Developmental and epileptic encephalopathy is a concept to acknowledge that many genetic disorders show developmental impairment as a direct consequence of the genetic mutation, in addition to the detrimental effect of the frequent epileptic activity on brain development [1]. In the era of next generation sequencing, increasing monogenic causes for developmental and epileptic encephalopathy are being discovered, providing us with new insight to its underlying patho-genetic mechanisms.

Syntaxin-binding protein 1 (STXBP1) (also known as MUNC18-1) is a member of the membrane trafficking proteins predominantly expressed in the brain, which is important for docking and fusion of the synaptic vesicles [2]. There is approximately 200 cases reported in the literature until date, mostly being de novo heterozygous mutations [3]. STXBP1 mutation was first described in 2008, in patients with early infantile epileptic encephalopathy with suppression-burst (EIEE), or Ohtahara syndrome [4]. Subsequent studies showed that STXBP1 mutation is responsible for approximately 22% of cases with Ohtahara syndrome, 6% of non-syndromic early onset epileptic encephalopathy and 2% of West syndrome [4-7]. Due to the expanding phenotype STXBP1 encephalopathy was suggested to be more appropriate [8].

Recognition and classification of diverse phenotypes arising in STXBP1 mutation is crucial to guide future management options. Here, we report 10 patients with STXBP1 mutation from a single center and summarize the detailed clinical features, with the aim to expand its phenotypic spectrum in Korean pediatric population.

Patients with STXBP1 mutation were identified from a cohort of 198 pediatric patients with developmental and epileptic encephalopathy of unknown etiology. Patient cohort was selected from the Division of Pediatric Neurology of the Seoul National University Children’s Hospital from September 2012 to May 2019. All the patients had no obvious etiology based on clinical features, neuroimaging and metabolic screening. The first-tier test included chromosomal microarray or targeted multi-gene panel. Whole exome sequencing was conducted as a first-tier test as well as a second-tier test, in selected cases based on the decision of the child neurology expert consortium. Variants were evaluated and classified according to the guideline proposed by American College of Medical Genetics (ACMG) [9]. ClinVar database was searched for past variant reports [10]. Population frequency of variants were determined using 1000genome, ExaC, and gNomad database.

STXBP1 sequencing was performed on DNA from probands and family members using the Sanger method to identify parental origin whenever possible. Information obtained from medical records include age at seizure onset, symptoms at onset, duration from onset to diagnosis, previous diagnosis and treatment, associated psychiatric and behavior symptoms, response to antiepileptic drug (AED) treatment and developmental outcome. Each patient’s electroencephalography (EEG) was obtained and analyzed. Seizure types and epileptic syndromes were classified according to the 2017 International League Against Epilepsy guidelines [1].

The study protocol was approved by the Institutional Review Board of Seoul National University Hospital (IRB No.H-2001-134-1096), and the study was conducted in accordance with relevant guidelines and regulations. Written informed consent by the patients was waived due to the retrospective nature of our study.

Here we identified 10 Korean patients with STXBP1 mutation, which included three novel mutations. We described detailed phenotypes and genotypes of the patients with STXBP1 encephalopathy. Due to its rarity, the clinical spectrum of STXBP1 encephalopathy is not yet well known, but recent large cohort study suggests that intellectual disability and epilepsy is the two main major components of STXBP1 encephalopathy.

Epilepsy was observed in 95% of STXBP1 encephalopathy patients. Among the patients with available information, about half was taking more than three AEDs and one-third was suffering from frequent seizure. On the other hand approximately one-third achieved seizure freedom [3]. Consistent with the above observation, nine patients (90%) in the current cohort showed epilepsy and all patients required more than two AEDs.

In the same study, moderate to severe intellectual disability was observed in 88.4% of patient. Notably, developmental delay was present before seizure onset in 64.3%, and 7% of patients were observed to have just developmental delay without any seizure. Other previous studies also observed that some degree of developmental delay was often observed prior to any seizure onset, thus it is considered an independent domain from epilepsy [3,12]. Indeed, in our cohort all patients showed profound developmental delay or severe intellectual disability and 30% of patients showed developmental delay before seizure onset.

However, it is often difficult to assess whether seizure activity or epileptiform discharges have any effect on developmental process or whether developmental delay is totally separate phenotype in STXBP1 encephalopathy. It was suggested that developmental regression is rarely seen and does not seem to be related to seizure activity [3]. In the current cohort, patient 6 and 7 showed clear regression during follow-up. Interestingly, both patients were different from typical patients presenting with early onset epileptic encephalopathy, as seizure was not their main feature and both showed predominantly non-epileptic movement symptoms. Patient 7 followed Rett syndrome like features with regression starting at age of 3 with autistic features, and patient 6 showed many resemblances to the phenotype previously described as ataxia-tremor-retardation syndrome [16]. Patient 6 showed clear motor function regression after seizure onset with worsened ataxia and newly developed dysphagia. Above two patients support that seizure activity and intellectual development are indeed independent domains.

STXBP1 encephalopathy was known to be most commonly associate with burst suppression and hypsarrhythmia, as it was initially described mostly in Ohtahara syndrome [11,12,17]. However recent large cohort study suggest most commonly described changes are focal or multifocal activities, seen in 64% of the cases [3]. Our patients also showed that focal or multifocal epileptiform discharges were the most common finding (60%), and classical burst-suppression patterns were seen in only proportion of the patients (40%). However, patients who were followed up long term showed that patients with burst-suppression pattern often evolve to hypsarrhythmia or non-specific focal patterns. Thus it is possible some patients did have burst-suppression pattern at one point but was never recorded. The range of abnormal EEG findings was wide in the current cohort, and they showed little correlation with their developmental status or seizure activity, in accordance to previous reports [3,18].

The reason why the clinical presentation of patients with STXBP1 mutation varies so much is yet unknown. Previous evidence supported the hypothesis that haploinsufficiency is the main pathogenic mechanism underlying STXBP1 encephalopathy, which may explain for such phenotypic heterogeneity [19]. However, there is growing evidence that STXBP1 mutation have more than one mechanism of pathophysiology. Recently, a homozygous STXBP1 mutation was found to cause Lennox-Gastaut syndrome, showing that STXBP1 also have a dominant-negative effect [20]. Thus mechanism of STXBP1 encephalopathy still needs much further research in the future.

Another important aspect of STXBP1 mutation to consider is mosaicism. Both somatic and germline mosaicism of STXBP1 mutation has been reported. It is interesting that focal epileptiform discharges are commonly seen in STXBP1 encephalopathy, especially since there were two reports of significant improvement of seizure with epilepsy surgery [8,21]. According to these reports, presence of focal cortical dysplasia have been confirmed by tissue pathology in both cases. One patient was confirmed to also harbor somatic mosaicism for homozygosity of STXBP1 mutations in the dysplastic tissue. Pathogenic role of somatic mosaicism on STXBP1 encephalopathy is still unknown, but this finding suggest they may play an important role in cortical development.

On the other hand, germline mosaicism can cause major problem in genetic diagnosis of STXBP1 mutation and in family genetic counseling. Germline mosaicism of STXBP1 has been reported previously from an unaffected parent of a patient [22,23]. Assumption of de novo mutation based on parental Sanger sequencing maybe incorrect, as it is unlikely to detect low-rate mosaicism [24] .Therefore it is possible that unrecognized parental STXBP1 mosaicisms are present among the current cohort as well. Recently, the high fold coverage of next-generation sequencing allow for the detection of even very low levels of mosaicism in the blood cells [25]. This has opened a new era of genetic testing, which will hopefully broaden our knowledge of mosaicism in STXBP1 encephalopathy.

Current study has several limiting factors. Selection bias is present due to primary identification of cases from pediatric clinics with significant epilepsy or developmental delay. Given the wide variety of phenotypes, it is plausible that there are cases of STXBP1 encephalopathy with milder symptoms or different phenotype were overlooked. There are also possibility that patients with focal cortical dysplasia or other cortical malformation harbor STXBP1 mutation and were never considered for genetic testing, in the absence of the classical features.

In conclusion, STXBP1 encephalopathy showed a wide clinical spectrum of phenotypes from severe epileptic encephalopathy such as Ohtahara syndrome or West syndrome to unknown neurodevelopmental retardation without epilepsy. Their seizure types and EEG findings are also diverse. Therefor it is important to consider STXBP1 mutation even in patients without seizure, or predominantly focal EEG changes without history of burst suppression. Further research is needed to discover the full range of phenotypes of STXBP1 encephalopathy in order to elucidate underlying disease mechanism.