STXBP1 Encephalopathy: Cannabidiol as a Treatment Option and Expansion of the Genotype-Phenotype Spectrum – Case Reports

Abstract

Introduction

STXBP1 encodes syntaxin-binding protein 1, a protein with a role in trafficking of syntaxin-1 to the synapse and synaptic neurotransmitter release. Mutations in STXBP1 are associated with a wide range of neurodevelopmental phenotypes, including epilepsy, intellectual disability, and movement disorders.

Case Series

We report 4 individuals with pathogenic variants in STXBP1, including 2 previously unreported variants, presenting with a range of severity. One severely affected child with refractory seizures and distressing dystonia responded well to treatment with cannabidiol (CBD), with both seizure cessation and improvement in tone.

Conclusion

STXBP1 variants are associated with significant phenotypic pleiotropy and a wide spectrum of severity. CBD may be beneficial for severely affected individuals with seizures and movement disorders that are refractory to conventional treatments.

Established Facts

• Mutations in STXBP1, encoding syntaxin-binding protein 1, have been associated with a wide range of neurodevelopmental phenotypes, including early-onset developmental epileptic encephalopathy, intellectual disorders, and movement disorders.

Novel Insights

• We report a case series of 4 patients with STXBP1 variants, including 2 novel, previously unpublished variants, with phenotypes ranging from relatively mild to severely disabling.

• Cannabidiol may alleviate both refractory seizures and intrusive dystonia for patients with STXBP1 developmental epileptic encephalopathy, significantly improving quality of life.

Introduction

The gene STXBP1 encodes syntaxin-binding protein 1. STXBP1 was first associated with human disorders in 2008, when pathogenic variants were identified in 5 patients with Ohtahara syndrome [1]. Since then, various other STXBP1 mutation phenotypes have been described including West syndrome, Lennox-Gastaut syndrome, early myoclonic epilepsy, Dravet syndrome (DS), unclassified early-onset epileptic encephalopathies, mental retardation with non-syndromic epilepsy, atypical Rett syndrome, intellectual disability without epilepsy, and ataxia-tremor-retardation syndrome without epilepsy [2].

To date, more than 500 cases of STXBP1 variants have been reported [3, 4]. Approximately 90% have seizures, 50% are reported to have motor impairment. Global developmental delay (GDD) and variable degrees of intellectual disability are reported most commonly (95%) [3].

We describe 4 patients with STXBP1 variants, 2 of the variants have not previously been reported in literature. They present with a spectrum of severity ranging from relatively mild GDD and easily controlled seizures to refractory seizures with severe dystonia. Despite several treatment strategies failing to control seizures and alleviate dystonia, our patient with the most severe phenotype responded positively to cannabidiol (CBD).

Case Report

Details on seizure semiology and investigation results are presented in Table 1. Patient 1 is a female of Malay ancestry who presented at 6 weeks of age with up to 20 seizures a day. She achieved seizure control on phenobarbitone, carbamazepine, and levetiracetam. However, at 5 months of age, she developed infantile spasms. This responded to prednisolone. She has not had any further seizures since.

Table 1.

Seizure semiology and investigation results

Case	Sz onset	Sz stop	Dystonia	Vision	Feeding	Sz semiology	EEG	MRI	STXBP1 variant	dbSNP/1000G, gnomAD/EVS	ACMG classification
1	6 weeks	5 months	No	Normal	Normal	FM, tonic, IES	6 weeks: normal	6 weeks: normal	c.1381_1390del	Absent	Pathogenic
							5 months: HA		p.Lys461Glyfs*82
2	7 weeks	5 years	Not intrusive +	Normal	NGT from 2.5 months	FM, tonic, IES, MC, GTC	7 weeks: multifocal SWD and BS	2 months: normal	c.1006C>T: p.(Gln336*)	rs1057519501/absent	Pathogenic
							4 months: HA
							5 years: slow background with intermittent diffuse fast activity, myoclonic jerks- no EEG correlate
3	3 weeks	On-going	++	Poor	Thickened feeds from 8 years	FM, tonic, atypical absences, head-drops, GTC	1 month: multifocal SWD, normal background	1 month: normal	c.325+2 T>A	Absent	Likely pathogenic
							9 years: diffuse slow delta to theta background with frequent multifocal independent SWD, myoclonic jerks, no EEG correlate	9 years: normal	p.?
4	1 month	3.5 years	+++	Poor	NGT from 4 months	FM, tonic, MC, IES	1 month: multifocal SWD and BS	2 months: normal	In-frame deletion (exon 16)	Absent	Pathogenic
							5 months: HA
							2 years: slow background with multifocal independent SWD

ACMG, American College of Medical Genetics; BS, burst suppression; EVS, Exome Variant Server; GTC, generalised tonic-clonic; FM, focal motor; HA, hypsarrhythmia; IES, infantile epileptic spasms; MC, myoclonic; N.A., not applicable; SWD, Spike-wave discharge; Sz, seizure; 1000G, 1000 Genomes.

Currently, at the age of 3 years, she has mild GDD but is making developmental progress. She has normal vision and hearing. She smiles and imitates words but is otherwise nonverbal. She has reduced axial tone and poor trunk control, but her peripheral tone and deep tendon reflexes are within normal limits. She walks independently. Clinical genetic testing via an epilepsy gene panel identified a previously reported heterozygous, frameshift 10-nucleotide deletion in STXBP1 (NM_003165.3: c.1381_1390del: p.Lys461Glyfs*82) [5, 6]. The inheritance of this deletion could not be determined as her parents declined genetic testing.

Patient 2 is a Chinese girl who first presented at 7 weeks of age. Her seizure frequency escalated rapidly to more than 20 a day. At 4 months of age, she developed infantile spasms. Peripheral hypertonia and oropharyngeal dysphagia were noted from 2.5 months of age and nasogastric feeding was initiated. Intermittent non-epileptic myoclonic jerks emerged at 5 years of age.

Although her seizures were previously refractory to phenobarbitone, prednisolone, vigabatrin, and zonisamide, as she grew older, her seizures reduced in frequency. Since the age of 5 years, her seizures have been controlled on zonisamide alone. She has not needed intervention for dystonia.

Currently, at 9 years of age, she demonstrates visual fixation and tracking. She has normal hearing. She is socially interactive and but is nonverbal. She is wheelchair bound but tolerates assisted standing for up to 2 h a day. She has thoracic kyphoscoliosis, generalized spasticity, tight tendon Achilles, and fixed knee flexion contractures bilaterally. She has intermittent dystonic stiffening and myoclonic jerks, but these are not considered to be intrusive. A de novo heterozygous nonsense substitution in STXBP1 (NM_003165.3: c.1006C>T: p.[Gln336*]) in exon 12 was identified through research sequencing using the TruSight One panel and confirmed by Sanger sequencing [7].

Patient 3 is a Chinese male who had seizure onset at 3 weeks of age. At his worst, his seizures occurred 20–25 times a day. Increased tone and brisk reflexes were noted from 2.5 months of age. As he got older, he developed intermittent choreiform movements and non-epileptic myoclonic jerks. He started having increasing difficulty with swallowing at 8 years of age, requiring thickened oral feeds.

Currently, at 11 years of age, he has 2 to 3 brief seizures daily. He has poor visual attention and is nonverbal. Due to significant axial hypotonia, he is unable to sit without support. Peripherally, he has dystonia with spasticity and significant muscle atrophy. He has lumbar scoliosis and bilateral hip subluxation.

He has had treatment trials with several antiseizure medications (ASMs) including phenobarbitone, bromhexine, carbamazepine, pyridoxine, and clobazam but achieved his best seizure control on his current combination of topiramate and levetiracetam. He is not on any other medication. Clinical genetic testing via epilepsy gene panel identified a heterozygous splice variant in STXBP1 (NM_003165.3: c.325+2 T>A). The inheritance of this variant could not be determined as his parents declined genetic testing. This variant has not previously been published. It has been submitted on ClinVar (Variation ID: 955041).

Patient 4 is a Chinese girl who presented at the age of 1 month. At her worst, she had more than 30 seizures a day. By 4 months of age, oropharyngeal dysphagia was evident and nasogastric feeding was started. At the age of 5 months, she developed infantile spasms. From 6 months of age, she developed significant spasticity and dystonic posturing. Each day, she would have 3–4 episodes of severe segmental or generalised dystonic attacks lasting 10–20 min each.

Despite trial of several ASMs including phenobarbitone, zonisamide, levetiracetam, and valproate, her seizures remained refractory. Her epileptic spasms proved refractory to both vigabatrin and prednisolone but showed partial response to the ketogenic diet. In addition, she continued to have severe dystonia despite trial of levodopa, trihexyphenidyl, gabapentin, clonidine, and tetrabenazine. At the age of 3 years, she was started on CBD (Epidiolex). The dose of this was gradually titrated up to 120 mg twice daily (15 mg/kg/day) and within 6 months, she was seizure-free. There was also striking improvement in her dystonia, allowing tapering and cessation of clonidine. She currently remains seizure-free on a combination of Epidiolex, levetiracetam, zonisamide, and valproate. As part of the requirement for named-patient use of CBD, she underwent frequent clinical review and regular laboratory tests to monitor for side effects. She experienced no side effects clinically and had no biochemical derangements from her test results.

Currently, aged 3 years 8 months, she does not demonstrate visual attention, but she is responsive to auditory stimulus, smiles and makes cooing sounds. She is seizure-free. She has low axial tone. She continues to have mixed generalised dystonia and spasticity; worse in the lower limbs, but this has improved, facilitating manipulation of her limbs for daily activities. She no longer has distressing dystonic attacks. Clinical genetic testing via epilepsy gene panel identified a de novo heterozygous deletion encompassing the entire exon 16 of the STXBP1 gene.

Discussion

STXBP1 is predominantly expressed in neurons. STXBP1’s well-established interaction with the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex protein, syntaxin-1, facilitates trafficking of syntaxin-1 to the synapse and regulates SNARE-complex formation [8, 9].

The mechanisms by which STXBP1 mutations lead to neurological dysfunction are poorly understood. However, disturbances in neurotransmitter release and synaptic dysfunction are likely to contribute to epileptic and motor symptoms in STXBP1 disorders. The existence of heterozygous deletions, frameshifts, splice-site, and nonsense mutations in relation with disorders suggest that haploinsufficiency and loss-of-function mechanisms are important. This is supported by the discovery that most missense mutations studied to date cause destabilization of the protein, leading to aggregation, degradation, and a reduction in cellular STXBP1 protein levels [2].

The patients presented in our report all have, in common, cognitive impairment, GDD, early-onset seizures, oropharyngeal dysphagia, and movement disorder, clinical features frequently associated with STXBP1 mutations. Patients 1, 2, and 4 presented with infantile spasms, consistent with a recent observation by Xian et al. [4] that protein truncating variants and deletions are nominally associated with infantile spasms.

Seizure course in STXBP1 encephalopathy has been proposed to follow 1 of 2 main trajectories – early seizure remission or drug-resistant epilepsy. Balagura et al. [10] reported in their series of patients that 76% of those who achieved seizure freedom did so within the first year of life. Xian et al. [4] also reported significant reduction in seizure frequency by 7 years of age. Our patients exhibit a wide spectrum of seizure severity, from easily controlled seizures within the first year of life, to seizure freedom on medication at 5 years to refractory seizures. While they all had neurodevelopmental impairment, this ranged from relatively mild delay (patient 1) to distressing dystonia and immobility from an early age (patient 4). As noted, by previous studies, there does not appear to be any correlation between their genotypes and the severity of their symptoms. Patient 1 harbours a recurrent STXBP1 variant (c.1381_1390del: p.Lys461Glyfs*82), but her phenotype differs significantly from the previously reported male with the same variant who presented with tonic-clonic seizures at 10 days of age [5].

Other genetic background may contribute to phenotypic pleiotropy in epilepsy genetics. In addition to pathogenic/likely pathogenic variants in STXBP1, patients 1 and 3 have some variants reported in other genes (online suppl. Table 1; for all online suppl. material, see https://doi.org/10.1159/000550657). The genes CNTNAP2 and SLC19A3 are both associated with recessive disorders and our patients only have heterozygous variants (carrier status). The other variants in GABBR2, KCNQ3, GRIN2A, KANSL1 are all rare variants with conflicting predictions on in silico algorithms. Functional studies have not been performed and they are currently categorized as variants of uncertain significance. It is possible that some of these variants may contribute to patient phenotype if it is established, in future, that they are disease-causing.

Our current limited understanding of the mechanisms through which STXBP1 mutations lead to their neurological manifestations has translated to our present lack of targeted treatment options. Strategies for seizure control remain empirical and ASMs that have been reported to be effective include levetiracetam, valproate, and vigabatrin [11]. Some patients respond to the ketogenic diet [12]. No specific treatment for early-onset disabling movement disorders has been reported to date.

The use of CBD to treat refractory seizures first received keen interest in 2010, following media reports that a young girl, Charlotte Fiji, with intractable seizures had a dramatic positive response when given a high CBD strain of marijuana [13]. Since then, several studies have demonstrated the positive effects of CBD on patients with DS, Lennox-Gastaut syndrome, tuberous sclerosis, and many other early-onset epileptic encephalopathies [13].

CBD’s antiseizure mechanism of action is not completely understood, it is hypothesized to modulate neuronal hyperexcitability and contribute to the regulation of intracellular calcium concentration through its actions on GPR55 (G protein-coupled receptor 55), serotonergic receptors, glycine receptors, and various ion channels. It is also a potent inhibitor of hepatic metabolism of various AEDs including clobazam, topiramate, and eslicarbazepine, thereby augmenting their effects [14].

Studies looking at safety and efficacy of CBD have reported adverse events in 35–45% of patients. These are mostly mild and transient, with some effects attributed to interaction with other drugs. The most frequent reported adverse reactions include somnolence, changes in appetite/body weight, gastrointestinal symptoms, irritability/behavioural change and transaminitis or thromobocytopaenia [14].

For STXBP1-encephalopathies, Bravo and Izquierdo first reported that adjunctive treatment with CBD reduced seizure frequency and markedly improved motor symptoms in a 19-year old female with STXBP1-related DS who had been treated with various ASMs [15]. A more recent report documented the effectiveness of CBD in a pair of monozygotic twins with early-onset seizures refractory to multiple ASMs, including valproate, clobazam, adrenocorticotrophic hormone, pyridoxine, and phenobarbitone. Both twins achieved seizure freedom on different formulations of CBD, enabling cessation of other ASMs [16]. Our patient showed good seizure control with CBD and concurrent reduction in dystonia severity, with no sign of commonly reported side effects clinically, or on laboratory testing.

In conclusion, much remains to be understood about how STXBP1 mutations lead to disease. The genotypic and phenotypic spectrum of STXBP1 mutations will continue to grow as more patients are identified by genetic screening; and perhaps, in future, a more comprehensive picture for genotype-phenotype correlation will surface. Better understanding of the mechanisms by which STXBP1 variants lead to symptoms will pave the way for advancing targeted treatment options. CBD has potential as a safe and effective treatment option in refractory epilepsy and dystonia in patients with STXBP1 mutations.

Statement of Ethics

These patients were recruited for genetic investigation via a study protocol reviewed and approved by the SingHealth Centralised Institutional Review Board, Singapore, approval reference 2015/2159. Written informed consent has been obtained from the parents/legal guardians of all patients for participation and publication.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This work was supported by grants 02/FY2016/P1/03-A14 from the SingHealth Duke-NUS Academic Medical Centre, the SingHealth Paediatrics Academic Clinical Programme, Tan Cheng Lim fund, and NMRC/CG1/006/2021 from the National Medical Research Council, Ministry of Health, Republic of Singapore.

Author Contributions

A.N., T.H.Y., S.L., J.K., and D.W.S.C. recruited and clinically characterized patients. A.N., E.C.T., and D.W.S.C. acquired funding. A.N., H.W., E.C.T., and D.W.S.C. were involved with investigation, data collection, and analysis. A.N., H.W., and E.C.T. contributed to data analysis. E.C.T. and D.W.S.C. verified data and had roles with supervision. A.N. and H.W. wrote the manuscript. All authors contributed to the critical review of the manuscript, had full access to all data in the study, and agreed with submission for publication.

Funding Statement

This work was supported by grants 02/FY2016/P1/03-A14 from the SingHealth Duke-NUS Academic Medical Centre, the SingHealth Paediatrics Academic Clinical Programme, Tan Cheng Lim fund, and NMRC/CG1/006/2021 from the National Medical Research Council, Ministry of Health, Republic of Singapore.

Data Availability Statement

De-identified data collected and presented in this study, including individual participant data, can be made available upon reasonable request after publication of this article. Data can be requested by contacting the corresponding author.