# NAPB and developmental and epileptic encephalopathy: Description of the electroclinical profile associated with a novel pathogenic variant

Cécile Mignon-ravix, Florence Riccardi, Géraldine Daquin, Pierre Cacciagli, Sylvie Lamoureux-toth, Laurent Villard, Nathalie Villeneuve, Florence Molinari

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Cécile Mignon-ravix, Florence Riccardi, Géraldine Daquin, Pierre Cacciagli, Sylvie Lamoureux-toth, et al.. NAPB and developmental and epileptic encephalopathy: Description of the electroclinical profile associated with a novel pathogenic variant. Epilepsia, 2023, 10.1111/epi.17603. hal-04072437

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BRIEF COMMUNICATION

# NAPB and developmental and epileptic encephalopathy: Description of the electroclinical profile associated with a novel pathogenic variant

Cécile Mignon-Ravix<fnref n="1" />

Florence Riccardi<fnref n="1" />,<fnref n="2" />

Géraldine Daquin<fnref n="3" />

Pierre Cacciagli<fnref n="4" />

Sylvie Lamoureux-Toth<fnref n="5" />

Laurent Villard<fnref n="1" />,<fnref n="6" /> ID

Nathalie Villeneuve<fnref n="7" />

Florence Molinari<fnref n="1" /> ID

Correspondence

Nathalie Villeneuve, AP-HM, Hôpital Timone Enfants, Service de Neurologie Pédiatrique, Marseille, France.
Email: nathalie.villeneuve@ap-hm.fr

Florence Molinari, Aix-Marseille Univ, INSERM, MMG, Marseille, France.
Email: florence.molinari@inserm.fr

Funding information

A*Midex, Grant/Award Number: AMX-19-IET-004; Agence Nationale de la Recherche, Grant/Award Number: ANR-17-EURE-0029

Abstract

Developmental and epileptic encephalopathies (DEE) are a group of neurodevelopmental disorders characterized by epileptic seizures associated with developmental delay or regression. DEE are genetically heterogeneous, and the proteins involved play roles in multiple pathways such as synaptic transmission, metabolism, neuronal development or maturation, transcriptional regulation, and intracellular trafficking. We performed whole exome sequencing on a consanguineous family with three children presenting an early onset (<6 months) with clusters of seizures characterized by oculomotor and vegetative manifestations, with an ocipital origin. Before 1 year of age, interictal electroencephalographic recordings were well organized and neurodevelopment was unremarkable. Then, a severe regression occurred. We identified a novel homozygous protein-truncating variant in the *NAPB* (N-ethylmaleimide-sensitive fusion [NSF] attachment protein beta) gene that encodes the βSNAP protein, a key regulator of NSF–adenosine triphosphatase. This enzyme is essential for synaptic transmission by disassembling and recycling proteins of the SNARE complex. Here, we describe the electroclinical profile of each patient during the disease course. Our findings strengthen the association between biallelic variants in *NAPB* and DEE and refine the associated phenotype. We suggest including this gene in the targeted epilepsy gene panels used for routine diagnosis of unexplained epilepsy.

KEYWORDS

developmental and epileptic encephalopathies, electroencephalogram, NAPB seizures, SNAP SNARE, whole exome sequencing

<footnote n="1">¹Aix-Marseille Univ, INSERM, MMG, Marseille, France</footnote>

<footnote n="2">²Département de Génétique Médicale, CHITS, Hôpital Ste Musse, Toulon, France</footnote>

<footnote n="3">³AP-HM, Hôpital Timone Enfants, Service de Neurophysiologie Clinique, Marseille, France</footnote>

<footnote n="4">⁴Biological Resources Center, CRB, Assistance Publique des Hôpitaux de Marseille, Marseille, France</footnote>

<footnote n="5">⁵Centre Hospitalier Avignon, Service de Pédiatrie, Avignon, France</footnote>

<footnote n="6">⁶AP-HM, Hôpital Timone Enfants, Service de Génétique Médicale, Marseille, France</footnote>

<footnote n="7">⁷AP-HM, Hôpital Timone Enfants, Service de Neurologie Pédiatrique, Marseille, France</footnote>
# 1 | INTRODUCTION

Developmental and epileptic encephalopathies (DEE) refer to a group of neurodevelopmental disorders characterized by epileptic seizures, often intractable, a severe developmental delay or regression, and a poor prognosis. With the development of next generation sequencing, DEE were shown to have a strong but highly heterogeneous genetic basis.¹

Neurotransmitter release at synapses is ensured by the fusion of presynaptic vesicles mediated by the soluble N-ethylmaleimide-sensitive fusion (NSF) protein attachment protein receptor (SNARE) complex following Ca²⁺ ion increase. After fusion, the SNARE complex disassembly relies on the NSF–adenosine triphosphatase (ATPase) associated with soluble NSF attachment protein (SNAP) adaptor proteins.² A large number of pathogenic variants have been described in proteins composing the SNARE complex and were shown to result in several neurodevelopmental syndromes including DEE.³ Recently, homozygous splice or protein-truncating variants have been described in the *NAPB* (NSF attachment protein beta; MIM#611270) gene, encoding the βSNAP protein, in four patients from three independent families presenting DEE (DEE107, MIM#620033).⁴⁻⁶ Here, we describe a consanguineous family composed of three siblings with early onset epilepsy and no specific interictal electroencephalogram (EEG) abnormalities. At the beginning of the second year of age, a developmental regression was observed leading to severe intellectual disability. Using whole exome sequencing (WES), we identified a novel homozygous deletion in the *NAPB* gene. The description of the electroclinical profile of each patient in this family allows refinement of the phenotype associated with pathogenic variants in this gene and confirmation of its involvement in DEE.

# 2 | PATIENTS AND METHODS

## 2.1 | Patients

Individuals who participated in this study were identified and clinically characterized in Marseille University Hospital (Hôpital d'Enfants de La Timone). DNA from participating family members was extracted from peripheral blood lymphocytes, stored, and delivered by the Biological Resources Center of La Timone Marseille Public University Hospital, according to the French regulation (CRB AP-HM Biobank; NF S96-900 & ISO 9001 v2015 certifications).

## 2.2 | Whole exome sequencing

WES was performed on the two affected brothers (II.3 and II.5), their father (I.1), and a nonaffected brother (II.1) using Twist Human Core Exome kit (Twist Bioscience) on an Illumina platform (CeGaT). For more details, see Appendix S1. Sequencing data were analyzed according to an autosomal recessive inheritance pattern. We selected variants that (1) affected the protein sequence, (2) had a minor allele frequency < .1% in the gnomAD database, (3) were predicted to be pathogenic or likely pathogenic by at least 3 of 5 in silico prediction programs, and (4) were expressed in cerebral tissue. Validation and segregation analysis of the *NAPB* variant was done using Sanger sequencing (data not shown).

# 3 | RESULTS

## 3.1 | Clinical case descriptions

The three affected children (II.2, 3, and 5) were born after a normal pregnancy from consanguineous healthy parents (Figure 1A). Two other siblings (II.1 and II.4) are healthy, with no history of seizures, epilepsy, or intellectual disability.

The clinical phenotype of the three affected patients evolved in three main phases. The first phase was an early onset epilepsy characterized by clusters of seizures starting at 1, 4, and 2 months of age for Patients II.2, 3, and 5 respectively (Table 1). Seizures had a low frequency (1–2 clusters per month) and were characterized by vegetative signs, few behavioral (wandering eyes, tonic upper limb extension, chewing, behavioral arrest) or motor symptoms (tonic upper limb extension), and facial erythrosis or cyanosis. Interictal EEGs were well organized for age, with rare posterior anomalies (II.2: from 1 to 18 months, Figure S1A; II.3: from 3 to 8 months, Figure S1B; II.5: from 6 to 12 months, Figure S1C). Initial neurodevelopment was unremarkable for the three affected children. The second phase started at approximately 7–10 months of age with an increase in seizure duration (1–3 min) and frequency (1–8 per day). Ictal EEG started with focal posterior spike discharges followed by suprasylvanian propagation (Figure 1). At the end of the seizure, discharge frequency decreased, with few impacts on the contralateral hemisphere (Figure 1). Interictal awake EEG recordings showed subnormal background activity (5–6 Hz, Figure S1A). During sleep, spikes or polyspikes were observed, followed by slow waves mainly in posterior regions (Figure S1). At the beginning of the second year, all children showed neurodevelopmental regression, mainly on social aspects.

The last phase began at approximately 3 years of age, with a modification of seizures that became mainly
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a) 0'

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b) 1'20

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c) 2'

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b) 35''

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c) 1'35

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b) 40''

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c) 1'28

<figure><img src="image_13.png" /><figcaption>**FIGURE 1** NAPB variant and associated electroencephalographam (EEG) description. (A) Family pedigree and genetic characteristics of individuals carrying the variant c.347_353delGGTTTAC in NAPB (NM_022080). Affected siblings (solid symbols) carry a homozygous deletion. +, wild-type allele; Del, deleted allele; NT, not tested. (B) Schematic representation of NAPB gene showing exons (white boxes), introns (black lines), and non-coding region (gray boxes). Localization of NAPB variants identified in our family (bold) and previously reported cases are shown. (C–E) Ictal video-EEG description of the three patients, at the beginning (a, *), during (b), and at the end of the seizure (c). Seizures were recorded awake at 10, 18, and 7 months for II.2 (C), II.3 (D), and II.5 (E), respectively. (C) Seizure lasts 3 min and starts with a right posterior subclinical discharge (1 min, 5 Hz, θ) getting richer in spikes (asterisk, Ca). Then, the discharge spreads to the entire right hemisphere, mixed with some slow waves with no impact on the left hemisphere (Cb). Finally, slow waves propagate to the left hemisphere (Cc). (D) Seizure lasts 1 min and 50 s and starts with a right temporal spike discharge (asterisk, Da), followed by a rapid suprasylvan discharge lasting 6 s (black line, Db). At the end of the seizure, diffuse bilateral slow 1.5-Hz (δ) waves predominate in the right anterior (Dc). (E) Seizure begins with a left posterior fast discharge, slowing into a spike discharge involving the left temporal area (Ea,b). At 2 min 10 s, the left posterior discharge stops, and a right posterior spike discharge appears (Ec). The end of the seizure recording is disturbed by artifact in the right suprasylvan (Ec). Times and scale are indicated for each recording. Low pass filter: 70 Hz. ECG, electrocardiogram.</figcaption></figure>

nocturnal and occurred monthly. Interictal EEG record-
ings during sleep were disorganized, with paroxysmal
abnormalities characterized by slow spikes-and-waves
(Figure S1A,B).

At last evaluation, all affected children had a global
neurodevelopmental disorder with delayed (II.2 and II.5)
or unacquired walking (II.3), and severe language and
social communication impairment. The affected girl II.2
died at 5.5 years of age from sudden unexpected death in
epilepsy (SUDEP) during sleep. The youngest affected boy

presented an autism spectrum disorder associated with
visual self-stimulation and stereotypies. Extensive meta-
bolic workup and magnetic resonance imaging performed
for each individual were normal (Table 1).

**3.2 | Sequencing results**

A targeted gene panel containing 144 genes known to be
involved in epileptic syndromes (Table S1) was sequenced
<table><caption>TABLE 1 Summary of genetic data and clinical characteristics of patients with NAPB variants.</caption><thead><tr><th rowspan="2">Pedigree ID</th><th colspan="2">Family described here</th></tr><tr><th>II.2, deceased at 5.5 years</th><th>II.3</th></tr></thead><tbody><tr><td>Sex, age at last evaluation</td><td>Female, 5 years and 3 months</td><td>Male, 11 years</td></tr><tr><td>Parental consanguinity</td><td>+</td><td>+</td></tr><tr><td>NAPB variant</td><td colspan="2">chr20-23375625-TGTAAACC-T<br>NM_022080:c.347_353delGGTTTAC, p.(Arg116Lysfs*19)</td></tr><tr><td>ACMG classification</td><td colspan="2">Likely pathogenic</td></tr><tr><td>Birth</td><td></td><td></td></tr><tr><td>Gestational age</td><td>40 weeks</td><td>40 weeks</td></tr><tr><td>Length, cm</td><td>53</td><td>Normal</td></tr><tr><td>Weight, g</td><td>4060</td><td>Normal</td></tr><tr><td>Head circumference, cm</td><td>36</td><td>Normal</td></tr><tr><td>Measurements at last evaluation</td><td></td><td></td></tr><tr><td>Height, cm</td><td>112.5</td><td>142</td></tr><tr><td>Weight, kg</td><td>21.5</td><td>34</td></tr><tr><td>Head circumference, cm</td><td>51</td><td>50.8</td></tr><tr><td>Development</td><td></td><td></td></tr><tr><td>Initial development</td><td>Normal psychomotor acquisition<br>Sitting, 9 months</td><td>Normal psychomotor acquisition<br>Sitting, 9 months<br>Walk with assistance, 15 months</td></tr><tr><td>Developmental regression</td><td>Yes, at 15 months</td><td>Yes, at 18 months</td></tr><tr><td>Global development</td><td>Delayed<br>Walk at 21 months<br>Absence of language<br>No self-feeding</td><td>Delayed<br>After regression: no sitting or walking<br>Absence of language<br>No self-feeding</td></tr><tr><td>Intellectual disability</td><td>Severe</td><td>Severe</td></tr><tr><td>Social communication</td><td>—</td><td>—</td></tr><tr><td>Autism spectrum disorder</td><td>+</td><td>(+)</td></tr><tr><td>Neurological findings</td><td></td><td></td></tr><tr><td>Seizures, onset</td><td>+, at 1 month</td><td>+, at 4 months</td></tr><tr><td>Seizure Type/Frequency</td><td></td><td></td></tr><tr><td></td><td><ul><li>Onset: focal seizures characterized by a right or a left tonic extension with cyanosis</li><li>&lt;10 months: focal seizures characterized by behavioral arrest, cyanosis, and tonic extension of the upper limbs (2 clusters per month)</li><li>1–3 years: focal seizures with increased frequency (8–10 seizures per day)</li><li>&gt;3 years: monthly nocturnal generalized seizures characterized by bilateral clonic-tonic jerks</li></ul></td><td><ul><li>Onset: focal seizures characterized by right or left clonic jerks (5–15 min) without cyanosis (low frequency)</li><li>9 months: focal seizures characterized by arrest of activity with erythrosis followed by right or left tonic extension of upper limbs, increasing frequency</li><li>1–4 years: focal seizures characterized by staring, uncontrollable eye movements from right to left, facial erythrosis, fast jerking of the upper limbs (1 per day to 1 per month)</li><li>&gt;4 years: nocturnal generalized seizures (1 or 2 per year)</li></ul></td></tr><tr><td>AED treatment</td><td>CBZ, VPA, CLB, TPM</td><td>PB, VPA, TPM, CLB</td></tr><tr><td>Brain MRI, age</td><td>Normal (10 months)</td><td>Normal (2 years)</td></tr></tbody></table>

Abbreviations: –, absence; +, presence; ACMG, American College of Medical Genetics and Genomics; AED, antiepileptic drug; CBZ, carbamazepine; CLB, clobazam; DZP, diazepam; FDS, Focal dyscognitive seizures; GTCS, generalized tonic-clonic/bilateral convulsive seizures; LEV, levetiracetam; MRI, magnetic resonance imaging; NA, not available; PB, phenobarbital; PHT, phenytoin; TPM, topiramate; VPA, valproate.
<table><thead><tr><th rowspan="2">II.5</th><th rowspan="2">Family described in Conroy et al.<sup>4</sup></th><th rowspan="2">Family described in Reuter et al.,<sup>5</sup> ER53330</th><th colspan="2">Family described in Zhao et al.<sup>6</sup></th></tr><tr><th>II.1</th><th>II.2</th></tr></thead><tbody><tr><td>Male, 4 years</td><td>Female, 6 years</td><td>Male, not indicated</td><td>Female, 13 years</td><td>Male, 8 years</td></tr><tr><td>+</td><td>+</td><td>+</td><td>—</td><td>—</td></tr><tr><td></td><td>chr20-23370665-G-T</td><td>chr20-23383635-C-T</td><td>chr20-23370902-C-T</td><td></td></tr><tr><td></td><td>NM_022080: c.479C&gt;A, (p.Ser160*)</td><td>NM_022080 c.173G&gt;A (p.Trp58*)</td><td>NM_022080.3:c.421-1G&gt;A</td><td></td></tr><tr><td></td><td>Likely pathogenic</td><td>Likely pathogenic</td><td>Likely pathogenic</td><td></td></tr><tr><td>40 weeks</td><td>Full term</td><td>NA</td><td>37 weeks</td><td>38 weeks</td></tr><tr><td>50</td><td></td><td>NA</td><td>Normal</td><td>Normal</td></tr><tr><td>4070</td><td>3100</td><td>NA</td><td>Normal</td><td>Normal</td></tr><tr><td>37</td><td></td><td>NA</td><td>&lt;−3 SD</td><td>NA</td></tr><tr><td>87 (+2 SD)</td><td></td><td></td><td>140 (−2 SD)</td><td>110 cm (−3 SD)</td></tr><tr><td>14.8 (+2.5 SD)</td><td></td><td></td><td>31.5 (−2 SD)</td><td>16.5 (−3 SD)</td></tr><tr><td>47.8</td><td>Progressive microcephaly</td><td>Microcephaly</td><td></td><td>Microcephaly (&lt;−3 SD)</td></tr><tr><td colspan="5">Normal psychomotor acquisition<br>Sitting, 9 months</td></tr><tr><td>Yes, at 15 months</td><td>Yes, at 8 months</td><td>NA</td><td>Yes, at 8 months</td><td>NA</td></tr><tr><td>Delayed</td><td>Delayed</td><td>NA</td><td>Delayed</td><td>Delayed</td></tr><tr><td>Walk at 2 years</td><td>Sitting with support</td><td></td><td>Walk unsteadily</td><td>No walking</td></tr><tr><td>Absence of language</td><td>Walk with assistance</td><td></td><td>Absence of language</td><td>Absence of language</td></tr><tr><td>No self-feeding</td><td>Absence of language</td><td></td><td>No self-feeding</td><td>No self-feeding</td></tr><tr><td>Severe</td><td>Severe</td><td>+</td><td>Severe</td><td>Severe</td></tr><tr><td>—</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>+</td><td>NA</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>+, at 2 months</td><td>+, at 5 months</td><td>+, NA</td><td>+, at 6 months</td><td>+, at 2 months</td></tr><tr><td colspan="5">• Onset: focal seizures characterized by unilateral right or left clonic jerks with rare cyanosis (1 cluster per month)</td></tr><tr><td colspan="5">• 7–12 months: focal seizures characterized by eye deviation, erythrosis, slight cyanosis, increasing frequency</td></tr><tr><td colspan="5">• 1–3 years: longer focal seizures with eye deviation, lip and face cyanosis but no shaking or tonus modification (1–7 seizures per day)</td></tr><tr><td colspan="5">• &gt;3 years: rare nocturnal generalized seizures (1 or 2 per year)</td></tr><tr><td>CBZ, PB, DZP</td><td>VPA, PHT (seizure reduction), CBZ (replace PHT)<br>TPM at 8 months; stopped GTCS.<br>3rd year: LEV (replace CBZ)</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Normal (2 months)</td><td>Normal (2 years)</td><td>NA</td><td>Normal (7 years)</td><td>NA</td></tr></tbody></table>
for each individual. A de novo missense variant located in exon 7 of the *SCN8A* (sodium voltage-gated channel, alpha subunit 8) gene was identified in Patient II.2 (Hg19 chr12:52094949G>C:NM_014191:c.950G>C:p. [Gly317Ala]).⁷ This variant was absent from the gnomAD (v2.1.1) and ClinVar databases, and was predicted to be damaging by 18 prediction tools out of 20 (https://varso.me.com/).⁸ Because this variant was absent from her affected siblings, exome sequencing was performed on the two affected boys (II.3 and II.5), their healthy brother (II.1) and father (I.1) to identify other pathogenic variants. Because of consanguinity, we focused our analysis on a recessive mode of inheritance, and variant filtering was performed as described in the methods. We found a homozygous 7-base-pair deletion in the two affected brothers, located in exon 5 of the *NAPB* gene (Hg19 chr 20:23375625TGTAAACC>T:NM_022080:c.347_353del:p. [Arg120Lysfs*19]; Figure 1B, Figure S2). We confirmed using sanger sequencing that this protein-truncating variant (PTV), which affects all *NAPB* isoforms, was homozygous for their affected sister (II.2), and heterozygous for both parents and their healthy child (II.1). It has never been described in the literature⁴⁻⁶ or gnomAD (v2.1.1), ClinVar, or LOVD (v3.0) databases (Figure 1A,B). No other homozygous or compound heterozygous variants that could explain the clinical phenotype were identified (Tables S2 and S3).

## 4 | DISCUSSION

This report of three additional patients strongly supports the gene-disease association between *NAPB* and DEE. Seven patients from four independent families have now been described with homozygous PTV (nonsense or splicing variants) in *NAPB* (Figure 1B, Table 1). All patients presented with early onset seizures (before 6 months of age) associated with a global neurodevelopmental disorder characterized by developmental regression at approximately 15 months of age leading to severe intellectual disability with language and social impairment. Noticeably, patients do not present any dysmorphic features or cerebral malformations and microcephaly is not always present (Table 1). Based on the 2022 International League Against Epilepsy classification, this syndrome is part of the DEE group.⁹ In this group, many syndromes show abnormal interictal epileptiform discharges such as suppression burst, multifocal discharges, or excessive slowing. Patients with an *NAPB* variant show very few interictal abnormalities, even when seizure frequency increases and developmental regression sets in. In the DEE group, only Dravet syndrome (DS) and the *CDKL5*-related epilepsy present frequent seizures with a normal interictal

EEG at the beginning of epilepsy. However, in DS, developmental progress slows at the end of the second year of age, whereas *NAPB* patients show developmental regression at the beginning of the second year.⁹,¹⁰ Moreover, we do not observe seizures triggered by fever, nor myoclonic or atypical absence seizures, which are often present in DS.⁹ Interestingly, increase of seizure frequency in *NAPB* patients is not linked to a seizure type modification or to occurrence of spasms, as it can be observed in *STXB1*-related epilepsy,¹¹,¹² nor to a profound EEG alteration. The *NAPB* epilepsy etiology is specific, with three successive stages: (1) early epilepsy onset with tonic focal seizures associated with facial erythrosis or cyanosis and normal neurodevelopment, (2) increased tonic seizure frequency and developmental regression, and (3) epileptic encephalopathy with nocturnal seizures.

Affected girl II.2 presented an earlier seizure onset (1 month) compared to her brothers and premature death at age 5.5 years from SUDEP during sleep. In addition to the PTV in *NAPB*, she had a likely pathogenic de novo variant in *SCN8A*. This gene encodes the Nav1.6 voltage-dependent sodium channel that initiates membrane depolarization for the generation of action potential. Pathogenic variants in *SCN8A* cause several neurological diseases, including DEE (MIM#614558).¹³ Usually, patients with *SCN8A*-related epilepsy respond well to sodium channel blockers such as carbamazepine (CBZ). Here, the patient's seizures were well controlled by valproate but CBZ had no effect (Table 1).⁷ At this stage, only functional studies on the *SCN8A* variant p.(Gly317Ala) could help to reveal precisely how it could contribute to the patient's clinical features. The cumulative effect of two variants in two epilepsy-related genes could explain the earlier onset of seizures and death.

*NAPA* and *NAPB* encode the alpha and beta SNAP proteins (α/βSNAP), two highly homologous cofactors of NSF-ATPase. This enzyme is essential for synaptic transmission, because it disassembles and recycles proteins of the SNARE complex.¹⁴ *NAPA* is ubiquitously expressed in mammals (Figure S3),¹⁵ and the knockout (KO) mouse model is embryonically lethal.¹⁶ The natural hypomorphic mouse model HYH (hydrocephalus with hop gait) contains a homozygous recessive missense mutation (p.M105I) in *Napa* resulting in decreased αSnap protein expression without affecting its function.¹⁴,¹⁶ These mice show a smaller cerebral cortex associated with an alteration of the neuronal cell fate but no epilepsy,¹⁶ synaptic transmission, or plasticity alterations.¹⁴ *NAPB* is almost exclusively expressed in the postnatal brain (Figure S3),¹⁴,¹⁵ and the βSnap-KO mice develop spontaneous recurrent epileptic seizures at postnatal day 11 (P11) followed by ataxia that could lead to death.¹⁴ Brain morphology is normal, and expression of the other SNARE proteins is unchanged. Basic
synaptic transmission and short-term plasticity in autaptic hippocampal neurons is normal together with synapses number.¹⁴ βSNAP has been less studied than αSNAP, but in vitro studies showed that α- and βSNAP have equivalent function, leading to the hypothesis that αSNAP could compensate for βSNAP absence.¹⁴,¹⁷ However, despite NAPA expression in the brain,¹⁵,¹⁶ the βSnap-KO develops recurrent epileptic seizures, suggesting that this compensation is no longer sufficient from P11 onward. The α- and βSNAP proteins are involved in the recycling of the SNARE protein complex to release them for a next round of docking and fusion events. If βSNAP is no longer available, recycling of the SNARE proteins could be disturbed, leading to a decrease of the synaptic vesicle fusion and to a decrease of neurotransmitter release, particularly during sustained neuronal stimulation. This mechanism has been suggested in epileptic encephalopathy linked to SNARE proteins, named “SNAREopathies,” mainly for STXBP1, SNAP25, and STX1B.³

In conclusion, the description of a fourth family that associates DEE and NAPB variants strongly supports that NAPB is a novel DEE-related gene. An extended study of the βSnap-KO mice and in vitro analyses of induced pluripotent stem-derived neurons¹⁸ would help to better understand the pathophysiological mechanism involved in NAPB-related DEE. Because multigene panels are still often used as first-tier testing,¹⁹,²⁰ we suggest including NAPB but strongly recommend using exome and genome sequencing to identify and describe more NAPB patients and better understand the associated phenotypes.

## AUTHOR CONTRIBUTIONS

Cécile Mignon-Ravix, Laurent Villard, and Florence Molinari designed the experiments, interpreted data, and wrote the initial manuscript. Cécile Mignon-Ravix analyzed and interpreted WES data and performed validation of variants. Cécile Mignon-Ravix, Nathalie Villeneuve, and Florence Molinari designed figures and tables. Florence Riccardi, Sylvie Lamoureux-Toth, and Nathalie Villeneuve performed clinical follow-up of the family. Géraldine Daquin, Sylvie Lamoureux-Toth, and Nathalie Villeneuve performed and interpreted EEG recording. Pierre Cacciagli extracted, stored, and delivered DNA from participating family members. All authors contributed to and revised the manuscript.

## ACKNOWLEDGMENTS

We are grateful to all family members who participated in this study. We thank the Genomics and Bioinformatics Platform of the Marseille Medical Genetics Center for their support and Auriane Caurette for technical assistance. This work was supported by Institut National de la Santé et de la Recherche Médicale, Aix-Marseille University,

and Assistance Publique des Hôpitaux de Marseille. F.R. has received 1-year financial support from the NeuroMarseille Institute through the French government program Investissements d'Avenir, Initiative d'Excellence d'Aix-Marseille Université via A*Midex funding (AMX-19-IET-004), and ANR (ANR-17-EURE-0029) for this work.

## CONFLICT OF INTEREST STATEMENT

None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

## ORCID

Laurent Villard https://orcid.org/0000-0001-6657-5008

Florence Molinari https://orcid.

org/0000-0001-5111-7215

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Mignon-Ravix C, Riccardi F, Daquin G, Cacciagli P, Lamoureux-Toth S, Villard L, et al. NAPB and developmental and epileptic encephalopathy: Description of the electroclinical profile associated with a novel pathogenic variant. Epilepsia. 2023;00:1-8. https://doi.org/10.1111/epi.17603