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To report the effect of the ketogenic diet (KD) on non-convulsive status epilepticus (NCSE) due to Angelman syndrome (AS) in two members of a large Georgian family affected by a novel frameshift variant in the UBE3A gene (NM_000462.3).
Methods
We evaluated two members of this family who were affected with clinical and EEG features of AS. Clinical history with special emphasis on development, seizure type, frequency, and treatment was reviewed. Routine and long-term video EEG monitoring were conducted, particularly during NCSE. A non-fasting inpatient KD protocol was implemented using blended food orally with full administration of 4:1 (fat to non-fat) ratio. Urine ketone bodies (KBs), measured with urine ketone acetone strips readings, reached 150 mg/dL in both patients.
Results
Patients had characteristic signs of AS and presented with epilepsy between the age of 2–4 years. As methylation tests were negative, next generation sequencing disclosed a c.2365del variant. For both, NCSE was revealed by cognitive deterioration and did not respond to anti-seizure medication. As recommended, IV pyridoxine, benzodiazepines, and valproic acid were administered, but without success. For both patients, NCSE resolved on the second-third day of KD initiation, before the appearance of ketonuria and resulting in improved communication, mood and sleep.
Conclusion
KD is safe and effective for the treatment of NCSE due to AS. Resolution before the appearance of ketone bodies points to a possible mechanism of action of KD.
Angelman syndrome (AS) is characterized by severe delay, speech impairment, movement or balance disorder, behavioural uniqueness and frequent epilepsy. Non-convulsive status epilepticus (NCSE) affects half of the patients [
Although de novo deletion of the 15q11.2-q13 region of the maternally inherited chromosome 15 is the main cause of AS, pathogenic variants in the maternally inherited UBE3A gene are disclosed in ∼10% of the patients.
We report a large Georgian family with an unreported frameshift variant in the UBE3A gene (NM_000462.3) in which NCSE responded dramatically to the ketogenic diet (KD).
1. Patients and methods
Clinical history was obtained from parents and medical charts, with special emphasis on development, history of seizures, and treatment.
1.1 DNA extraction and sequencing
Four affected patients were sampled and underwent genomic CGH-array. Genomic DNA of each index patient and his or her parents were extracted from peripheral leukocytes using the Promega DNA Kit, and a panel of 56 ID/DD-related genes (including UBE3A) was analysed according to the standard procedure [
]. Sanger sequencing was performed for index patients and other family members to validate the disclosed variant.
1.2 Evaluation and treatment of NCSE
Trials with IV pyridoxine, benzodiazepines, and valproic acid were done in ICU during NCSE recorded with video electroencephalogram (EEG). Following parents informed consent, a non-fasting inpatient 4:1 ratio KD protocol was implemented using blended food orally and reaching full administration on the fourth day. Urine acetoacetic acid, measured with urine strips, reached 150 mg/dL in both patients.
The study was approved by the ethics committee of MediClubGeorgia Medical Centre.
2. Results
2.1 Genetics
Five individuals were affected: two sisters, 10y and 9y old (patient (Pt.)1 and Pt.2) belonging to generation IV (IV-1, IV-2) while a 5-year-old brother (V-1) and a 3-year-old sister (Pts.3 and 4) belonged to generation V (V-2) (Fig. 1). One individual with developmental delay (IV-18) could not be enroled in the molecular genetic study.
All affected patients had a novel UBE3A frameshift variant (NM_000462.3) caused by the deletion of G at the position c.2365 in exon 3, inherited from their asymptomatic mother, creating a premature stop codon with a truncated protein (p.Glu798Serfs*32).
The mode of inheritance is consistent with that of AS, provided that the paternal first cousin (IV-18) of the mother (IV-10) of affected siblings was also affected. Since all affected individuals share the same UBE3A variant, it is likely the cause of the clinical phenotype.
2.2 Clinical description
Patients were born at full term following an uneventful pregnancy. They started sitting independently at 8–15 months and walking at 3–5 years They could not articulate more than two syllables, and displayed a typical AS phenotype: happy behaviour with frequent smiling and laughing, and ataxic gait. The two oldest patients developed epilepsy.
Pt. 1, a 10-y old female, began at 2 years to have episodic tremor/jerks that occasionally caused falling backward. At 5 years they were diagnosed myoclonic seizures and levetiracetam (LEV) gave a dramatic response, with seizure-freedom since then.
However, at 5.5, she started losing developmental skills: decreased alertness with poor communication and agitation. She was constantly in a “bad” mood, not responsive to any commands, with poor sleep. EEG detected continuous, regular, 1.5–2 Hz spike/polyspike-slow wave discharges which confirmed NCSE. IV diazepam, valproic acid, and pyridoxine failed. After discussion of further treatment options, parents preferred ketogenic diet over conventional antiepileptic drugs. Non-fasting inpatient 4:1 ratio KD was initiated.
On the third day, mood, behaviour, and communication became better: she responded to her name and could communicate better with gestures and eye gaze, sleep improved and EEG showed a major decrease of spike-wave activity (Fig. 2.B). KBs were first noticed in urine on the fourth day of KD.
B: On the 3rd day of the KD. NCSE has disappeared.
C: The improvement is maintained one month after the initiation of KD treatment.
Patient 2, 4 year-old.
A: So-called hypsarrhythmia-like pattern, similar to tracing in myoclonic status: high-amplitude, generalized, irregular delta waves intermixed with multifocal spikes and sharp waves.
B: On the 3rd day of the KD, background activity has improved, spikes have disappeared.
C: One month after initiation of KD treatment, the tracing is typical for AS.
Pt. 2 was a 9-y old female. At 3 years 6 months she had 5 episodes in 24 h of febrile seizures. Two months later she developed non-febrile seizures with loss of consciousness and tone, lasting 2–3 min, once to twice a month. No medication was given.
At the age of 4 years and 1 month, decreased alertness, loss of eye contact and interest in the environment, and deterioration of motor activity, including laughing were noticed. EEG detected a continuous diffuse high amplitude, asynchronous spike and slow-wave activity reminiscent of hypsarrhythmia which confirmed NCSE. Based on its success for her sister, she began KD. On the second day, parents noticed that the child “switched on”, became active, alert, and interested in toys and music, with her usual bursts of laughter, and began to crawl. EEG recorded on the third day confirmed the disappearance of spiking activity, while KBs appeared in the urine only on the fifth day of the diet.
After 1 year of treatment period the ketogenic diet was discontinued in both patients due to difficulty to keep a stringent diet in socially active kids. It was replaced by carbohydrate restriction followed by low glycaemic index treatment. NCSE did not recur during the 5 years follow-up period with annual EEG monitoring showing typical EEG pattern for AS, but parents mentioned that it is during the KD that their level of alertness and behaviour had been the best.
3. Discussion
We report a large Georgian family with four patients affected by AS with an unreported UBE3A frameshift variant likely to create a premature stop codon. It truncates the C-terminal part of the UBE3A protein which is essential for polyubiquitin chain formation, suggesting that this frameshift variant is pathogenic [
In silico modeling of the cryptic E2∼ubiquitin–binding site of E6-associated protein (E6AP)/UBE3A reveals the mechanism of polyubiquitin chain assembly.
]. UBE3A pathogenic variants are frequent in familial cases, with a relatively mild phenotype compared to deletion-type AS also deleting genes of the GABA-A receptor complex [
]. During deterioration, Pts. 1 and 2 displayed different EEG patterns: Pt.1 showed the usual regular, 1.5–2 Hz spike-slow wave/polyspike-slow wave discharges (Fig. 2), while Pt.2 showed a less synchronous aspect reminding hypsarrhythmia or the tracing associated to myoclonic status epilepticus [
Myoclonic status in non-progressive Encephalopathies.
in: Delgado Escueta AV Guerrini R Medina MT Genton P Bureau M Dravet CH Advances in neurology, myoclonic epilepsies. 95. Lippincott Williams & Wilkins,
Philadelphia, PA2004: 153-156
] (Fig. 2). This pattern has been reported as a diagnostic pitfall since these infants show marked regression or delay in development with jerks, and vigabatrin given for suspected infantile spasms usually worsens the myoclonic seizures [
]. A distinctive feature in our patient 2 is that this pattern occurred at the age of 4y without any motor seizure, whereas almost all reported cases were noticed before the age of two years [
], few did require additional therapy. The KD has long been used successfully for patients with a recent deterioration of seizure control and neurological status [
For our two patients, the 4:1 KD stopped NCSE within a few days, and EEG background dramatically changed with beneficial effects on behaviour, mood, and sleep.
While the direct anticonvulsant action of KBs remains unclear and controversial [
], it is noteworthy that for our two patients and several reported in the literature (up to 20% - see supplementary data), seizure activity stopped before the appearance of ketosis. Although this does not rule out the possibility that there was a subcellular ketone body-related metabolic shift that may have accounted for the antiepileptic activity, reports on the low glycaemic index diet efficacy underline the possible effects of glucose restriction by itself on seizure control.
Like the brain, the muscle may use both aerobic and/or anaerobic sources for energy metabolism. In the muscle, particularly the myocardium, energy supply for the resting state and basal activity is provided by KBs. However, in case of sudden effort increasing energy consumption, the sudden rise in energy supply is mainly provided by aerobic sources, thus glucose to sustain the effort [
]. Regarding the brain, the very small amount of glycogen available is sufficient to ensure a few minutes of major activity (for a review, see Obel et al. [
]) needed for seizure onset. In the context of glycogen depletion generated by the KD, brain effort would be hampered because of lack of glucose source in all parenchyma's, including the brain. Such a hypothesis could also account for the antiepileptic effect of the “Low glycaemic index” diet.
Whatever the mechanism, our case study shows that KD is safe and efficient for the treatment of NCSE due to AS.
4. Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
The authors declare that there are no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We would like to acknowledge Beth Zupec-Kania, RDN, CD (Ketogenic Therapies, LLC) and Ana Mingorance, PhD (Orphan Drug Discovery Strategy) for their valuable suggestions and advices in the development of this manuscript.
In silico modeling of the cryptic E2∼ubiquitin–binding site of E6-associated protein (E6AP)/UBE3A reveals the mechanism of polyubiquitin chain assembly.
Myoclonic status in non-progressive Encephalopathies.
in: Delgado Escueta AV Guerrini R Medina MT Genton P Bureau M Dravet CH Advances in neurology, myoclonic epilepsies. 95. Lippincott Williams & Wilkins,
Philadelphia, PA2004: 153-156
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