SCN1B‐linked early infantile developmental and epileptic encephalopathy

Abstract Objective Patients with Early Infantile Epileptic Encephalopathy (EIEE) 52 have inherited, homozygous variants in the gene SCN1B, encoding the voltage‐gated sodium channel (VGSC) β1 and β1B non‐pore‐forming subunits. Methods Here, we describe the detailed electroclinical features of a biallelic SCN1B patient with a previously unreported variant, p.Arg85Cys. Results The female proband showed hypotonia from birth, multifocal myoclonus at 2.5 months, then focal seizures and myoclonic status epilepticus (SE) at 3 months, triggered by fever. Auditory brainstem response (ABR) showed bilateral hearing loss. Epilepsy was refractory and the patient had virtually no development. Administration of fenfluramine resulted in a significant reduction in seizure frequency and resolution of SE episodes that persisted after a 2‐year follow‐up. The patient phenotype is more compatible with early infantile developmental and epileptic encephalopathy (DEE) than with typical Dravet syndrome (DS), as previously diagnosed for other patients with homozygous SCN1B variants. Biochemical and electrophysiological analyses of the SCN1B variant expressed in heterologous cells showed cell surface expression of the mutant β1 subunit, similar to wild‐type (WT), but with loss of normal β1‐mediated modification of human Nav1.1‐generated sodium current, suggesting that SCN1B‐p.Arg85Cys is a loss‐of‐function (LOF) variant. Interpretation Importantly, a review of the literature in light of our results suggests that the term, early infantile developmental and epileptic encephalopathy, is more appropriate than either EIEE or DS to describe biallelic SCN1B patients.


Introduction
Voltage-gated sodium channels (VGSCs) are responsible for the rising phase and propagation of action potentials in excitable cells. 1 VGSCs were purified as heterotrimeric complexes of a and b subunits from mammalian brain. 2 This work showed that a central a subunit forms the ion-conducting pore and is associated with two different b subunits. 3 Originally characterized as "auxiliary," b subunits are now known to be dynamic, multifunctional molecules that engage in diverse and essential roles in multiple tissues. 4,5 Their ability to participate in both conducting and nonconducting roles makes VGSC b subunits unique among voltage-gated ion channel subunits.
The breadth of b subunit function hinges on the key structural motif common to all members of this family of proteins: an immunoglobulin (Ig) loop enabling them to function as cell adhesion molecules (CAMs). [4][5][6] CAM-mediated adhesive functions are critical to brain development, including the processes of neurite outgrowth, axon pathfinding, fasciculation, and cell migration. 4,5 Integrity of the Ig loop is also critical for b1-mediated VGSC modulation in vivo, 7 making this domain multi-functional. In their roles as ion channel modulators, not only of VGSCs but also of voltage-gated K + channels, [8][9][10][11] b subunits make important contributions to the regulation of neuronal firing. As substrates for sequential cleavage by b-(BACE) and c-secretases, b subunits contribute to the regulation of VGSC a subunit gene expression. 12,13 Taking all of these roles into consideration, it is not surprising that variants in the genes encoding VGSC b subunits are linked to pathophysiology.
Patients with Early Infantile Epileptic Encephalopathy (EIEE) 52 (OMIM 617350) have inherited, homozygous variants in the gene SCN1B, encoding the VGSC b1 and b1B non-pore-forming subunits. 5 Inherited, heterozygous SCN1B variants have been linked to generalized epilepsy with febrile seizures (FS) plus, temporal lobe epilepsy (TLE), and cardiac arrhythmias, including Brugada syndrome and atrial fibrillation. 5,14 Homozygous SCN1B variants have been reported in seven epilepsy patients to date, with clinical descriptions suggestive of Dravet syndrome (DS). [15][16][17] Scn1b null mice have a phenotype that is similar to DS. 18,19 Thus, this small patient cohort was presumed to represent a subset of DS. Here, we describe a 4-year electroclinical follow-up of an eighth reported SCN1B patient with a previously unreported variant, c.253C > T (p.Arg85Cys), located in the extracellular Ig loop domain. The electroclinical profile suggests a diagnosis of early infantile developmental and epileptic encephalopathy (DEE), a severe neurodevelopmental disorder often beginning in infancy that is characterized by intractable seizures and pronounced developmental impairment, a more severe form of epilepsy and developmental delay than DS. Electrophysiological analysis of the variant shows b1 subunit loss-of-function (LOF) in spite of normal cell surface expression of the protein. These data are similar to previous work with the SCN1Bp.Cys121Trp variant, also located in the Ig loop domain, showing normal cell surface expression, but LOF in terms of sodium current (I Na ) modulation 7,20 and for which a homozygous mouse model shows a phenotype that is similar to Scn1b null mice. 21 Importantly, our results provide new information about autosomal recessive inheritance in epilepsy (reviewed in 22) and suggest reconsideration of the linkage of SCN1B variants to early infantile DEE rather than to EIEE or DS.

Case study
The female proband was born from consanguineous parents, first cousins of Belgian origin. The familial pedigree was highly suggestive of GEFS+ (Fig. 1). The father and his sister presented with febrile seizures (FS) in childhood. Three cousins of the parents had a non-detailed history of FS or epilepsy.
After an uncomplicated pregnancy, a global hypotonia was noted from birth. At 2 months of age, the proband was unable to hold her head, had no visual interactions, and began to present frequent erratic myoclonus triggered by transition phases of sleep, fever, and hot bath. Long-term (24 h) video electroencephalogram (EEG) monitoring at that time revealed frequent bilateral central spikes (Fig. 2). Spikes were isolated or occurred in bursts lasting a few seconds followed by high-voltage slow waves followed sometimes by unilateral or bilateral myoclonus (Fig. 3). Myoclonus was also observed without simultaneous spikes. Less frequently, there were bilateral temporal spikes. One cluster of myoclonus was followed by a focal to bilateral clonic seizure (Fig. 4), and thousands of epileptic and nonepileptic myoclonus (Fig. 3) were captured during this first long-term video EEG. Despite the frequent central and temporal spikes, structure of sleep stages was overall preserved with the presence of sleep spindles.
Despite the levetiracetam (50 mg/kg/d) treatment, erratic bursts of myoclonus persisted. The proband experienced a first fever-induced myoclonic status epilepticus (SE) at 3 months, which lasted for 30 min. Several episodes of focal to bilateral tonic-clonic seizures followed. Central spikes and waves with some generalized spikes were still observed on EEG. Addition of valproate (30 mg/kg/d) with clobazam (0.5 mg/kg/d) resulted in a slight decrease in both spikes and seizures. At 7 months, introduction of the ketogenic diet and topiramate (5 mg/kg/d) decreased myoclonus frequency and intensity. The proband became slightly more interactive with social smiling but with persistent, severe global hypotonia.
Eight myoclonic or hemiclonic febrile SE occurred within the first 2 years of life. At 24 months, EEG was unchanged, with thousands of myoclonus isolated or in bursts during the awake state or during sleep that often led to arousal. The epilepsy remained refractory, with developmental plateauing. The proband was treated at that time with a combination of valproic acid (30 mg/kg/d), topiramate (5 mg/kg/d), clobazam (0.5 mg/kg/d), and ketogenic diet. Fenfluramine (FFA) (0.6 mg/kg/d) was started as an add-on at 28 months. No further SE has occurred since that time, despite weaning of the ketogenic diet at age 3. Moreover, a significant reduction in seizure frequency was observed (Fig. 5). The 24-h video EEG performed 1 year after addition of FFA revealed no myoclonus during the awake state, even when the background EEG remained unchanged. During sleep, several epileptic and non-epileptic myoclonus were recorded; however, they were less severe than observed prior to FFA administration and did not lead to arousal of the child. Taken together, these observations suggest that FFA was effective in reducing epileptic severity. Nevertheless, her motor and cognitive development remained severely impaired with little improvement since the beginning of epilepsy: interaction is present with social smiling and vocalization, but the child is still unable to hold her head at 5 years, with a severe global hypotonia.
The work-up showed normal metabolic analysis and normal brain MRI. Auditory brainstem response (ABR) indicated brainstem involvement, since wave V vanished bilaterally at 50 dB, while waves I and III were still identified at 30 dB. Evoked otoacoustic emissions were preserved, and both the behavioral audiometry and the auditory steady-state responses estimated a bilateral hearing loss with a hearing threshold around 50 dB (Fig. 6).
Electrocardiogram, holter ECG, and cardiac echography were normal (data not shown). A gene panel testing of DNA amplified by multiplex PCR (Ampliseq â ) and re-sequenced by next-generation sequencing on Ion PGMâ found a homozygous SCN1B variant c.253C > T (p.Arg85Cys) in the proband. Both parents and her older sister were found to be heterozygous carriers (Fig. 1). Other family members were not tested for the presence of the variant.

b1-p.Arg85Cys is expressed at the cell surface
Previous work with the SCN1B-p.Cys121Trp variant, linked to GEFS+, showed normal cell surface expression in heterologous cells, but LOF in terms of I Na modulation. 7,20 A biallelic Scn1b-p.Cys121Trp mouse model showed a phenotype that is similar to DS. 21 In contrast, heterologous expression experiments with the variant, SCN1Bp.Arg125Cys, linked to a diagnosis of DS, showed that the mutant b1 protein was retained inside the cell and     subsequently unavailable at the plasma membrane to modulate I Na . 15 Growing the cells at a lower temperature, to permit proper protein folding, resulted in cell surface expression and normal I Na modulation. 15 To investigate the subcellular localization of this newly identified DEE-linked SCN1B variant, p.Arg85Cys, which is located in the Ig loop domain ( Fig. 7A and B), 23 we performed cell surface biotinylation experiments comparing the polypeptide, b1p.Arg85Cys, to wild-type (WT) b1 protein (Fig. 7A). Experiments were performed in Chinese hamster lung (CHL) fibroblasts that were stably transfected with soluble enhanced green fluorescent protein (eGFP), WT b1, or b1-p.Arg85Cys. The WT b1 and b1-p.Arg85Cys cDNA constructs each contained a C-terminal, in-frame, V5-epitope tag followed by a 2A endoproteolytic sequence and eGFP on the 3' end of the construct for detection of equimolar expression of a fluorescence marker protein. Total protein and neutravidin-selected cell surface proteins were analyzed by western blot with anti-V5 antibody. An antibody to HSP90 was used as an internal control to detect non-cell surface proteins. Results shown in Figure 7C suggest that, similar to WT b1, b1-p.Arg85Cys localizes to the cell surface. To confirm this result by another method, b1 subcellular localization in the same cell lines used in Figure 7B was assessed by confocal immunofluorescence microscopy. Cells were stained with fluorescently labeled wheat germ agglutinin (WGA), to identify the plasma membrane, and anti-V5 antibody, to label WT or mutant b1 subunits. Colocalization between anti-V5 and WGA was observed in both the WT b1 and b1-p.Arg85Cys lines, confirming that the patient variant, b1-p.Arg85Cys, can traffic to the plasma membrane (Fig. 7D). Orthogonal views of a single z-stack from immunofluorescence microscopy are shown in Fig. 7E. No other overt differences in subcellular localization were observed between WT b1 and b1-p.Arg85Cys.

Electrophysiological analysis
We next investigated the ability of b1-p.Arg85Cys to modulate I Na . Human Na v 1.1 channels (hNa v 1.1) were chosen for this experiment due to their association with EIEE and DS. 24 HEK-hNa v 1.1 cells were transiently transfected with WT b1, b1-p.Arg85Cys, or eGFP for voltage-clamp analysis. Representative I Na density traces, showing transient and persistent currents, evoked by a 50-msec test pulse to 0 mv following a prepulse to À120 mV are shown in Figure 8A and quantified in Figure 8B and C. WT b1 co-expression with hNa v 1.1 significantly increased transient and persistent I Na density compared to hNa v 1.1 alone. In contrast, co-expression of b1p.Arg85Cys with hNa v 1.1 did not significantly change these values. Figure 8B and E show quantification of the mean s values for the fast and slow components, respectively, of I Na inactivation. WT b1 significantly reduced the fast, but not slow component, while b1-p.Arg85Cys had no significant effect on either value. Results shown in Figure 9 and Table 1 indicate that neither WT b1 nor b1-p.Arg85Cys subunits affect the voltage dependence of hNa v 1.1-generated I Na .

Expression vectors
Human WT b1-V5-2AeGFP and b1-p.Arg85Cys-V5-2AeGFP cDNAs were generated by gBLOCK from Integrated DNA Technologies. Both b1 constructs contained an in-frame, C-terminal V5-epitope tag followed by a 2A endoproteolytic sequence and eGFP on the 3' end of the construct. A control construct expressing eGFP only was amplified from the WT construct using gene-specific primers and gel-purified. cDNAs were inserted into pENTR-SD/D TOPO Gateway entry vector by a TOPO reaction according to the manufacturer's instructions. All constructs were moved to the Gateway-compatible destination vector, pcDNAdest40, using a LR clonase reaction according to the manufacturer's instructions.

Cell surface biotinylation and western blot analyses
Stable cell lines were grown in 100-mm tissue culture plates until 90-100% confluent. Cell surface proteins were biotinylated using the Cell Surface Protein Isolation Kit (Pierce) according to the manufacturer's protocol. Loading buffer containing 1% sodium dodecyl sulfate, 1 mmol/L bmercaptoethanol, and 0.2% dithiothreitol was added to samples and heated for 10 min at 85°C. Samples were separated on 10% Tris-Glycine polyacrylamide gels, as noted in the figure legends, then transferred to nitrocellulose membrane (16 h, 55 mA, 4°C) and probed with antibodies as noted in the figure legends. Incubations with anti-V5 and secondary antibody were completed using a SnapID (Millipore-Sigma) with 10-min incubations. Anti-HSP90 was incubated overnight at 4°C in 2% milk in Tris-buffered saline with 0.1% tween (TBST). Secondary antibodies to detect anti-HSP90 were incubated at room temperature for 1 h. Immunoreactive bands were detected using West Femto chemiluminescent substrate (GE Health Sciences) and imaged on an iBrightFL1000 (Invitrogen).

Immunocytochemistry and confocal microscopy
CHL cells stably overexpressing WT b1-V5-2AeGFP or b1p.Arg85Cys-V5-2AeGFP constructs, as indicated in the figure legend, were incubated with WGA for 10 min at 4°C. Cells were washed three times for 5 min each in Dulbecco's phosphate-buffered saline (DPBS) at 4°C and immediately fixed with 4% paraformaldehyde at room temperature for 15 min. Following fixation, cells were quickly washed three times in DPBS and blocked in 90% DPBS, 10% goat serum, and 0.3% triton X-100 for 1 h at room temperature in a dark, humidified chamber. Cells were incubated in anti-V5 overnight in a dark, humidified chamber. Cells were washed three times for 10 min each with DPBS, incubated in secondary antibody (Alexa Fluor TM 647) for 2 h in a dark, humidified chamber at room temperature, and then washed three times for 10 min in

Electrophysiological analysis
HEK-hNa v 1.1 cells were transiently transfected as described above. Electrophysiological recordings were per-formed~12 h following final plating. Isolated I Na was recorded using standard whole-cell patch clamp, with previously described conditions. 25  Holding potential was À80 mV. Voltage-clamp analysis was performed using pClamp 10 (Molecular Devices) and SigmaPlot 11 (Systat software). Statistical analyses, t-test, and one-way ANOVA, were performed using SigmaPlot. Statistical significance was determined by P > 0.05.

Discussion
Here, we provide the first detailed electroclinical investigation of an epilepsy patient with bilateral hearing loss presenting with a previously unreported, inherited homozygous SCN1B missense variant, p.Arg85Cys, suggesting that this genotype is linked to an early infantile DEE rather than typical DS. Moreover, a significant reduction of seizure severity and of the fever-induced SE was observed after FFA add-on that persisted after a 2year follow-up. Functional analysis of b1-p.Arg85Cys protein in heterologous cells showed loss of Na v 1.1-generated I Na modulation in spite of normal levels of expression of the mutant protein at the cell surface. described SCN1B LOF variant at this position, b1p.Arg85His, has been linked to atrial fibrillation familial type 13 in heterozygous patients. 26 Similar to b1p.Arg85Cys shown here, b1-p.Arg85His was found to be expressed at the cell surface but lost the ability to modulate Na v 1.5-generated I Na when expressed in a heterologous system. 26 Previous work has shown that b1mediated effects on current modulation are cell-type specific. 5 Additionally, the heterologous cells utilized in these studies are nonpolarized and do not form specialized membrane microdomains such as axon initial segments or intercalated discs. Future work that characterizes these patient variants in neurons or cardiomyocytes may provide additional insights into disease mechanisms.
Patients with homozygous SCN1B variants are very rare. Seven patients have been reported so far, all diagnosed with DS. [15][16][17] However, a review of the literature suggests that six of these seven published cases do not strictly fit DS criteria. In DS, first seizures usually begin around 6 to 12 months of age, with prolonged febrile, generalized, clonic, or hemiclonic seizures, and a normal EEG background until 2 years of age. Development shows signs of decline after 2 years of age. Disease is progressively marked by additional seizures of multiple etiologies, ataxia, and pyramidal signs. 27,28 The first SCN1B DEE patient described by Patino et al. (SCN1B-p.Arg125Cys) 15 was similar to the present case but with some clinical characteristics in favor of DS, for example, the presence of refractory FS, myoclonus, and seizure triggers like vaccination, fever, and infection. Nevertheless, the disease evolution of the SCN1B-p.Arg125Cys patient was worse than the usual DS course. The EEG was abnormal from 3 months of age, with central discharges. While the neonatal period was not reported, a tetrapyramidal syndrome with global hypotonia was noted at 13 months. The reported absence of SE was not typical of DS. The patient died due to aspiration pneumonia at 14 months. The second patient, described by Ogiwara et al. (SCN1B-p.Ile106Phe), 17 was more similar to DS. While initial seizures were not provoked by fever, feversensitive seizures of various types, including myoclonic seizures, appeared around 6 months of age. Developmental decline occurred earlier than typical DS. The course of the disease was marked by a global developmental delay with progressive ataxia and intractable epilepsy. Interictal EEG was initially normal, but started to display multifocal spikes and slow waves, occurring singly or in bursts, around 1 year of age. In a less clinically detailed paper, Ramadan et al. reported five other cases of SCN1B-linked EE from three consanguineous families (SCN1B-p.Tyr119-Asp and SCN1B-c.449-2Ala > Gly, which are likely splice site variants). 16 No seizure triggers, like fever or vaccination, were mentioned. The ages of EEG completion and seizure types were not systematically specified. Epilepsy onset and developmental decline appeared early (1 or 2 months old) in four of the five cases. One of the three families had a different phenotype with microcephaly, dysmorphic features, and various malformations.
In the present patient case, development was already abnormal at birth. Epilepsy started early, at 3 months of age, with abundant EEG discharges. The course of the disease was severe with profound motor and cognitive delay. While the observed prolonged febrile myoclonic or hemiclonic seizures were more concordant with DS, electroclinical evidence suggests that SCN1B-linked DEE is more severe, with an earlier onset of seizures and developmental delay. This idea is consistent with the negative result of the study of Kim et al, looking for SCN1B variants in a cohort of DS patients 29 as well as with the finding of neuronal pathfinding deficits in Scn1b null mice at postnatal day 10 prior to seizure onset. 30 Thus, we propose that the term, "early infantile DEE," is more appropriate than EIEE, which is classically used in patients with a severe early onset epilepsy and profound developmental delay. The term "epileptic encephalopathy" refers to conditions where the epileptic abnormalities themselves are thought to contribute to the progressive disturbance of cerebral function, such that early effective intervention may improve developmental outcome. 31 Whether the patient reported here, and more generally all patients with EIEE, fit this definition of epileptic encephalopathy is highly debatable because the neurological prognosis of these patients seems predominantly caused by preexisting brain dysfunction 31 and not by the severity of the epilepsy. Our case illustrates this concept: development was already abnormal from birth, before the onset of epilepsy, and despite the resolution of febrile SE and significant improvement of seizure frequency with FFA add-on, we did not observe a significant improvement in neurological development. Because neurological prognosis is predominantly caused by a developmental encephalopathy of genetic origin, early infantile DEE seems a more appropriate term than EIEE to describe the electroclinical picture of this patient. The addition of FFA (0.6 mg/kg/d) to the anticonvulsant regimen of our patient resulted in a significant and prolonged reduction in seizure frequency with increased quality of life (Fig. 5), but no significant improvement in motor or cognitive development. FFA was developed originally as an appetite suppressant but was withdrawn from human use after reports of increased risk of cardiac valvulopathy and pulmonary hypertension in adult patients treated with high-dose FFA for obesity. 32 The potential utility of FFA in epilepsy emerged in the 1980s with its use in intractable self-induced epilepsy 33,34 and in the 1990s with a remarkable effect on a group of patients who were shown later to present with SCN1A-DS variants. 35 The positive benefit/risk ratio profile of low-dose FFA treatment in patients with DS was confirmed in a recently reported phase 3 study. 36,37 FFA antiepileptic activity is thought to arise via enhanced serotonergic neurotransmission by augmenting carrier-mediated synaptic release of 5-HT and by preventing reuptake. 38,39 In our patient, low-dose FFA provided resolution of febrile SE and sustained, clinically meaningful, daily seizure reduction that persisted after a 2-year follow-up. FFA was well tolerated with no clinical and/or echocardiographic signs of cardiac valvulopathy or pulmonary hypertension. These results suggest that FFA may provide a promising treatment option in patients with homozygous SCN1B-linked early infantile DEE.
Early infantile DEE resulting from homozygous SCN1B LOF variants may be similar to a new class of variants linked to a recurrent, missense SCN1A variant, p.Thr226Met, which is associated with a more severe clinical phenotype than typical DS. In SCN1A-linked early infantile DEE, seizures and epileptic spasms are observed beginning at an average of 9 weeks of age, with concomitant development of hyperkinetic movement disorders, including choreoathetosis, dystonia, myoclonus, and perioral hyperkinesia. Patients have greater developmental impairments than DS, are nonambulatory, and the majority require feeding tubes. 40 Heterologous expression of the mutant Na v 1.1-p.Thr226Met protein showed negative shifts in the voltage dependence of both activation and inactivation of I Na compared to WT Na v 1.1, which can be interpreted as both gain-and loss-of-function. 41 To conclude, we present an eighth case of a homozygous variant in SCN1B with a phenotype of early infantile DEE, rather than DS, and a prolonged response to FFA.
The pathogenic role of this variant has been assessed in vitro and, while FFA appears to be particularly efficient in resolution of febrile SE and reduction in seizure frequency in this patient, further studies are needed to identify new therapeutic options to improve the neurological development of this devastating disease. Importantly, a review of the literature in light of our results suggests that early infantile DEE is a more appropriate terminology than either EIEE or DS for SCN1B-linked epileptic encephalopathy.