The study conforms to the Declaration of Helsinki. All research was approved by the ethics committee of Genotek Ltd. (05/2019). The patients’ parents provided written informed consent to studies and publication of clinical information and sequencing data.
Among all of the families there was no history of seizures or intellectual disability. All patients were diagnosed with EIEE9 when PCDH19 (NM_001184880.2) mutations were found.
Patient 1 (3 years old, female, 23 kg, 103 cm)
The pregnancy and perinatal history were unremarkable. The girl developed without abnormalities to the age of 3 years. At 3 years the girl developed generalized onset motor tonic-clonic seizures with apnoea at night against a background of fever. A month later the seizures were repeated against the background of normal temperature. Seizures during wakefulness first developed at 3 years and 2 months. Seizures persisted despite treatment with valproic acid.
Intelligence was normal. Speech was restricted to phrases. Ophtalmologic examination revealed no pathology. Ultrasound examination revealed hepatosplenomegaly. A blood test showed elevated lactate level (2,6 mmol/L, normal < 1,7 mmol/L).
Video electroencephalogram (VEEG) was performed for 2.5 h in states of active and passive wakefulness, sleep, and during functional tests (Fig. 1). The basic rhythm was represented by a regular, stable, modulated alpha rhythm with a frequency of 7–8 Hz and an amplitude up to 70 μV. This rhythm was recorded in the occipital regions with distribution to the posterior temporal and parietal regions of the hemispheres. Rhythmic theta-activity with tendency to hypersynchronization and regional delta-slow accentuation in the right temporal region with polyphasic potentials, but with no typical epileptiform activity were revealed at wake state. During rhythmic photostimulation, a rhythm following reaction was not observed. Photoparoxysmal response has not been recorded. A 3-min hyperventilation test revealed a moderate disorganization of cortical rhythm in the form of diffuse slow-wave activity of the theta range without a significant increase in amplitude. At the end of the test, there was rapid restoration of background. Sleep was modulated in phases; physiological patterns of sleep were differentiated. During sleep, regional epileptiform activity in the form of sharp-and-slow wave discharges in the temporal regions was revealed. K-complexes in the form of high-amplitude flashes of slow biphasic and polyphase waves with an amplitude of up to 190 μV were periodically recorded.
The brain magnetic resonance imaging (MRI) revealed a region of gliosis in the left frontal lobe.
The patient received antiepileptic treatments (valproic acid 600 mg/day, topiramate 25 mg/day, levetiracetam 1000 mg/day) without significant improvement.
Patient 2 (7 years old, female, 26 kg, 130 cm)
The pregnancy was complicated by a threat of preterm birth at 16 weeks and chlamydia. Up to 3 years and 2 months, the girl developed according to her age with periodic tension of the neck muscles which started at 7 months. At the age of 3 years and 2 months the first febrile seizure occurred in the form of tonic tension of the extremities at night (generalized onset motor tonic-clonic seizures). Seizures persisted despite treatment with valproic acid (500 mg/day). Clusters of seizures repeated every 5–10 days. Aggression and hysterical reactions were observed after the seizures. However, intellectual abilities were age appropriate. Ophthalmologic examination revealed no pathology.
The VEEG was performed for 2.5 h in states of active and passive wakefulness, sleep, and functional tests (Fig. 2). The basic rhythm was represented by a regular, stable, modulated alpha rhythm with a frequency of 8–9,5 Hz. Rhythmic 5–6 Hz theta activity in the frontal regions and theta accentuation in the vertex region, but with no typical epileptiform activity were revealed at wake state. Sleep was modulated in phases; physiological patterns of sleep were differentiated (K-complexes, vertex-potentials, sleep spindles). Moderate fusiform brush-like beta-activity accentuations and atypical K-complexes with spike-like insertions were revealed in frontal regions.
With antiepileptic (valproic acid 600 mg/day, levetiracetam 500 mg/day) and antipsychotic (aminophenylbutyric acid 250 mg/day) treatment at the time of analysis, seizures were not observed.
Genetic studies
After obtaining informed consent for the genetic analyses, targeted exome enrichment and sequencing were performed using patient and parental genomic DNA extracted from circulating leukocytes.
Exome sequencing
Exome sequencing of samples was carried out by Genotek Ltd. Genomic DNA from peripherial blood samples was extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturerʼs protocol. DNA libraries were prepared using the QIAseq FX DNA library kit (Qiagen, Hilden, Germany). Target enrichment, sequencing and quality control were performed as described previously [16]. To estimate pathogenicity, data were extracted from the dbNSFP, Clinvar, OMIM, and HGMD databases. The variants were analyzed in silico using Scale-Invariant Feature Transform (SIFT) and Polymorphism Phenotyping v2 (PolyPhen-2). Mutant allele frequencies were extracted from the 1000Genomes, ExAC, and Genotek databases. Pathogenicity was evaluated in accordance with international recommendations of ACMG (American College of Medical Genetics and Genomics), CAP (College of American Pathologists), and AMP (Association for Molecular Pathology).
Sanger sequencing
All variants found by exome sequencing were confirmed by Sanger sequencing. Additionally, we used Sanger sequencing to confirm the presence of PCDH19 mutations in the parents of probands. BigDye Terminator Cycle Sequencing Kit v3.1 (Thermo Fisher Scientific) was used to label amplicons with fluorescent labels. Sanger sequencing was performed on ABI PRISM 3500 Genetic Analyzer (Applied Biosystems) in accordance with the protocol of manufacturer.
All described variants are under consideration in the ClinVar database.