The HCN1 p.Ser399Pro variant causes epileptic encephalopathy with super-refractory status epilepticus

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.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate a cationic current that stabilizes the neuronal membrane potential against excitatory or inhibitory input and regulates neuronal network excitability 1,2 .HCN1 (NM_021072), one of the four genes that encode HCN channels 1 , is highly expressed in the neocortex, hippocampus, and brainstem of the central nervous system 3 .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 encephalopathy 4 .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 DEE [5][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-centroparietal 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 described 11 .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 age 5 .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 variant 5 and summarized the clinical manifestations of previously reported cases 10 (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 variant 5,10 .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 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 gainof-function HCN1 variants 9 .The p.S399P variant is also a loss-offunction variant.However, status epilepticus is caused not by a specific HCN1 variant but by a variety of variants 9 .Both upregulation and downregulation of HCN channels have been associated with epileptic activity in animal models 5,12 .A previous study indicated that patients with the p.G391D variant showed the most severe phenotypes among HCN1 DEE 5 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 S6 3 .The amino acid glycine 391, located on the intracellular interface of the S6 transmembrane domain, is adjacent to the p.S399P variant 5 .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 In addition, whole-cell patch-clamp recordings showed no current in cells separately transfected with each variant, suggesting that these are loss-of-function variants 5 .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 medications 5 .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 seizures 13 .In Hcn1 M294L mice carrying a homolog of the HCN1 p.M305L variant; phenytoin, lamotrigine, retigabine, and carbamazepine increased frequency, whereas levetiracetam, diazepam, sodium valproate, and ethosuximide significantly reduced spike frequency 14 .Sodium channel blockers such as carbamazepine or phenytoin may aggravate seizures in Dravet syndrome with SCN1A variants 15 .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 are hosted at the Human Genome Variation Database at https://doi.org/10.6084/m9.figshare.hgv.3311.

Fig. 1
Fig. 1 Electroencephalogram (EEG) and axial T2-weighted brain magnetic resonance imaging (MRI) of the patient.A An ictal EEG during right hemiclonic seizures at 4 months of age shows spike-wave discharges with onset in the left fronto-centro-parietal region.B Brain MRI at 4 months of age, showing normal findings.C At 8 months of age, after a second episode of convulsive status epilepticus, brain MRI shows severe frontal-dominant cortical and white matter atrophy with ventricular enlargement.

Table 1 .
Phenotypic manifestations of two patients with the p.S399P variant and a summary of previously reported cases.EIEE early infantile epileptic encephalopathy, EIMFS epilepsy of infancy with migrating focal seizures, FBTCS focal to bilateral tonic-clonic seizure, FS febrile seizures, GOF gain of function, LOF loss of function, MRI magnetic resonance imaging, TCS tonic-clonic seizure.