, San Diego, CA) with 101 base-paired end reads. Exome data

processing, variant calling, and variant annotation were performed as previously described11. Variant pathogenicity was predicted using SIFT, Polyphen-2, CADD and M-CAP (Table S1). Whole

exome sequencing of the patient’s leukocyte-derived genomic DNA identified an _HCN1_ variant: NM_021072.4, c.1195T>C, p.(Ser399Pro). Trio-based Sanger sequencing confirmed that this was a

de novo variant. The variant was absent in 38 K JPN and gnomAD v2.1.1 (accessed on 22nd Feb 2023), as well as in our 408 in-house control exomes (all Japanese). The same variant was

previously reported in a patient with infantile epileptic encephalopathy who had prolonged febrile seizures and intractable apneic tonic–clonic seizures starting at 4 months of age5.

Therefore, the variant was classified as pathogenic according to the ACMG-AMP Guidelines (PS1, PS2, PM2, and PP3). The clinical and molecular genetic studies were performed in accordance

with the Declaration of Helsinki and were approved by the institutional review board of Yamagata University Faculty of Medicine, Showa University School of Medicine, and Yokohama City

University School of Medicine. The patient’s parents provided written informed consent. We compared the features of the present patient with those of a previous patient carrying the same

genetic variant5 and summarized the clinical manifestations of previously reported cases10 (Table 1). The present patient showed infantile-onset DEE with repetitive focal seizures, recurrent

refractory status epilepticus, and progressive brain atrophy. Among previously reported patients with _HCN1_ variants, brain atrophy was observed in only one patient with a p.G391D

variant5,10. Therefore, the phenotype in the present patient appears to be more severe than that of a previous patient with the same p.S399P variant. This phenotypic variability may be

caused by genetic modifiers such as a prominent family history of febrile seizures even though no other variants in the known epilepsy-related genes were identified through whole exome

sequencing of this patient. Progressive brain atrophy may also have been caused by acute encephalopathy or encephalitis. However, because MRI changes were not observed during the acute phase

of status epilepticus in the patient, it was unlikely that he had acute encephalopathy or encephalitis. Recurrent status epilepticus, refractory to intravenous anticonvulsant

administration, was one of the characteristic features observed in this patient. Administration of general anesthesia was necessary to abolish a prolonged seizure lasting 36 hours. In

previous studies, status epilepticus was reported in 5/18 patients with loss-of-function _HCN1_ variants and in 1/13 patients with gain-of-function _HCN1_ variants9. The p.S399P variant is

also a loss-of-function variant. However, status epilepticus is caused not by a specific _HCN1_ variant but by a variety of variants9. Both upregulation and downregulation of HCN channels

have been associated with epileptic activity in animal models5,12. A previous study indicated that patients with the p.G391D variant showed the most severe phenotypes among _HCN1_ DEE5

patients, similar to the present patient with the p.S399P variant. HCN subunits have six transmembrane domains (S1–S6), including a positively charged voltage sensor (S4) and the

ion-conducting pore region between S5 and S63. The amino acid glycine 391, located on the intracellular interface of the S6 transmembrane domain, is adjacent to the p.S399P variant5.

Transient expression of _HCN1_ variants in Chinese hamster ovary cells showed that the protein levels of both the p.G391D and p.S399P variants were significantly decreased compared with

those of wild-type proteins. In addition, whole-cell patch-clamp recordings showed no current in cells separately transfected with each variant, suggesting that these are loss-of-function

variants5. Interestingly, when cells were cotransfected with wild-type and p.G391D variant constructs, a strong reduction in current density was observed, suggesting that p.G391D has a

dominant-negative effect on heteromeric channel function. Similarly, severe DEE in the present patient may have been caused by a dominant-negative effect of the p.S399P variant. The

epileptic seizure frequency in patients with _HCN1_ variants ranges from no to daily seizures, and half of patients demonstrate resistance to anti-seizure medications5. High-dose

phenobarbital and potassium bromide were partially effective for the present patient. In murine models of p.G391D and p.M153I, administration of the sodium channel antagonists lamotrigine

and phenytoin resulted in paradoxical induction of seizures; however, administration of sodium valproate did not lead to convulsive seizures13. In _Hcn1_M294L mice carrying a homolog of the

_HCN1_ p.M305L variant; phenytoin, lamotrigine, retigabine, and carbamazepine increased spike frequency, whereas levetiracetam, diazepam, sodium valproate, and ethosuximide significantly

reduced spike frequency14. Sodium channel blockers such as carbamazepine or phenytoin may aggravate seizures in Dravet syndrome with _SCN1A_ variants15. These blockers might also aggravate

seizures in patients with _HCN1_ variants; therefore, early genetic diagnosis could be crucial for appropriate pharmacotherapy selection. HGV DATABASE The relevant data from this Data Report

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thank the patient and his family for their participation in this study. This work was supported by the Japan Agency for Medical Research and Development (AMED) (grant numbers JP22ek0109486,

JP22ek0109549, JP22ek0109493) and the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Scientific Research (B) (grant number JP20H03641) and (C) (grant number

JH21K06819). We thank Edanz (https://jp.edanz.com/ac) for editing the English text of a draft of this manuscript. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Child Neurology,

National Hospital Organization Nishiniigata Chuo Hospital, Niigata, Japan Yu Kobayashi, Jun Tohyama, Kei Yamada, Moemi Hojo, Eijun Seki, Masaki Miura & Noriko Soma * Department of

Pediatrics, Niigata Prefecture Hamagumi Medical Rehabilitation Center for Disabled Children, Niigata, Japan Noriyuki Akasaka * Department of Pediatrics, Niigata University Medical and Dental

Hospital, Niigata, Japan Takeshi Ono * Department of Pediatrics, Showa University School of Medicine, Tokyo, Japan Mitsuhiro Kato * Department of Biochemistry, Hamamatsu University School

of Medicine, Hamamatsu, Japan Mitsuko Nakashima & Hirotomo Saitsu * Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan Naomichi Matsumoto

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Scholar CONTRIBUTIONS Y.K. cared for the patient and drafted the manuscript. N.A., K.Y., M.H., E.S., N.S., and T.O. cared for the patient. M.K. helped draft the manuscript and critically

revised the manuscript for important content. M.N., H.S., and N.M. were responsible for genomic analysis, critically revised the manuscript for important content, and helped draft the

manuscript. J.T. cared for the patient and revised the manuscript. All authors contributed to the writing of the final manuscript. CORRESPONDING AUTHOR Correspondence to Jun Tohyama. ETHICS

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http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Kobayashi, Y., Tohyama, J., Akasaka, N. _et al._ The _HCN1_ p.Ser399Pro variant

causes epileptic encephalopathy with super-refractory status epilepticus. _Hum Genome Var_ 10, 20 (2023). https://doi.org/10.1038/s41439-023-00247-8 Download citation * Received: 24 April

2023 * Revised: 05 June 2023 * Accepted: 05 June 2023 * Published: 23 June 2023 * DOI: https://doi.org/10.1038/s41439-023-00247-8 SHARE THIS ARTICLE Anyone you share the following link with

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