PPP3CA gene-related developmental and epileptic encephalopathy: Expanding the electro-clinical phenotype

Purpose: The objective of this study is to characterize the electro-clinical phenotype of individuals affected by the rare PPP3CA gene-related developmental and epileptic encephalopathy (DEE). Methods: We provide a detailed electro-clinical description of four previously unreported subjects, with unremarkable structural brain MRI and a normal screening for inborn errors of metabolism, who carry pathogenic variants within the regulatory domain of the PPP3CA gene, which encodes for calcineurin. We also conducted a literature review via PubMed and SCOPUS (up to December 2023) to collect all the studies reporting clinical details of subjects with PPP3CA pathogenic variants within the regulatory domain. Results: Our in-depth investigation reveals two distinct electro-clinical phenotypes with unique interictal and ictal patterns. Pathogenic variants within the calmodulin-binding domain result in childhood-onset epilepsy with focal and generalized seizures, developmental and intellectual impairments. Pathogenic variants within the regulatory domain lead to early onset drug-resistant severe epilepsy and potentially fatal outcomes. Comparative analysis with existing literature corroborates the notion that truncating mutations, prevalent in the regulatory domain but also possible in the calmodulin-binding domain, consistently associate with more profound disabilities and drug-resistant epilepsy. Conclusion: Our study emphasizes the critical role of pathogenic variants ’ type and location on the severity of PPP3CA-related DEE. We also speculate, based on peculiar EEG patterns, on potential pathophysiological mechanisms involving calcineurin dysfunction and calcium homeostasis. In order to improve our understanding of this rare DEE, we need both collaborative efforts to gather larger cohorts and further experimental studies.


Introduction
Calcineurin is a crucial protein phosphatase that mediates calciumdependent signaling by affecting the phosphorylation of numerous proteins [1].It is encoded by the PPP3CA gene, which is widely expressed in the brain [2].Calcineurin comprises a catalytic domain (calcineurin A, CnA) and a regulatory domain, which in turn includes three domains: calmodulin binding (CaMB), calcineurin B binding (CnBB) and an auto-inhibitory (AI) domain [3,4].The activation of calcineurin involves the binding of calcium to calmodulin and calcineurin B, which causes a conformational change that removes the AI domain from the catalytic site [1,5].The first report of PPP3CA-gene related developmental and epileptic encephalopathy (DEE) linked calcineurin dysfunction and altered calcium homeostasis to defects in synaptic vesicle cycling [6].As a consequence this DEE was associated with other epilepsy syndromes of genetic etiology like those caused by pathogenic variants in genes involved in synaptic function such as DNM1, STXBP1, and SNAP25 [6].A first genotype-phenotype correlation identified two main types of pathogenic variants: missense loss-of-function mutations in the catalytic domain, associated with West syndrome, and missense gain-of-function mutations in the AI domain, which were linked to multiple congenital anomalies and intellectual disability with or without epilepsy [4].However, further clinical studies showed that the epileptic phenotype exhibited greater complexity than typical West syndrome, encompassing various seizure types, often drug-resistant, such as epileptic spasms, focal, tonic, myoclonic, generalized tonic-clonic and atonic seizures [6].
In addition, subsequent reports highlighted the need to distinguish, among loss-of-function pathogenic variants, between missense mutations in the catalytic domain and truncating mutations in the regulatory domain.In fact, missense mutations in the catalytic domain disrupt enzyme function, whereas truncating mutations in the regulatory domain appear to dramatically reduce the amount of functional calcineurin, resulting in more severe phenotypes characterized by an earlier onset of drug-resistant seizures and more severe intellectual disability [8,9,10].
To comprehensively characterize and to better understand this rare DEE, it is crucial to thoroughly describe the electroclinical phenotype of new patients including those with pathogenic variants in the calmodulin-binding domain, a scenario apparently not yet reported in the current literature.A targeted analysis of interictal and ictal EEG patterns is essential and has not been addressed in the cases described so far, although considered necessary by several authors in order to identify common and distinctive features [11]

Materials and methods
We described four subjects (three females) studied at three different Italian centers: the Pediatric Neurology and Neurophysiology Unit of the University of Padua, the Child Neuropsychiatry Unit of Santa Chiara Hospital in Trento and the Department of Pediatric Neurology of Bolzano.Moreover, one patient was evaluated in the follow-up at Pediatric Child Neurology Clinic in Innsbruck, Austria.We selected these four subjects on the basis of the following inclusion criteria: an electroclinical diagnosis of developmental and epileptic encephalopathy with varying degrees of severity; unremarkable brain magnetic resonance imaging (MRI) and expanded screening for inborn errors of metabolism; an extensive genetic analysis (Next Generation Sequencing custom panels or whole exome sequencing) demonstrating a pathogenic variant of the PPP3CA gene.Two subjects harbored PPP3CA pathogenic variants (one missense and one truncating) within the calmodulin binding domain, the other two carried truncating PPP3CA pathogenic variants in We included in this review only the studies that report clinical details of subjects who suffered from developmental and epileptic encephalopathy and harbored a PPP3CA mutation within the regulatory domain.

Results
Medical history and electroclinical data for each participant are summarized in Table 1.

Subject 1
The first patient is a girl, delivered by cesarean section at 37 gestational weeks following a twin pregnancy achieved through in vitro fertilization.The girl had turricephaly, wide nasal wings, a blissful mouth, full lips, and micro-retrognathia.She achieved independent walking at age 20 months and semantic language use at three years.A progressive hypokinetic movement disorder with rigidity, bradykinesia and camptocormia was observed.A structured cognitive assessment (Wechsler Intelligence Scale for Children IV -WISC IV) performed at 9 years of age confirmed severe cognitive impairment with Full-Scale Intelligence Quotient (FSIQ) uninterpretable and General Ability Index of 34.While no autistic traits were identified, there was a notable tendency toward emotional dysregulation and severe behavioral intermittent explosive disorder.Genetic analysis, through a Next Generation Sequencing custom panel, revealed a de novo, not previously reported, truncating, heterozygous variant of the PPP3CA gene (p.Lys393Ter) within the calmodulin binding domain.The variant was classified as pathogenic according to American College of Medical Genetics (ACMG) guidelines.
The girl had onset of epilepsy at 4 years of age, with focal to bilateral motor clonic seizures (see videos in Supplementary materials), focal emotional seizures, atypical absences, generalized reflex tonic seizures and later, within one year from the onset, with focal motor hemiclonic status epilepticus.
Interictal EEG (Fig. 1A) showed generalized slow background activity with frequent spike-wave complexes in bilateral frontotemporal regions, sometimes forming unilateral or bilateral bursts.During drowsiness (Fig. 2A), high-voltage slow waves alternated with low voltage background activity and spike/polyspike-wave complexes at bilateral frontal regions.Sleep EEG revealed reduced slow abnormalities, featuring frontal spikes, polyspikes, and generalized interictal epileptiform discharges.Ictal EEG during focal clonic motor seizures exhibited initial triphasic slow waves with fast activity, evolving into hemispheric theta activity and focal electrical patterns that alternated between hemispheres or diffused bilaterally (Fig. 3A).
Currently, at 13 years of age, the patient has drug-resistant epilepsy in partial control on a polytherapy with clobazam, valproic acid, lacosamide, cannabidiol.Focal motor hemiclonic status epilepticus was recurrently observed and treated with second-line drugs (intravenous phenytoin, intravenous lacosamide).She implanted a vagus nerve stimulator in summer 2023 (intensity 2 mA, frequency 20 Hz, pulse width 500 microsec, duty cycle 10%) but this did not prevent her from presenting with two others focal motor status epilepticus, both responsive to intravenous benzodiazepines or phenytoin.

Subject 2
The second patient is a girl, born at 38 gestational weeks through a normal delivery following an uneventful pregnancy.In terms of dysmorphisms, the child displayed turricephaly.Head control was achieved at 4 months, and she started walking independently at 21 months.Neurological examination showed persistent global hypotonia and clumsiness since the first evaluation at 3 years of age.At age 8 years, a comprehensive functional assessment using the Griffith Mental Development Scales revealed a global developmental delay (general ageequivalent scores of 24 months instead of the 98 months chronological age) and showed widespread impairments in all developmental areas (Age-equivalent scores: Gross motor function 32 months, Personal-Social-emotional 24 months, Language and Communication 28 months, Eye and Hand Coordination Performance 36 months, Foundations of Learning 39 months).The child exhibited a hyperkinetic movement disorder, significant hyperactivity, autistic traits, and emotional dysregulation.Genetic analysis, through a Next Generation Sequencing custom panel, identified a de novo missense heterozygous variant in the PPP3CA gene (p.Leu412Pro) within the calmodulin binding domain.The variant wasn't previously reported in literature and in the Human Gene Mutation Database and, according to ACMG guidelines, was classified as likely pathogenic.
At age 3 years, the girl had onset of epilepsy with focal emotional seizures and generalized myoclonic seizures in wakefulness and by generalized tonic seizures in sleep.She never experienced status epilepticus.Interictal EEG during wakefulness (Fig. 1B) showed generalized slowing with frequent slow waves, sharp waves, and spike-wave complexes in bilateral frontal and left temporal regions, often forming left temporal focal sequences or bilateral bursts.In drowsiness and sleep (Fig. 2B), bilateral frontal regions exhibited high-voltage slow waves or spike-wave complexes alternating with monomorphic theta activity or generalized voltage background activity.Over three years, interictal EEG showed progressive slowing of background activity with lower voltage.Tonic seizures during sleep were characterized by a bilateral high-amplitude triphasic slow wave, followed by low-voltage fast activity, and a buildup of rhythmic slow waves and spike-wave complexes in frontal regions.
At the current age of 9, the patient experiences episodic generalized seizures (overall myoclonias) despite treatment with valproic acid and rufinamide.

Subject 3
The third patient is a girl, born at 39 gestational weeks following an uneventful pregnancy but concluded with a precipitous labor.No dysmorphic features were observed.The infant failed to achieve head control, displaying generalized hypotonia and limited eye contact.Genetic analysis, through a Next Generation Sequencing custom panel, uncovered a de novo heterozygous frameshift variant in the PPP3CA gene (p.Ser419fs*) within the regulatory domain, resulting in the synthesis of a truncated protein.The variant, according to ACMG guidelines, was classified as pathogenic.
The girl had onset of epilepsy in the neonatal period (within 24 hours of birth) with multifocal motor seizures, followed by apnea, cyanosis and desaturation.By 4 months, the condition progressed into an epileptic encephalopathy with spasms.The child unfortunately died at 6 months during an episode of status epilepticus.Interictal EEG findings prior to 4 months depicted multifocal interictal epileptiform discharges predominantly involving fronto-temporal regions during wakefulness (Fig. 1C), activating during sleep.After age 4 months, an electrographic pattern consistent with hypsarrhythmia emerged, marked by predominant epileptiform abnormalities in bilateral fronto-temporal areas.
During the neonatal period, multifocal seizures originating independently from the two temporal regions were recorded, demonstrating an alternating discharge between hemispheres.This discharge was succeeded by apneas recorded via pneumogram and prolonged periods of suppression of electrical activity.Subsequently, after 4 months, epileptic spasms were documented with their specific electrical correlate of isolated triphasic slow waves with superimposed fast activity (Fig. 3B).

Subject 4
The fourth patient is a boy, born at 38 gestational weeks through a difficult delivery with the application of a vacuum extractor after an uneventful pregnancy.From the age of 3 months, signs of visual disorganization emerged, characterized by a lack of fixation and visual pursuit.Global postural-motor disorganization manifested with limited spontaneous motor activity in the upper limbs and absent activity in the lower limbs, along with more pronounced axial hypotonia.Over the years, a profound intellectual disability developed, marked by poor social interaction and communication with a complete absence of language.The patient exhibited flaccid postural tetraparesis, accompanied by a peculiar evolution of muscle tone across development from hypotonia to hypertonia in the extremities, as well as hyperkinetic movement disorder (including dyskinesias, dystonia and segmental non-epileptic myoclonus).Additionally, the patient experienced episodes of desaturation requiring oxygen dependency, recurrent respiratory infections, hyperhidrosis, and temperature dysregulation in the extremities.A whole exome sequencing revealed a de novo heterozygous frameshift mutation in the PPP3CA gene (p.Ser419fs*) within the regulatory domain, leading to the production of a truncated protein.The variant, according to ACMG guidelines, was classified as pathogenic.Epilepsy onset was characterized by focal motor seizures at 3 months, followed by epileptic spasms at 6 months, focal non-motor seizures at 8 months and generalized tonic seizures at 10 months, the latter becoming the predominant seizure type (see videos in Supplementary materials).The patient consistently exhibited significant drug-resistance with several daily seizures and died at the age of 6 years during an episode of refractory status epilepticus.
Initially, the interictal EEG in wakefulness showed generalized slowing with dominant slow waves in occipital regions and an unrecognizable, non-reactive posterior rhythm.Over time, background activity became poorly organized, with low voltage in posterior regions, theta activity in paramedian regions, and irregular delta activity in fronto-temporal regions (Fig. 1D).Multifocal epileptiform discharges were observed in frontal, central, and temporal regions.During drowsiness and light sleep (Fig. 2C), there was increased delta activity and spike-wave complexes in frontal regions.In deeper sleep, a "burst suppression-like" pattern emerged, characterized by frontal discharges and bursts of slow and sharp waves followed by 2 to 5-second epochs of diffusely low voltage activity (Fig. 2D).In wakefulness, focal non-motor seizures with eyelid blinking and fixed upward gaze were recorded, associated with bilateral rhythmic slow waves at 2.5 Hz, predominantly in fronto-temporal regions (Fig. 3C).During sleep, tonic seizures began with a sudden "shock-like" movement, followed by hypertonia of the limbs, eye and mouth opening with upward gaze, eyelid blinks, sialorrhea, and altered breathing.The ictal EEG revealed a bilateral highamplitude triphasic slow wave, followed by low-voltage fast activity for about 30 seconds, and then a build-up of rhythmic slow waves in frontal regions.Some seizures were initially associated with brief asystole (1-2 seconds) (Fig. 3D).

Genotype-phenotype correlations
Based on the above, we aimed to determine if it was possible to phenotypically distinguish within a group of subjects with a pathogenic variant in the regulatory domain of calcineurin, those whose variant fell within the calmodulin-binding domain from those whose variant fell outside (Supplementary Fig 1).This phenotypic characterization highlighted two potentially distinct clinical phenotypes with a certain continuum between them.However, definitive conclusions cannot be drawn due to the small cohort of patients.
From an overall clinical point of view, the two subjects with pathogenic variants in the calmodulin binding domain manifested a moderate form of epilepsy with onset in late childhood (at 3 and 4 years of age), global developmental delay, subsequent severe intellectual disability, hypo-and hyperkinetic movement disorders, dysmorphic features and behavioral disturbances, notably emotional dysregulation, hyperactivity and autistic traits.Noteworthy is the comparatively milder neurological severity observed in the case with the missense mutation as opposed to the case with the truncating mutation.
From an epileptological perspective, the epilepsy onset was characterized by focal motor seizures (often clonic) No epileptic spasms were observed, but sporadic typical ictal EEG patterns were detected.Subsequently, focal non-motor emotional seizures, focal motor hemiclonic status epilepticus and generalized seizures (atypical absences, myoclonic and tonic seizures) emerged.
Regarding the EEG features, the two subjects displayed in wakefulness (Fig. 1) generalized slow background activity, frontotemporal sequences of epileptiform discharges and bilateral bursts.During drowsiness (Fig. 2), they displayed on the frontal regions sequences of high-voltage slow waves alternated with sequences of spike/polyspikewave complexes.During sleep (Fig. 3), they exhibited bursts of generalized interictal epileptiform discharges alternated with monomorphic theta activity or globally low voltage background activity.
In contrast, the two subjects harboring truncating mutations within the regulatory domain (affecting the same amino acid residue) were characterized since birth by multiple dysmorphic features, compromised head control, generalized hypotonia and limited ocular engagement.Disease progression was marked by global postural-motor disorganization, flaccid postural tetraparesis, pronounced axial hypotonia, a distinctive evolution of muscle tone from hypotonia to hypertonia in the extremities, profound intellectual disability, complete absence of language.Additional features encompassed a hyperkinetic movement disorder (comprising dyskinesias, dystonia, and segmental non-epileptic myoclonus), episodes of desaturation necessitating oxygen support, recurrent respiratory infections and autonomic dysregulation.
From an epileptological perspective, they both presented an early onset epilepsy (neonatal in one case and at 3 months in the other) with a fatal outcome.At the onset the epilepsy was characterized by multifocal motor seizures, often clonic, albeit followed by apnea, cyanosis, and desaturation.The clinical picture evolved into an epileptic encephalopathy with spasms followed, in the second subject, initially by focal non-motor seizures and subsequently by generalized myoclonic and predominantly tonic seizures, sometimes associated at the very beginning with a brief asystole.
Regarding the EEG features, both subjects exhibited in wakefulness a poorly organized background activity with irregular delta activity on fronto-temporal regions and multifocal epileptiform discharges.Hypsarrhythmia, when present, was marked by predominant epileptiform abnormalities in bilateral fronto-temporal areas (Fig. 1).During drowsiness, they displayed on frontal regions sequences of high-voltage slow waves alternated with sequences of spike/polyspike-wave complexes (Fig. 2).In sleep, subject 3 and 4 displayed a distinctly discontinuous pattern, reminiscent of a "burst suppression-like" pattern (Fig. 3).

Literature review
Our literature review identified 10 previously described cases in which mutations affect the regulatory domain.In Table 2 we reported the key elements to outline the electro-clinical phenotype.More extensive clinical data are provided in Supplementary Table 1.All these previously described variants are clustered within a short protein segment between the calmodulin binding domain and the inhibitory domain, and none of them is located within the calmodulin-binding domain.
The only patient with a missense mutation (p.Ala447Thr) [6] appears to present a similar clinical picture to our patient 2 (harboring a missense mutation within the calmodulin-binding domain), characterized by the onset of epilepsy at 3.5 years with multifocal discharges, tonic-clonic and tonic generalized seizures, severe intellectual disability, limited vocabulary with short sentences (lots of perseverations), autistic features and an ataxic gait.,This individual exhibits more pronounced dysmorphisms (coarse facial features, thick lips and a large tongue, hoarse voice, hypertelorism) observed only in subjects 1 and 4 (Table 1).
As for the other cases with truncating mutations, they all exhibit severe phenotypes, consistent with our cases.The age of seizure onset ranged between 6 weeks to 2 years, but with all cases except 2 experiencing seizures in the first 6 months of life.The described seizures are predominantly focal seizures, spasms associated with hypsarrhythmia, tonic generalized seizures and are often drug-resistant.EEG findings include a slow background activity during wakefulness and often discontinuous activity during sleep with multifocal epileptiform anomalies, predominantly fronto-temporal.The overall neurological picture is compromised, characterized by severe intellectual disability, significant impairments in relational and linguistic skills and a motor profile with flaccid quadriplegia but with a possible peculiar muscle tone evolution from hypotonia to hypertonia in the extremities.

Discussion
Our study further explores the electro-clinical phenotypes associated with pathogenic variants in the PPP3CA gene.We thoroughly characterized the phenotypes of four subjects: two with variants in the calmodulin-binding domain-previously unreported in the literature-and two with variants in the regulatory domain but outside the calmodulin-binding domain.We also compared the latters subjects with 10 previously described cases, all carrying variants in the regulatory domain.
The overall pattern shows that epilepsy onset can be late (in subjects with missense or truncating variants in the calmodulin-binding domain) or early (in subjects with truncating variants in the regulatory domain), typically presenting with focal motor seizures.Late-onset cases often include focal non-motor seizures with predominant emotional symptoms (e.g., fear) and generalized seizures.Interestingly, one subject with a truncating variant developed recurrent focal motor hemiclonic status epilepticus (not previously reported for PPP3CA cases) and more severe neurological impairment compared to a subject with a missense variant in the same domain.
Early-onset cases (all characterized by truncating variants) may involve motor focal seizures with autonomic dysfunctions, progressing to epileptic encephalopathy with spasms, followed by focal non-motor seizures and predominantly tonic generalized seizures.
A critical aspect of our study is the detailed characterization of EEG features beyond the different seizure types.Patients with variants in the calmodulin-binding domain exhibited interictal EEG features such as slow background activity, frequent slow waves, and epileptiform discharges in bilateral frontotemporal regions.In contrast, those with mutations in the regulatory domain displayed multifocal epileptiform discharges and severely altered background activity both during wakefulness, with a tendency toward hypsarrhythmia, and in sleep, with a discontinuous pattern.

Table 2
Genetic, clinical and neurophysiological data of previously reported patients with PPP3CA-related DEE and a mutation localized within the regulatory domain.
While our study contributes to elucidate the electro-clinical spectrum of PPP3CA-related DEE, the limited number of cases highlights the rarity of this condition.Collaborative efforts are needed to accumulate larger cohorts for more comprehensive analysis and to draw definitive conclusions.

Possible pathophysiological mechanisms
Furthermore, our attempt to further characterize the electro-clinical features and correlate genotype with phenotype leads us to propose some speculative hypotheses regarding the potential pathogenic mechanism.Since calcineurin is crucial for neuronal adaptation to high intracellular calcium concentrations [1,2,3], this unique DEE may serve as a model to support the fundamental role of calcium homeostasis in epileptogenesis.It also provides an opportunity to study the intricate interplay between intra-and extracellular calcium concentration, calcium-dependent proteins, and epileptogenesis.
We believe it is essential to unravel the pathophysiological underpinnings through experimental in vitro and animal studies, especially considering that currently available antiepileptic drugs do not target these pathways.Certain EEG features we observed might offer a framework for exploration.Notably, patients with truncating variants within the regulatory domain may present a discontinuous pattern during sleep, resembling a burst-suppression pattern.
Elegant experimental studies conducted through intracellular recordings of neurons and glia, extracellular calcium measurements and EEG recordings in animal models during the induction of burstsuppression using isoflurane [13] have revealed that the burst-suppression pattern is associated with a state of cortical hyperexcitability, likely induced by a global increase in extracellular Ca2+ concentrations.The bursts are evoked by even subliminal stimuli that reach the cortex and the quasi-rhythmicity of the bursts seems to result from an interplay between cortical hyperexcitability and a post-burst refractory period, which is also Ca2+ dependent [13].
Contextualizing these findings within the electro-clinical framework of the subjects described, we can hypothesize two possible mechanisms for the burst-suppression-like pattern observed during sleep.On one hand, this could be a peculiar "EEG trait" of the syndrome: significant calcineurin dysfunction may render the cells incapable of implementing compensatory homeostatic mechanisms on calcium concentration, elevating extracellular calcium levels and inducing burst suppression.On the other hand, this peculiar DEE might nonspecifically increase cortical hyperexcitability, making the bursts resemble "ictal" patterns (induced by subliminal stimuli) followed by compensatory suppression periods.
In this context, it is noteworthy that two of the described subjects (subject 1 and subject 4) benefited from cannabidiol therapy, a drug that appears to both reduce extracellular calcium influx and inhibit intracellular calcium release.Clearly, these hypotheses are purely speculative and will require validation through appropriate in vitro and in vivo experimental studies.

Conclusion
In conclusion, our study reveals distinct clinical trajectories associated with PPP3CA variants, ranging from early-onset severe phenotypes to late-onset moderate forms.This underscores the critical influence of mutation location within the gene on the severity of developmental and epileptic encephalopathy.Notably, we describe for the first time subjects with pathogenic variants within the calmodulin-binding domain and, unlike previously published studies, our research meticulously characterizes both interictal and ictal EEG features.Consistent with existing literature, our findings confirm the correlation between the degree of protein loss of function and the severity of PPP3CA-related DEE, highlighting important clinical implications for prognosis and management.The primary limitation of our study is the small number of cases, reflecting the rarity of this condition.Collaborative efforts are essential to gather larger cohorts for more comprehensive analyses and to draw definitive conclusions.Finally, we propose potential pathophysiological mechanisms, suggesting that calcineurin dysfunction in these unique DEE cases may serve as a model to understand the fundamental role of calcium homeostasis in epileptogenesis.This underscores the need for experimental in vitro and animal studies to uncover the underlying pathophysiological mechanisms, especially given that current antiepileptic drugs do not target these pathways.

Declaration of competing interest
None of the authors has any conflict of interest to disclose.

Fig. 2 .
Fig. 2. Interictal EEG features in drowsiness and sleep.The figure depicts representative epochs (30 s) of the interictal EEG recorded in drowsiness (Panel A and C) and sleep (Panel B and D) in our four subjects.In drowsiness, all subjects displayed sequences of high-voltage slow waves alternated with sequences of spike/ polyspike-wave complexes at bilateral frontal regions.Patient 1 -Panel A: high-pass filter 70 Hz, low-pass filter 0.5 Hz, notch filter 50 Hz, amplitude 170 uV/ cm.Patient 4 -Panel C: high-pass filter 70 Hz, low-pass filter 0.5 Hz, notch filter 50 Hz, amplitude 120 uV/cm.In sleep the first two subjects (Patient 1 and Patient 2) exhibit bursts of generalized interictal epileptiform discharges alternated with monomorphic theta activity or globally low voltage background activity (Patient 2 depicted in Panel B).On the other hand, Patient 4 (Panel D) displayed a distinctly discontinuous pattern, reminiscent of a "burst suppression-like" pattern.Patient 2 -Panel B: high-pass filter 70 Hz, low-pass filter 0.5 Hz, notch filter 50 Hz, amplitude 150 uV/cm.Patient 4 -Panel D: high-pass filter 70 Hz, low-pass filter 0.5 Hz, notch filter 50 Hz, amplitude 120 uV/cm.

Table 1
Genetic, clinical and neurophysiological data of the four reported patients. 10]