Genetic exploration of Dravet syndrome: two case report

Dravet syndrome (DS), previously known as severe myoclonic epilepsy of infancy (SMEI), is an infantile-onset developmental and epileptic encephalopathy (DEE) characterized by drug resistance, intractable seizures, and comorbidities including intellectual disability, behavioral problems, sleep disturbances, gait disturbances, and an increased risk of sudden unexpected death in epilepsy [1, 2]. The incidence of DS is approximately 1 in every 15,700 births [3]. The first symptom of DS is seizures in the first year of life, followed by developmental delay [1]. This first seizure is either generalized tonic–clonic or focal (occasionally hemiclonic) clonic, and in more than half of the cases, it is a febrile seizure, making it difficult to distinguish from a self-limiting febrile seizure. Infection, hot environment, exhaust, sunlight, or exercise can initiate an attack of DS [4, 5]. Approximately 80% of patients with DS carry a pathogenic variant of the sodium channel alpha 1 subunit (SCN1A) gene resulting in haploinsufficiency Nav1.1, the alpha-1 subunit of the sodium channel. PCDH19, SCN2A, SCN8A, SCN1B, GABRA1, GABRB3, GABRG2, KCNA2, CHD2, CPLX1, HCN1A, and STXBP1 variants may also be involved in DS or DS-like phenotypes. Accordingly, genetic testing is required to identify other genes that play a role in the DS phenotype and to expand genotype-DS phenotype correlations to enhance the future management of this disease [6]. In the last decade, next-generation sequencing (NGS) technology has been able to analyze a set of genes (targeted panel sequencing), exome [(whole exome sequencing (WES)], or genome [whole genome sequencing (WGS)] in a single sequencing process, making it possible to diagnose rare diseases such as early childhood epilepsy [7]. Identification of the genetic basis of DS can provide additional information regarding pathophysiology, prognosis, and individual drug therapy options according to the patient’s condition.

Research on the identification of DS genetic mutations using NGS has never been done in Indonesia. In 2010, we conducted a study to identify pathogenic variants of the SCN1A gene using the Sanger sequencing method and successfully reported cases of novel SCN1A mutations in Indonesia in patients with severe myoclonic epilepsy in infancy (SMEI) and borderline SMEI (SMEB). The first boy identified with SMEI experienced a variety of seizures, including his first febrile seizure and general tonic–clonic seizure at 7 months of age, and later suffered from myoclonic seizures, left-sided hemiconvulsions, also focal convulsions without fever, along with delayed speech development. The second patient with SMEB had his first febrile seizures with GTCS after immunization at 3 months old, then later on experienced status epilepticus, GTCS, and atonic convulsions without fever [13]. We also conducted another research on the spectrum of generalized epilepsy with febrile seizure plus (GEFS+) focusing on clinical manifestations and SCN1A gene mutations. That study analyzed a total of 34 patients who suffered from SMEI (7 patients), SMEB (7 patients), febrile seizure plus (FS+) and absence/myoclonic/atonic/partial seizures (11 patients), and FS+ (9 patients) [14].

However, the research that we have done uses the Sanger sequencing genetic examination, which is expensive and takes considerable time. Additionally, it is unable to find any other gene besides SCN1A in patients with DS. A study by Djémié et al. in Belgium reported the discovery of 28 pathogenic variants of the SCN1A gene using the NGS method which were previously missed or undiagnosed using Sanger sequencing [7]. To link DS cases more effectively, we are attempting to conduct NGS genetic tests, specifically WES and WGS.

Dravet syndrome (DS) was infrequently reported in Indonesia due to its difficulty in diagnosis, misdiagnosis as febrile seizures or other epilepsy syndromes, or lack of follow-up and genetic testing in our country. According to the to the International League Against Epilepsy (ILAE) [15], the diagnostic criteria for this condition should consist of a number of the following symptoms: (1) a family history of epilepsy or febrile seizures; (2) normal development before seizures onset; (3) seizure before 1 year of age; (4) EEG with generalized spike and polyspike waves; (5) pleomorphic epilepsy (myoclonic, focal, clonic, absence, and generalized seizures); (6) focal abnormalities or early photosensitivity; (7) psychomotor retardation after 24 months; (8) exacerbation of seizures with increased body temperature; and (9) the appearance of subsequent ataxia, pyramidal signs or interictal myoclonus after the beginning of psychomotor slowing. Both of our patients had seizures beginning with increased body temperature and regression of development after seizure onset, which were resistant to the majority of anticonvulsant medications. The seizures began as generalized tonic–clonic seizures, followed by absence seizures. Both of our patients also experienced subsequent ataxia and pyramidal signs. Thus, they were suspected of having DS and were advised to undergo genetic testing.

Infants with DS have normal physical and psychomotor development at the time of their first seizure, which typically occurs between the ages of 5 and 8 months. In our case series, both of our patients experienced their first seizure at the age of 3 months [16, 17]. In the first year of life, the most common form of seizure is febrile tonic–clonic. Some patients may experience myoclonic and dyscognitive seizures infrequently. Frequently, protracted seizures result in status epilepticus. In the first year of life, seizures are precipitated by fever/illness, immunization, and cleansing [16]. As the infant develops, he or she will experience a variety of seizure types, as well as fever and emotional stress, flashes of light, and overexertion being seizure triggers. The child with DS will develop hypotonia, ataxia, incoordination, and pyramidal signs, dysautonomia events, cognitive impairment, and behavioral disturbances such as attention deficit, hyperactivity, or autistic characteristics [15]. Some of the conditions above are very consistent with what happened to our patients.

The EEG performed during the early phases of the disease is normal. However, as the child grows, generalized spike waves with isolated or brief discharges of fast polyspike waves may be present [15, 18]. In the first case, we found diffuse epileptiform irritative abnormality with a normal background, whereas in the second case, initially it was found normal, then a few months later it became abnormal irritative epileptiform with a normal background.

Genetic testing is developing rapidly and playing a significant role in the specific diagnosis and management of epilepsy [19, 20]. Several genes with pathogenic mutations produce DS or DS-like phenotypes, which inevitably require different drug therapy approaches. Genes that cause DS can be grouped based on how they work: specifically, three sodium channel-related genes (SCN2A, SCN8A, and SCN1B), one potassium channel-related gene (KCNA2), three gamma-aminobutyric acid receptors (GABAR) genes (GABRA2, GABRB3, and GABRG2), a cyclic nucleotide gated cation channel gene (HCN1), and other functional genes including CHD2, CPLX1, and STXBP1. Approximately 80% of patients with DS have a pathogenic variant of the SCN1A gene, from which the majority of SCN1A variants are de novo, but 10% of people inherit the SCNA1 mutation from one or both parents [6]. Both of our patients had a mutation in the SCN1A gene, which is the most common mutation seen in DS.

Furthermore, TTC21B and SCN9A mutations were also found in our first patient. A study conducted by Suls et al. also reported a four generation Bulgarian family with epilepsy, revealing a heterozygous 400 kb deletion on chromosome 2q24 that included the SCN1A and TTC21B genes [21]. The patients exhibited variable phenotypes, but all experienced generalized tonic–clonic seizures around the first year of life, with some presenting myoclonic or absence seizures. Febrile seizures occurred in three of the four patients during infancy. Notably, one patient had mild mental retardation, another had psychomotor slowing, and a third had mental retardation from early infancy; all showed reduced seizures on medication. The findings in that study parallel the situation observed in our initial patient case. Meanwhile, a study by Singh et al. identified a heterozygous mutation in the SCN9A gene in two patients diagnosed with DS [22]. One of these patients also exhibited a mutation in the SCN1A gene. The study provided evidence suggesting that the SCN9A gene on chromosome 2q24 could potentially serve as a modifier for DS. Among 109 patients with DS, 8% were found to have an SCN9A mutation. This included six patients with double heterozygosity for SCN9A and SCN1A mutations and three patients with only heterozygous SCN9A mutations, supporting the notion of a multifactorial inheritance pattern [22]. The previous research confirmed the severity of clinical symptoms in our first patient, whom we identified mutations in the SCN1A, SCN9A, and TTC21B genes.

In the last decade, there has been a very rapid development of neurogenetic science and diagnostic technology. NGS is the latest method of genetic examination that allows for the discovery of causal mutations, including de novo, novel, and familial mutations related to epilepsy syndromes that have variable phenotypic features [23]. The first generation of DNA sequencing using the Sanger method could only examine one gene at a time and had limitations especially when examining large genomic regions, so the NGS method is more widely used today [7, 23]. A study conducted by Kim et al. in Seoul reported an increase in diagnostic yield using WES after targeted panel sequencing with negative results in infantile onset epilepsy by 8%. This result suggests that WES assays increase the opportunity to search for new epilepsy genes and uncover less well-known epileptic phenotypes from known neurological diseases [24]. The WES examination also allows for the discovery of de novo or inherited mutations if the patient and both parents are examined [25].

According to the recommendations of the North American consensus panel, clobazam and valproic acid are the first-line therapies for antiepileptic drugs, followed by stiripentol, topiramate and levetiracetam. Patients with a suboptimal response to clobazam and valproic acid have been advised to consider the ketogenic diet as a second-line treatment [17]. SCN1A is a gene that codes for sodium channel channels, so drugs that work as sodium channel blockers, such as lamotrigine, phenytoin, carbamazepine, oxcarbazepine, lacosamide, and rufinamide, are contraindicated in patients with DS because they can increase the frequency of seizures [4]. After the failure of first- and second-line therapy, surgical therapies, such as vagus nerve stimulation (VNS), were moderately agreed upon and should be considered [17]. Besides medication, controlling infections and body temperature variations also showed to decrease the frequency of seizures and severity of the disease [18]. Initially, the first patient received oxcarbazepine and the second patient got phenytoin, which had been contraindicated to patients with DS. Futhermore, after eliminating medications that were contraindicated, both patients’ outcome improved.

In this study, we discovered unique mutations that have never been documented before, particularly in Indonesia, where NGS analysis of DS genetic variants has never been done. However, the limitation of this study, is that the information comes from two cases only. Further research is needed to explore more cases from Indonesia population.

In summary, our case series utilizing next-generation sequencing (NGS) unveils the intricate genetic landscape of Dravet syndrome (DS) in two Indonesian pediatric cases. By using WGS and WES, we identified distinct mutations in the SCN1A gene, as well as contributions from genes, such as TTC21B and SCN9A. The power of WGS lies in its ability to uncover rare pathogenic variants, including a 552.9 Kb deletion in the 2q24.3 region. These findings emphasize the importance of comprehensive genetic testing beyond SCN1A, providing valuable insights for personalized management and tailored therapeutic interventions in patients with DS. Our study underscores the potential of NGS in advancing genotype–phenotype correlations and enhancing diagnostic precision for effective disease management. Furthermore, we found that the clinical condition of the first patient was worse than that experienced by the second patient. This difference suggests that the more severe the genetic mutation detected, the more severe the clinical manifestations of the patient.