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Julie Xian, Kim Marie Thalwitzer, Jillian McKee, Katie Rose Sullivan, Elise Brimble, Eryn Fitch, Jonathan Toib, Michael C Kaufman, Danielle deCampo, Kristin Cunningham, Samuel R Pierce, James Goss, Charlene Son Rigby, Steffen Syrbe, Michael Boland, Benjamin Prosser, Nasha Fitter, Sarah M Ruggiero, Ingo Helbig, Delineating clinical and developmental outcomes in STXBP1-related disorders, Brain, Volume 146, Issue 12, December 2023, Pages 5182–5197, https://doi.org/10.1093/brain/awad287
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Abstract
STXBP1-related disorders are among the most common genetic epilepsies and neurodevelopmental disorders. However, the longitudinal epilepsy course and developmental end points, have not yet been described in detail, which is a critical prerequisite for clinical trial readiness. Here, we assessed 1281 cumulative patient-years of seizure and developmental histories in 162 individuals with STXBP1-related disorders and established a natural history framework.
STXBP1-related disorders are characterized by a dynamic pattern of seizures in the first year of life and high variability in neurodevelopmental trajectories in early childhood. Epilepsy onset differed across seizure types, with 90% cumulative onset for infantile spasms by 6 months and focal-onset seizures by 27 months of life. Epilepsy histories diverged between variant subgroups in the first 2 years of life, when individuals with protein-truncating variants and deletions in STXBP1 (n = 39) were more likely to have infantile spasms between 5 and 6 months followed by seizure remission, while individuals with missense variants (n = 30) had an increased risk for focal seizures and ongoing seizures after the first year. Developmental outcomes were mapped using milestone acquisition data in addition to standardized assessments including the Gross Motor Function Measure-66 Item Set and the Grasping and Visual-Motor Integration subsets of the Peabody Developmental Motor Scales.
Quantification of end points revealed high variability during the first 5 years of life, with emerging stratification between clinical subgroups. An earlier epilepsy onset was associated with lower developmental abilities, most prominently when assessing gross motor development and expressive communication. We found that individuals with neonatal seizures or early infantile seizures followed by seizure offset by 12 months of life had more predictable seizure trajectories in early to late childhood compared to individuals with more severe seizure presentations, including individuals with refractory epilepsy throughout the first year. Characterization of anti-seizure medication response revealed age-dependent response over time, with phenobarbital, levetiracetam, topiramate and adrenocorticotropic hormone effective in reducing seizures in the first year of life, while clobazam and the ketogenic diet were effective in long-term seizure management. Virtual clinical trials using seizure frequency as the primary outcome resulted in wide range of trial success probabilities across the age span, with the highest probability in early childhood between 1 year and 3.5 years.
In summary, we delineated epilepsy and developmental trajectories in STXBP1-related disorders using standardized measures, providing a foundation to interpret future therapeutic strategies and inform rational trial design.
Introduction
STXBP1-related disorders are among the most common genetic epilepsies and neurodevelopmental disorders, with an estimated frequency of 1:30 000.1,2 The phenotypic landscape is characterized by neurodevelopmental delay in 95% and epilepsy in more than 80% of individuals.3 However, the wide range of neurodevelopmental features has made it difficult to delineate phenotypic subgroups,4–9 map genotype-phenotype associations, and understand treatment response. Accordingly, the significant heterogeneity within STXBP1-related disorders has limited understanding of developmental progression and the range of clinical outcomes.
STXBP1 encodes the syntaxin-binding protein (MUNC18-1), a key organizer of the neuronal SNARE complex, the molecular machinery driving synaptic vesicle fusion.10,11 While the main disease mechanism is haploinsufficiency, dominant negative effects have been suggested as an alternative disease mechanism.12–14 Phenotypic signatures associated with specific genetic variants in STXBP1 have only recently been described,3 and efforts to elucidate the genomic architecture and explain the clinical heterogeneity are ongoing. Nevertheless, STXBP1 represents a focused target for gene therapy and clinical trials, with treatment strategies including chemical chaperones, antisense oligonucleotides and viral gene therapies on the horizon.14,15
Understanding longitudinal clinical presentations and trajectories in STXBP1-related disorders remains an ongoing challenge. First, despite numerous cross-sectional studies on outcomes,7,16,17 the variability resulting from cohort samples and study design differences has challenged interpretation of findings within and across studies. In addition, larger cohort studies have been limited to broad phenotypic descriptions that did not include longitudinal trajectories.3 Furthermore, a major challenge in moving STXBP1-related disorders towards clinical trial readiness has been the use of varying outcome measures for clinical end points.18–21 Lastly, the heterogeneous pattern of seizures limits precise understanding of optimal time windows for clinical trial design.22,23 Accordingly, there remains a critical and unmet need to systematically assess longitudinal clinical data and validate disease and subgroup-specific measures.
To address this gap, we aimed to assess the trajectory of epilepsy and neurodevelopment through validated outcome scales, stratifying trajectories by clinical and genetic subgroups across the age span in individuals with STXBP1-related disorders.
Materials and methods
Inclusion of individuals with STXBP1-related disorders
We assessed the genetic and clinical information of 162 individuals with STXBP1-related disorders, including 104 individuals at Children’s Hospital of Philadelphia and 58 additional individuals from the Ciitizen Natural History Study Patient Registry (Supplementary Table 1). Only variants considered pathogenic or likely pathogenic according to American College of Medical Genetics and Genomics (ACMG) criteria or de novo variants were included.
Reconstruction and assessment of epilepsy and developmental histories
We retrospectively reconstructed 765 patient-years of epilepsy histories across 101 individuals with STXBP1-related disorders for whom detailed seizure information was available in the medical records. Using a previously published outcome scale derived from the Pediatric Epilepsy Learning Health System (PELHS)-championed framework for seizure severity,3,24 we captured >20 seizure types and monthly seizure frequencies (SF), quantified as: multiple daily seizures (>5 per day, SF score = 5), several daily seizures (2–5 per day, SF score = 4), daily seizures (SF score = 3), weekly seizures (SF score = 2), monthly seizures (SF score = 1) and no seizures (SF score = 0) (Supplementary Fig. 1). For individuals in whom seizures were noted to be present but seizure frequency was unknown, we calculated the median seizure frequency within the cohort during the respective month. Seizure types were captured using the Human Phenotype Ontology (HPO), a controlled dictionary for harmonization of phenotypic data.25,26
To assess development, we included analysis of (i) milestone acquisition documented in the medical records or available in the Ciitizen Natural History Registry (Supplementary Table 1); and (ii) outcomes captured from routine clinical care through validated standardized measures and classification scales. The Gross Motor Function Measure 66—Item Sets (GMFM-66-IS) was completed by a physical therapist to measure motor function in areas such as rolling, sitting, crawling, standing, walking, running and jumping.27 The Grasping and Visual-Motor Integration subtests of the Peabody Developmental Motor Scales—Second Edition (PDMS-2) was completed by an occupational therapist to assess fine motor function.28 Raw scores, percentiles and age equivalents were calculated for the PDMS-2 while the GMFM-66-IS provided a single interval level score.
Gross motor, fine motor and language abilities were categorized using five-level classifications systems. The Gross Motor Function Classification System Extended and Revised (GMFCS-ER) describes how children are able to self-initiate movements in activities such as sitting, transfers and mobility.21,29 The Manual Ability Classification System (MACS) was developed for children aged 4 to 18 years old and describes their ability to use their hands with objects during activities of daily living, with a focus on the use of both hands together.30 The Communication Function Classification System (CFCS) describes everyday communication performance including sender roles, receiver role, pace of communication, degree of familiarity with a communication partner, age-appropriateness, and the use of augmentative and alternative communication.31 For all classification scales, Level I indicates high levels of function while Level V indicates decreased levels of function. The reliability and content validity of the GMFCS-ER, MACS and CFCS have been supported in multiple investigations.30–32
Longitudinal seizure frequency forecasting and quantification of seizure predictability
Given the heterogeneity of epilepsy in STXBP1, we aimed to stratify subgroups based on the variability in seizures across the age span. We developed a longitudinal seizure frequency forecasting model to characterize the predictability of the epilepsy course. In brief, we compared seizure frequencies across monthly time intervals between all combinations of individual pairs during the first 12 months of life and derived a measure of phenotypic resemblance in epilepsy histories (Supplementary material). We then predicted each individuals’ epilepsy trajectory after the first year of life based on the known epilepsy histories in the subgroup of 10 individuals that most closely resembled the individual in the first year of life. Forecasted predictions were compared to a distribution of randomly generated seizure frequencies and permutation testing of 100 000 for each individual estimation was performed to evaluate ‘better than chance forecasting’.
Comparing the difference between predicted and actual seizure trajectories after the first year of life enabled us to identify subgroups defined by seizure predictability (i.e. degree of difference between forecasted and actual seizure frequencies across months). The stratification of subgroups then allowed us to analyse features of seizure histories in the first year of life that were enriched in individuals with unpredictable seizures later in life compared to individuals with predictable seizure trajectories.
Comparative effectiveness and characterization of treatment response
Seizure end points were analysed alongside anti-seizure medications (ASMs) and treatment strategies, which were systematically extracted from the medical records in our local cohort. The rationale behind this analysis was that the effect of a novel drug should have additional benefit over existing treatment strategies. As previously described,3,33 efficacy was characterized by reduction in seizure frequency for short-term effect and maintaining seizure freedom, defined by no seizures across consecutive months, for long-term treatment effect. The relative effectiveness was determined through assessing the months in which periods of seizure reduction or seizure freedom coincided with certain ASMs compared to months in which no change in seizure frequency or a worsening of seizure frequency was noted (Supplementary material). We compared treatments across seizure types and characterized the per cent reduction in seizures in temporal relation to treatment initiation.
A framework for virtual clinical trials
Finally, we aimed to identify time windows during which a treatment effect that decreases seizure burden would have the highest probability of being detected. Accordingly, we developed a framework for modelling clinical trials, analysing different age ranges across 765 total patient-years of epilepsy histories. For each virtual trial, we sampled 20 individuals with ongoing seizures at the trial start and simulated a 6-month and 12-month period of 10%, 15% and 20% seizure reduction (Supplementary material). The per cent reduction in seizures was calculated based on the cumulative sum of seizure frequencies at the start of the window, and the simulated treatment effect across each trial window was performed by decreasing seizure frequencies across the trial period. We used the synthetic control method to statistically evaluate the simulated effect,34,35 comparing the distribution of seizure frequencies following reduction of seizures against the observed distribution of frequencies in the group. We ran 1000 virtual trials for each month across the age span and defined a novel measure, the Observed Frequency of Trial Success (OFTS), or the proportion of trials out of 1000 in which a significant effect was detected. This allowed us to analyse optimal windows during which a treatment response would most likely be observed in a real-world trial when using seizure frequency as the primary outcome.
Statistical analysis
All computational analyses were performed using the R Statistical Framework.36 The Kaplan-Meier estimate was used to measure the probability of seizure risk and seizure remission, defined by at least 12 consecutive months of seizure freedom,37 while the log-rank sum test was used to determine significance between subgroups. The Wilcoxon rank sum test was used for statistical comparison of distributions in seizure frequencies between clinical subgroups in addition to distributions of raw scores for the GMFM-66-IS and PDMS-2 in assessing developmental outcomes. Association between epilepsy onset with developmental outcomes was assessed using the Wilcoxon Rank Sum test, Pearson correlation coefficient, or Spearman correlation coefficient depending on the measure. Fisher’s exact test was used for assessing phenotypic correlations between clinical and genetic subgroups and comparative effectiveness of treatment strategies (Supplementary material).
Results
STXBP1-related disorders are characterized by neurodevelopmental differences and a dynamic seizure pattern over time
We assessed the clinical landscape across 162 individuals with STXBP1-related disorders and reconstructed longitudinal disease histories across a total observation time of 1281 patient-years. The median observation time was 5.1 years [interquartile range (IQR) 2.75–11.3 years], with a minimum of 3 months and maximum of 44.2 years (Table 1 and Supplementary Fig. 2). We reconstructed epilepsy histories across 9174 total patient-months in 101 individuals for whom detailed seizure data were documented. Of these individuals, 72 individuals (71.3%) had a past or current history of seizures while 29 (28.7%) did not have any seizures. More than 98% of individuals in our cohort had developmental delay or intellectual disability, with the first reported indication of delay at a median of 7 months (n = 30, IQR 6–9 months). The median age of genetic diagnosis was 1.9 years (IQR 6 months to 5.5 years), with the earliest at 3 weeks.
Demographics | |
Male | 81/162 (50.0%) |
Female | 81/162 (50.0%) |
Age distribution | |
Age at assessment (n = 162) | Median 5.1 years (IQR 2.8–11.3 years) Range 3 months–44.2 years |
Seizure onset (n = 72/101) | Median 2 months (IQR 1–7 months) Range neonatal onset–11.1 years data |
Age at genetic diagnosis (n = 99, data available) | Median 1.9 years (IQR 6 months–5.5 years) Range 3 weeks–38.0 years |
Genetic information | |
Genetic variants in STXBP1 | – |
Missense variants | 46 individuals |
p.Arg406Cys/His/Ser | 9 individuals |
p.Arg292Cys/His/Pro | 8 individuals |
p.Arg551Cys/His | 4 individuals |
PTV/dela | 55 individuals |
In-frame insertions/deletions | 3 individuals |
Epilepsy data | |
Seizure types (n = 72/101) | – |
Focal-onset seizures | 48 individuals |
Neonatal seizures | 31 individuals |
Infantile/epileptic spasms | 29 individuals |
Early infantile seizures | 27 individuals |
Developmental data | |
Milestones assessment (age range of cohort assessed) | – |
Head control | 119 individuals (IQR 2.3–9.9 years) |
Roll over | 135 individuals (IQR 2.7–10.0 years) |
Sit unassisted | 133 individuals (IQR 2.1–9.3 years) |
Grasp and hand control | 109 individuals (IQR 2.7–10.0 years) |
Walk assisted or unassisted | 151 individuals (IQR 3.0–10.7 years) |
Few words/sentences | 103 individuals (IQR 2.4–11.0 years) |
Clinical exams (age at assessment) | – |
GMFM-66-IS | 45 individuals (IQR 1.6–5.4 years) |
GMFCS-ER | 41 individuals (IQR 1.8–7.4 years) |
PDMS-2 | 21 individuals (IQR 1.2–3.8 years) |
MACS | 9 individuals (IQR 2.8–9.5 years) |
CFCS | 23 individuals (IQR 3.0–11.8 years) |
Demographics | |
Male | 81/162 (50.0%) |
Female | 81/162 (50.0%) |
Age distribution | |
Age at assessment (n = 162) | Median 5.1 years (IQR 2.8–11.3 years) Range 3 months–44.2 years |
Seizure onset (n = 72/101) | Median 2 months (IQR 1–7 months) Range neonatal onset–11.1 years data |
Age at genetic diagnosis (n = 99, data available) | Median 1.9 years (IQR 6 months–5.5 years) Range 3 weeks–38.0 years |
Genetic information | |
Genetic variants in STXBP1 | – |
Missense variants | 46 individuals |
p.Arg406Cys/His/Ser | 9 individuals |
p.Arg292Cys/His/Pro | 8 individuals |
p.Arg551Cys/His | 4 individuals |
PTV/dela | 55 individuals |
In-frame insertions/deletions | 3 individuals |
Epilepsy data | |
Seizure types (n = 72/101) | – |
Focal-onset seizures | 48 individuals |
Neonatal seizures | 31 individuals |
Infantile/epileptic spasms | 29 individuals |
Early infantile seizures | 27 individuals |
Developmental data | |
Milestones assessment (age range of cohort assessed) | – |
Head control | 119 individuals (IQR 2.3–9.9 years) |
Roll over | 135 individuals (IQR 2.7–10.0 years) |
Sit unassisted | 133 individuals (IQR 2.1–9.3 years) |
Grasp and hand control | 109 individuals (IQR 2.7–10.0 years) |
Walk assisted or unassisted | 151 individuals (IQR 3.0–10.7 years) |
Few words/sentences | 103 individuals (IQR 2.4–11.0 years) |
Clinical exams (age at assessment) | – |
GMFM-66-IS | 45 individuals (IQR 1.6–5.4 years) |
GMFCS-ER | 41 individuals (IQR 1.8–7.4 years) |
PDMS-2 | 21 individuals (IQR 1.2–3.8 years) |
MACS | 9 individuals (IQR 2.8–9.5 years) |
CFCS | 23 individuals (IQR 3.0–11.8 years) |
CFCS = Communication Function Classification System; GMFCS-ER = Gross Motor Function Classification System Extended and Revised; GMFM-66-IS = Gross Motor Function Measure; MACS = Manual Ability Classification System; PDMS-2 = Peabody Developmental Motor Scales.
aProtein-truncating variants (PTV)/deletions (dels) included splice sites, frameshifts and whole and partial gene deletions. One individual with a synonymous variant predicted to affect splicing was included in PTV/dels.
Demographics | |
Male | 81/162 (50.0%) |
Female | 81/162 (50.0%) |
Age distribution | |
Age at assessment (n = 162) | Median 5.1 years (IQR 2.8–11.3 years) Range 3 months–44.2 years |
Seizure onset (n = 72/101) | Median 2 months (IQR 1–7 months) Range neonatal onset–11.1 years data |
Age at genetic diagnosis (n = 99, data available) | Median 1.9 years (IQR 6 months–5.5 years) Range 3 weeks–38.0 years |
Genetic information | |
Genetic variants in STXBP1 | – |
Missense variants | 46 individuals |
p.Arg406Cys/His/Ser | 9 individuals |
p.Arg292Cys/His/Pro | 8 individuals |
p.Arg551Cys/His | 4 individuals |
PTV/dela | 55 individuals |
In-frame insertions/deletions | 3 individuals |
Epilepsy data | |
Seizure types (n = 72/101) | – |
Focal-onset seizures | 48 individuals |
Neonatal seizures | 31 individuals |
Infantile/epileptic spasms | 29 individuals |
Early infantile seizures | 27 individuals |
Developmental data | |
Milestones assessment (age range of cohort assessed) | – |
Head control | 119 individuals (IQR 2.3–9.9 years) |
Roll over | 135 individuals (IQR 2.7–10.0 years) |
Sit unassisted | 133 individuals (IQR 2.1–9.3 years) |
Grasp and hand control | 109 individuals (IQR 2.7–10.0 years) |
Walk assisted or unassisted | 151 individuals (IQR 3.0–10.7 years) |
Few words/sentences | 103 individuals (IQR 2.4–11.0 years) |
Clinical exams (age at assessment) | – |
GMFM-66-IS | 45 individuals (IQR 1.6–5.4 years) |
GMFCS-ER | 41 individuals (IQR 1.8–7.4 years) |
PDMS-2 | 21 individuals (IQR 1.2–3.8 years) |
MACS | 9 individuals (IQR 2.8–9.5 years) |
CFCS | 23 individuals (IQR 3.0–11.8 years) |
Demographics | |
Male | 81/162 (50.0%) |
Female | 81/162 (50.0%) |
Age distribution | |
Age at assessment (n = 162) | Median 5.1 years (IQR 2.8–11.3 years) Range 3 months–44.2 years |
Seizure onset (n = 72/101) | Median 2 months (IQR 1–7 months) Range neonatal onset–11.1 years data |
Age at genetic diagnosis (n = 99, data available) | Median 1.9 years (IQR 6 months–5.5 years) Range 3 weeks–38.0 years |
Genetic information | |
Genetic variants in STXBP1 | – |
Missense variants | 46 individuals |
p.Arg406Cys/His/Ser | 9 individuals |
p.Arg292Cys/His/Pro | 8 individuals |
p.Arg551Cys/His | 4 individuals |
PTV/dela | 55 individuals |
In-frame insertions/deletions | 3 individuals |
Epilepsy data | |
Seizure types (n = 72/101) | – |
Focal-onset seizures | 48 individuals |
Neonatal seizures | 31 individuals |
Infantile/epileptic spasms | 29 individuals |
Early infantile seizures | 27 individuals |
Developmental data | |
Milestones assessment (age range of cohort assessed) | – |
Head control | 119 individuals (IQR 2.3–9.9 years) |
Roll over | 135 individuals (IQR 2.7–10.0 years) |
Sit unassisted | 133 individuals (IQR 2.1–9.3 years) |
Grasp and hand control | 109 individuals (IQR 2.7–10.0 years) |
Walk assisted or unassisted | 151 individuals (IQR 3.0–10.7 years) |
Few words/sentences | 103 individuals (IQR 2.4–11.0 years) |
Clinical exams (age at assessment) | – |
GMFM-66-IS | 45 individuals (IQR 1.6–5.4 years) |
GMFCS-ER | 41 individuals (IQR 1.8–7.4 years) |
PDMS-2 | 21 individuals (IQR 1.2–3.8 years) |
MACS | 9 individuals (IQR 2.8–9.5 years) |
CFCS | 23 individuals (IQR 3.0–11.8 years) |
CFCS = Communication Function Classification System; GMFCS-ER = Gross Motor Function Classification System Extended and Revised; GMFM-66-IS = Gross Motor Function Measure; MACS = Manual Ability Classification System; PDMS-2 = Peabody Developmental Motor Scales.
aProtein-truncating variants (PTV)/deletions (dels) included splice sites, frameshifts and whole and partial gene deletions. One individual with a synonymous variant predicted to affect splicing was included in PTV/dels.
Seizures were most prominent in the first year of life (Fig. 1A). The median age of epilepsy onset was 2 months (IQR 1–7 months). Sixty-one of 72 individuals (84.7%) had seizure onset in the first year of life (Fig. 1B): 31 individuals had neonatal onset, 30 had infantile onset after the first month with 80% cumulative onset by 10 months of life and 90% by 1.6 years. Onset and trajectory of seizures were highly variable, with relatively few months in which the proportion of all individuals with seizures exceeded 50% (Fig. 1A, inset). The most common seizure types in the overall cohort were focal-onset seizures (n = 48, f = 0.67) and infantile spasms (n = 29, f = 0.40), of which 39 individuals and 29 individuals had onset in the first year of life, respectively. Of individuals who had neonatal seizures, 22/31 (71.0%) had focal-onset seizures, 10 (32.3%) developed tonic seizures and 14 (45.2%) developed spasms following the neonatal period.

Overall landscape of seizures in STXBP1-related disorders. (A) Longitudinal distribution of seizure types across the age span. The heterogeneous pattern of epilepsy progression in the first year of life (inset), showing the proportion of individuals with seizures at a certain frequency during each respective month. (B) Cumulative onset of seizures in individuals with epilepsy. (C) Seizure remission indicated by risk of having refractory seizures over time, defined by not achieving 12 consecutive months of seizure freedom. (D) Stratification by seizure types in STXBP1 demonstrate a dynamic pattern of risk across the first 10 years of life with infantile spasms most prominent in the first 6 months of life and other seizure types including focal-onset seizures and tonic seizures more broadly distributed.
While seizures were the most prominent in the first year of life, stratification by seizure types demonstrated a dynamic pattern of epilepsy over time (Fig. 1D and Table 2). The risk for infantile spasms was greatest between 2 and 6 months of life (n = 29), with a median onset of 5 months and 90% cumulative onset by 9 months. While the seizure onset risk for focal seizures (n = 48, median 2 months, IQR neonatal to 7.5 months, 90% cumulative onset by 2.3 years) and tonic seizures (n = 20, median 4 months, IQR 0.75 months—1.53 years, 90% cumulative onset by 3.1 years) were primarily in the first three years of life, the risk for bilateral tonic-clonic seizures presented later in life (n = 15, median 3.92 years, IQR 7.5 months to 9.5 years, 90% cumulative onset by 12.2 years). In addition to the age-dependent distribution of seizure onset, 41 (56.9%) individuals presented with more than one seizure type, with infantile spasms (n = 22) and focal impaired awareness seizures (n = 13) as the most frequent types occurring with at least one additional seizure type.
. | Percentile . | |||
---|---|---|---|---|
. | 25th . | 50th . | 75th . | 90th . |
Seizure outcomes | ||||
Seizure onset (n = 72) | Neonatal onset | 2 months | 7 months | 1.6 years |
Focal-onset seizures (n = 48) | Neonatal onset | 2 months | 7.5 months | 2.3 years |
Infantile/epileptic spasms (n = 29) | 1 month | 4 months | 5 months | 6 months |
Tonic seizures (n = 20) | 0.8 months | 4 months | 1.5 years | 3.1 years |
Bilateral tonic-clonic seizures (n = 15) | 7.5 months | 3.9 years | 9.5 years | 12.2 years |
PTV/dela (n = 39) | Neonatal onset | 2 months | 3 months | 1.7 years |
Missense variants (n = 30) | Neonatal onset | 1 month | 6.3 months | 1.1 years |
Overall seizure remissionb (n = 38/61) | 6 months | 1.2 years | 4.0 years | 10.1 years |
Focal-onset seizures (n = 27) | 3 months | 7 months | 2.9 years | 6.7 years |
Infantile/epileptic spasms (n = 18) | 5 months | 8 months | 1.1 years | 3.5 years |
Tonic seizures (n = 12) | 4.8 months | 8 months | 3.6 years | 6.1 years |
Bilateral tonic-clonic seizures (n = 8) | 1.3 years | 3.6 years | 7.3 years | 8.6 years |
PTV/del (n = 26) | 6 months | 11 months | 4.0 years | 9.0 years |
Missense variants (n = 11) | 7 months | 2.6 years | 5.1 years | 10.2 years |
Developmental outcomes | ||||
Milestone acquisition | ||||
Head control (n = 31) | 3 months | 6.3 months | 1.0 year | 3.0 years |
Roll over (n = 103) | 5 months | 7.6 months | 11 months | 1.2 years |
Sit unassisted (n = 84) | 10 months | 1.0 year | 1.4 years | 2.0 years |
Grasp and hand control (n = 60) | 7 months | 10 months | 1.0 year | 2.0 years |
Walk assisted or unassisted (n = 81) | 1.5 years | 2.4 years | 3.5 years | 5.3 years |
Few words/sentences (n = 22) | 1.6 years | 2.0 years | 3.3 years | 7.0 years |
Standardized assessments | ||||
GMFM-66-IS (n = 45 individuals, 57 exams) | ||||
0–1 year (n = 9 exams) | 19.1 | 25.8 | 27.3 | 29.2 |
1 year-2 years (n = 15 exams) | 23.7 | 33.9 | 40.2 | 41.9 |
2–5 years (n = 20 exams) | 25.8 | 35.8 | 46.1 | 61.6 |
5 + years (n = 13 exams) | 54.9 | 58.9 | 61.2 | 66.6 |
PDMS-2 (n = 21 individuals) | ||||
Grasping (n = 23 exams) | 24.5 | 33.5 | 39.0 | 41.8 |
Visual motor integration (n = 23 exams) | 26.5 | 34.5 | 53.0 | 64.5 |
Classification scales | ||||
GMFCS-ER (n = 41) | Level I: n = 5, Level II: n = 16, Level III: n = 4, Level IV: n = 12, Level V: n = 5 | |||
MACS (n = 9) | Level I: n = 0, Level II: n = 4, Level III: n = 2, Level IV: n = 2, Level V: n = 1 | |||
CFCS (n = 23) | Level I: n = 0, Level II: n = 1, Level III: n = 3, Level IV: n = 5, Level V: n = 14 |
. | Percentile . | |||
---|---|---|---|---|
. | 25th . | 50th . | 75th . | 90th . |
Seizure outcomes | ||||
Seizure onset (n = 72) | Neonatal onset | 2 months | 7 months | 1.6 years |
Focal-onset seizures (n = 48) | Neonatal onset | 2 months | 7.5 months | 2.3 years |
Infantile/epileptic spasms (n = 29) | 1 month | 4 months | 5 months | 6 months |
Tonic seizures (n = 20) | 0.8 months | 4 months | 1.5 years | 3.1 years |
Bilateral tonic-clonic seizures (n = 15) | 7.5 months | 3.9 years | 9.5 years | 12.2 years |
PTV/dela (n = 39) | Neonatal onset | 2 months | 3 months | 1.7 years |
Missense variants (n = 30) | Neonatal onset | 1 month | 6.3 months | 1.1 years |
Overall seizure remissionb (n = 38/61) | 6 months | 1.2 years | 4.0 years | 10.1 years |
Focal-onset seizures (n = 27) | 3 months | 7 months | 2.9 years | 6.7 years |
Infantile/epileptic spasms (n = 18) | 5 months | 8 months | 1.1 years | 3.5 years |
Tonic seizures (n = 12) | 4.8 months | 8 months | 3.6 years | 6.1 years |
Bilateral tonic-clonic seizures (n = 8) | 1.3 years | 3.6 years | 7.3 years | 8.6 years |
PTV/del (n = 26) | 6 months | 11 months | 4.0 years | 9.0 years |
Missense variants (n = 11) | 7 months | 2.6 years | 5.1 years | 10.2 years |
Developmental outcomes | ||||
Milestone acquisition | ||||
Head control (n = 31) | 3 months | 6.3 months | 1.0 year | 3.0 years |
Roll over (n = 103) | 5 months | 7.6 months | 11 months | 1.2 years |
Sit unassisted (n = 84) | 10 months | 1.0 year | 1.4 years | 2.0 years |
Grasp and hand control (n = 60) | 7 months | 10 months | 1.0 year | 2.0 years |
Walk assisted or unassisted (n = 81) | 1.5 years | 2.4 years | 3.5 years | 5.3 years |
Few words/sentences (n = 22) | 1.6 years | 2.0 years | 3.3 years | 7.0 years |
Standardized assessments | ||||
GMFM-66-IS (n = 45 individuals, 57 exams) | ||||
0–1 year (n = 9 exams) | 19.1 | 25.8 | 27.3 | 29.2 |
1 year-2 years (n = 15 exams) | 23.7 | 33.9 | 40.2 | 41.9 |
2–5 years (n = 20 exams) | 25.8 | 35.8 | 46.1 | 61.6 |
5 + years (n = 13 exams) | 54.9 | 58.9 | 61.2 | 66.6 |
PDMS-2 (n = 21 individuals) | ||||
Grasping (n = 23 exams) | 24.5 | 33.5 | 39.0 | 41.8 |
Visual motor integration (n = 23 exams) | 26.5 | 34.5 | 53.0 | 64.5 |
Classification scales | ||||
GMFCS-ER (n = 41) | Level I: n = 5, Level II: n = 16, Level III: n = 4, Level IV: n = 12, Level V: n = 5 | |||
MACS (n = 9) | Level I: n = 0, Level II: n = 4, Level III: n = 2, Level IV: n = 2, Level V: n = 1 | |||
CFCS (n = 23) | Level I: n = 0, Level II: n = 1, Level III: n = 3, Level IV: n = 5, Level V: n = 14 |
CFCS = Communication Function Classification System; GMFCS-ER = Gross Motor Function Classification System Extended and Revised; GMFM-66-IS = Gross Motor Function Measure; MACS = Manual Ability Classification System; PDMS-2 = Peabody Developmental Motor Scales.
aProtein-truncating variants (PTV)/deletions (dels) included splice sites, frameshifts and whole and partial gene deletions.
bSeizure remission was defined by at least 12 months of consecutive seizure freedom.
. | Percentile . | |||
---|---|---|---|---|
. | 25th . | 50th . | 75th . | 90th . |
Seizure outcomes | ||||
Seizure onset (n = 72) | Neonatal onset | 2 months | 7 months | 1.6 years |
Focal-onset seizures (n = 48) | Neonatal onset | 2 months | 7.5 months | 2.3 years |
Infantile/epileptic spasms (n = 29) | 1 month | 4 months | 5 months | 6 months |
Tonic seizures (n = 20) | 0.8 months | 4 months | 1.5 years | 3.1 years |
Bilateral tonic-clonic seizures (n = 15) | 7.5 months | 3.9 years | 9.5 years | 12.2 years |
PTV/dela (n = 39) | Neonatal onset | 2 months | 3 months | 1.7 years |
Missense variants (n = 30) | Neonatal onset | 1 month | 6.3 months | 1.1 years |
Overall seizure remissionb (n = 38/61) | 6 months | 1.2 years | 4.0 years | 10.1 years |
Focal-onset seizures (n = 27) | 3 months | 7 months | 2.9 years | 6.7 years |
Infantile/epileptic spasms (n = 18) | 5 months | 8 months | 1.1 years | 3.5 years |
Tonic seizures (n = 12) | 4.8 months | 8 months | 3.6 years | 6.1 years |
Bilateral tonic-clonic seizures (n = 8) | 1.3 years | 3.6 years | 7.3 years | 8.6 years |
PTV/del (n = 26) | 6 months | 11 months | 4.0 years | 9.0 years |
Missense variants (n = 11) | 7 months | 2.6 years | 5.1 years | 10.2 years |
Developmental outcomes | ||||
Milestone acquisition | ||||
Head control (n = 31) | 3 months | 6.3 months | 1.0 year | 3.0 years |
Roll over (n = 103) | 5 months | 7.6 months | 11 months | 1.2 years |
Sit unassisted (n = 84) | 10 months | 1.0 year | 1.4 years | 2.0 years |
Grasp and hand control (n = 60) | 7 months | 10 months | 1.0 year | 2.0 years |
Walk assisted or unassisted (n = 81) | 1.5 years | 2.4 years | 3.5 years | 5.3 years |
Few words/sentences (n = 22) | 1.6 years | 2.0 years | 3.3 years | 7.0 years |
Standardized assessments | ||||
GMFM-66-IS (n = 45 individuals, 57 exams) | ||||
0–1 year (n = 9 exams) | 19.1 | 25.8 | 27.3 | 29.2 |
1 year-2 years (n = 15 exams) | 23.7 | 33.9 | 40.2 | 41.9 |
2–5 years (n = 20 exams) | 25.8 | 35.8 | 46.1 | 61.6 |
5 + years (n = 13 exams) | 54.9 | 58.9 | 61.2 | 66.6 |
PDMS-2 (n = 21 individuals) | ||||
Grasping (n = 23 exams) | 24.5 | 33.5 | 39.0 | 41.8 |
Visual motor integration (n = 23 exams) | 26.5 | 34.5 | 53.0 | 64.5 |
Classification scales | ||||
GMFCS-ER (n = 41) | Level I: n = 5, Level II: n = 16, Level III: n = 4, Level IV: n = 12, Level V: n = 5 | |||
MACS (n = 9) | Level I: n = 0, Level II: n = 4, Level III: n = 2, Level IV: n = 2, Level V: n = 1 | |||
CFCS (n = 23) | Level I: n = 0, Level II: n = 1, Level III: n = 3, Level IV: n = 5, Level V: n = 14 |
. | Percentile . | |||
---|---|---|---|---|
. | 25th . | 50th . | 75th . | 90th . |
Seizure outcomes | ||||
Seizure onset (n = 72) | Neonatal onset | 2 months | 7 months | 1.6 years |
Focal-onset seizures (n = 48) | Neonatal onset | 2 months | 7.5 months | 2.3 years |
Infantile/epileptic spasms (n = 29) | 1 month | 4 months | 5 months | 6 months |
Tonic seizures (n = 20) | 0.8 months | 4 months | 1.5 years | 3.1 years |
Bilateral tonic-clonic seizures (n = 15) | 7.5 months | 3.9 years | 9.5 years | 12.2 years |
PTV/dela (n = 39) | Neonatal onset | 2 months | 3 months | 1.7 years |
Missense variants (n = 30) | Neonatal onset | 1 month | 6.3 months | 1.1 years |
Overall seizure remissionb (n = 38/61) | 6 months | 1.2 years | 4.0 years | 10.1 years |
Focal-onset seizures (n = 27) | 3 months | 7 months | 2.9 years | 6.7 years |
Infantile/epileptic spasms (n = 18) | 5 months | 8 months | 1.1 years | 3.5 years |
Tonic seizures (n = 12) | 4.8 months | 8 months | 3.6 years | 6.1 years |
Bilateral tonic-clonic seizures (n = 8) | 1.3 years | 3.6 years | 7.3 years | 8.6 years |
PTV/del (n = 26) | 6 months | 11 months | 4.0 years | 9.0 years |
Missense variants (n = 11) | 7 months | 2.6 years | 5.1 years | 10.2 years |
Developmental outcomes | ||||
Milestone acquisition | ||||
Head control (n = 31) | 3 months | 6.3 months | 1.0 year | 3.0 years |
Roll over (n = 103) | 5 months | 7.6 months | 11 months | 1.2 years |
Sit unassisted (n = 84) | 10 months | 1.0 year | 1.4 years | 2.0 years |
Grasp and hand control (n = 60) | 7 months | 10 months | 1.0 year | 2.0 years |
Walk assisted or unassisted (n = 81) | 1.5 years | 2.4 years | 3.5 years | 5.3 years |
Few words/sentences (n = 22) | 1.6 years | 2.0 years | 3.3 years | 7.0 years |
Standardized assessments | ||||
GMFM-66-IS (n = 45 individuals, 57 exams) | ||||
0–1 year (n = 9 exams) | 19.1 | 25.8 | 27.3 | 29.2 |
1 year-2 years (n = 15 exams) | 23.7 | 33.9 | 40.2 | 41.9 |
2–5 years (n = 20 exams) | 25.8 | 35.8 | 46.1 | 61.6 |
5 + years (n = 13 exams) | 54.9 | 58.9 | 61.2 | 66.6 |
PDMS-2 (n = 21 individuals) | ||||
Grasping (n = 23 exams) | 24.5 | 33.5 | 39.0 | 41.8 |
Visual motor integration (n = 23 exams) | 26.5 | 34.5 | 53.0 | 64.5 |
Classification scales | ||||
GMFCS-ER (n = 41) | Level I: n = 5, Level II: n = 16, Level III: n = 4, Level IV: n = 12, Level V: n = 5 | |||
MACS (n = 9) | Level I: n = 0, Level II: n = 4, Level III: n = 2, Level IV: n = 2, Level V: n = 1 | |||
CFCS (n = 23) | Level I: n = 0, Level II: n = 1, Level III: n = 3, Level IV: n = 5, Level V: n = 14 |
CFCS = Communication Function Classification System; GMFCS-ER = Gross Motor Function Classification System Extended and Revised; GMFM-66-IS = Gross Motor Function Measure; MACS = Manual Ability Classification System; PDMS-2 = Peabody Developmental Motor Scales.
aProtein-truncating variants (PTV)/deletions (dels) included splice sites, frameshifts and whole and partial gene deletions.
bSeizure remission was defined by at least 12 months of consecutive seizure freedom.
We then assessed seizure remission, defined as at least 12 consecutive months of seizure freedom. In individuals with at least 1 year of observation time following onset (n = 61), 38 individuals had seizure remission, independent of seizure type, with the median offset at 1.21 years (IQR 6 months to 4.0 years) (Fig. 1C and Table 2). The age at offset varied across seizure types, with median offset for focal-onset seizures (n = 27) and epileptic spasms at 7 months and 8 months, respectively (IQR 3 months to 2.92 years; IQR 5 months to 1.08 years), while the median offset for bilateral tonic-clonic seizures was 3.63 years (IQR 1.31 years to 7.25 years). While seizures reoccurred in 20 individuals following remission, our cohort represented a primarily paediatric population of STXBP1-related disorders, and we were not able to make robust conclusions regarding seizure reoccurrence, including childhood to adulthood.
Epilepsy trajectories diverge when stratifying by genetic variants in STXBP1
Given recognition of emerging phenotypic signatures associated with specific variants in STXBP1, we then assessed the variant spectrum in our cohort. Forty-six (44.2%) individuals had missense variants, 15 (14.6%) had frameshift variants, 14 (13.6%) had nonsense variants, 14 had splice site variants (13.6%), 12 (11.7%) had whole or partial gene deletions and three (2.9%) had in-frame insertions or deletions. Individuals with nonsense, splice site and frameshift variants and whole or partial gene deletions were grouped as protein-truncating variant (PTV)/deletions (del) for further analyses (n = 55, 52.9%). Thirty-six individuals had recurrent variants, including the three most frequent genetic hot spots identified in STXBP1: p.Arg406Cys/His/Ser (n = 9), p.Arg292Cys/His/Leu/Pro (n = 8) and p.Arg551Cys/His (n = 4).
First, we delineated the longitudinal epilepsy history in individuals with PTV/del (n = 39 with seizures) versus missense variants (n = 30 with seizures) (Fig. 2A). We found the greatest difference in seizure frequency distributions between 10 months and 2 years of life, during which time individuals with missense variants were more likely to have more frequent seizures than individuals with PTV/del. This observed difference was primarily driven by the higher frequency of focal-onset seizures, and a similar effect was also observed between 3.5 to 4 years and 5 to 6 years of life with higher frequencies in focal-onset seizures and tonic seizures associated with missense variants (Fig. 2C). For individuals with PTV/del, a significant difference was observed between 5 and 6 months of life, when infantile spasms were the most prominent (P = 0.03, Fig. 2B). The risk of intractable seizures was higher in individuals with missense variants (P = 0.006). When assessing recurrent missense variants,12,14 we found that individuals with p.Arg292Cys/His/Pro were at greater risk for neonatal seizures and individuals with p.Arg551Cys/His were more likely to have ongoing seizures after 12 months of life; however, these findings were driven by a limited number of individuals (Supplementary Fig. 3).

Difference in seizure histories between individuals with protein-truncating variants (PTV) and deletions versus missense variants in STXBP1. (A) Progression of epilepsy on a monthly basis in a subgroup of individuals with seizures at any point in the first 3 years of life (n = 30 missense, n = 39 PTV). Longitudinal significance of the difference in the distribution of overall seizure frequencies in each respective month across the two subgroups are shown below. (B) Distribution of common seizure types across individuals with PTV/deletions, contrasted with (C) the distribution of seizure types across individuals with missense variants in STXBP1 in the first 10 years of life.
Development in STXBP1 is highly variable, with stratification by disease severity markers
When analysing milestones assessed in at least 20 individuals (Fig. 3A and B), we found a range of percentages of achieved milestones, spanning from ability to use non-verbal communication (n = 20, f = 1.00), to ability to roll over (n = 121/135, f = 0.90), ability to sit unsupported (n = 96/133, f = 0.72), ability to walk (n = 86/151, f = 0.57) and walk unassisted (n = 45/99, f = 0.45), to ability to use sentences and verbal communication (n = 14/102, f = 0.14). We analysed the cumulative onset of commonly assessed milestones (Fig. 3A), namely, the ability to control head posture, roll over, sit unassisted, grasp, walk unassisted and initiate words and use verbal communication in individuals who achieved each respective milestone. Development across the domains was primarily within the first 5 years of life, and of individuals noted to achieve each respective milestone, the median age of head control was at 6.3 months (n = 31, IQR 3 months to 1 year, 90% cumulative onset at 3 years), roll over at 7.6 months (n = 103, IQR 5–11 months, 90% cumulative onset at 1.2 years), sit unassisted (n = 84, IQR 10 months to 1.4 years, 90% cumulative onset at 2.0 years), grasp (n = 60, IQR 7 months to 1.0 years, 90% cumulative onset at 1.96 years), walk (n = 81, IQR 1.5–3.47 years, 90% cumulative onset at 5.3 years) and initiate words (n = 22, IQR 1.6–3.3 years, 90% cumulative onset at 1.2 years) (Table 2).

Developmental outcomes and end points in STXBP1-related disorders. (A) Cumulative milestone acquisition by age in a subset of individuals who achieved each respective milestone in STXBP1-related disorders. Dashed lines indicate the age at when 75% of typically developing children achieve each respective milestone as reported by the Center for Disease Control and Prevention (CDC). The distribution of gross motor, fine motor and language development is primarily in the first 5 years of life (inset, showing age achieved indicated by each respective colour versus age at which individuals were last assessed in grey). (B) Proportion of milestones achieved in the overall cohort, showing a wide range of percentages with verbal communication and language at the lower end. (C) Distribution of 57 GMFM-66-IS scores over time across 45 individuals. The GMFM-66-IS is a criterion referenced assessment, with a range of 0% to 100% for the total score; a maximum score of 100% is considered equivalent to the measure that would be achieved by a typically developing 5-year-old. (D) Distribution of PDMS-2 raw scores for grasping and visual motor integration over time across 21 individuals. The PDMS-2 is a norm referenced assessment, with a range of 0 to 100 for each subset raw score. PDMS-2 scores in STXBP1 map to an age equivalent of less than 14 months, even in individuals after 2 years of life (Supplementary Fig. 4). Raw scores for the GMFM-66-IS and PDMS-2 are shown to delineate the range of outcomes that would not be captured from standard scores due to floor effects. GMFM-66-IS = Gross Motor Function Measure; PDMS-2 = Peabody Developmental Motor Scales.
Next, we assessed developmental trajectories using standardized outcome measures and classification scales (Fig. 3C and D and Table 2). For gross motor classification, five individuals were Level I on the GMFCS-ER, 16 individuals Level II, four individuals Level III, 12 individuals Level IV and five individuals had a Level V classification. Only nine individuals were assessed for MACS to characterize ability to navigate and perform in daily life, for which no individuals had a Level I classification, four were Level II, two Level III, two Level IV and one individual was Level V. Classification of language skills using the CFCS revealed that no individuals had a Level I classification, one was Level II, three Level III, five Level IV and 14 were Level V. Stratification of GMFM-66-IS scores by age ranges revealed a wide range of outcomes, with the greatest variability between the second and fifth year of life (median score of 35.8, IQR 25.75–46.05) and an observed plateau of gross motor function outcome on the GMFM-66-IS in late childhood (median score of 58.9, IQR 54.9–61.2). When quantifying fine motor development using the PDMS-2 measure, we found that individuals with STXBP1 had a range of scores for grasping and visual motor integration that mapped to an age equivalent of <14 months, even after 2 years of life (Supplementary Fig. 4). PDMS-2 percentiles demonstrated a floor effect after the first year of life, when visual motor integration dropped below the first percentile and the range for grasping remained less than the 20th percentile.
Lastly, we aimed to map the range of developmental outcomes associated with markers of seizure severity. We found that later epilepsy onset was associated with milestone acquisition of independent ambulation and verbal communication abilities (Supplementary Table 2 and Supplementary Fig. 5); individuals with epilepsy onset after the neonatal period were more than 5-fold more likely to initiate words [OR 95% confidence interval (CI) 1.5–26.2], achieved in 43% of individuals than compared to only 12% of those with neonatal onset. Similarly, individuals with seizure onset after the first month of life were 6-fold more likely to be able to achieve independent ambulation (OR 95% CI 1.9–22.0), achieved in 68% compared to 25% of those with neonatal onset. In contrast, individuals with an earlier epilepsy onset had overall higher GMFCS (P = 0.02), indicating lower levels of gross motor function. We found that individuals with a current or past history of epilepsy and individuals with spasms had lower GMFM-66-IS scores compared to individuals without any history of epilepsy (P = 0.01) and without spasms (P = 0.04), respectively (Supplementary Fig. 6). We did not find a significant difference in GMFM-66-IS scores between individuals with ongoing seizures and individuals who were seizure-free.
Longitudinal forecasting of seizures reveals subgroups with different trajectories
We then characterized the range of epilepsy trajectories and identified subgroups defined by seizure predictability. The first aim was to delineate subgroups based on seizure variability. To this end, we developed a longitudinal seizure frequency forecasting model that quantified the likelihood of seizure frequency outcomes after the first year of life, for individuals with complete histories in the first year (n = 78). Predictions compared to a distribution of randomized frequencies through permutation testing demonstrated ‘better than chance forecasting’ across 87% of the cohort. The remaining individuals with unpredictable trajectories primarily included individuals whose seizures remained refractory throughout childhood but also individuals who had periods of seizure freedom despite predictions to have ongoing seizures.
We then compared the difference between predicted and actual seizure trajectories (Fig. 4A) after the first year and derived a measure of variability across the cohort. This allowed us to characterize two subgroups: the ‘high-fidelity’ subgroup included individuals with predictable trajectories (n = 53), including 26 individuals who remained seizure-free up until time of assessment, while the ‘low-fidelity’ subgroup was defined by individuals with unpredictable trajectories (n = 25), including the individuals whose seizures could not be forecasted better than chance (Fig. 4B). This suggests that up to two-thirds of individuals with STXBP1 follow a predictable trajectory that can be reasonably inferred from the first year of life.

Longitudinal modelling of seizure histories in the first year of life in STXBP1 revealed two subgroups with unpredictable and predictable epilepsy trajectories later in life. (A) Observed and predicted monthly seizure trajectory for one individual. The seizure history during the first 12 months were used to define a reference cohort of most phenotypically similar individuals. Seizure frequencies in the reference subgroup were then used to predict the individual’s trajectory. The difference in observed and predicted seizure frequencies across months was used to characterize the variability. (B) Variability was assessed across the cohort (n = 78), with two subgroups: high-fidelity subgroup defined by predictable trajectories versus low-fidelity subgroup defined by unpredictable trajectories, showing seizure frequencies in the first year of life that resulted in high or low fidelity predictions of longitudinal epilepsy trajectories later in life. (C) Seizure types leading to risk of having unpredictable seizures later in life.
The second aim of this analysis was to identify epilepsy characteristics in the first year predictive of unpredictable seizures later in life. We found that features suggestive of a future predictable trajectory included neonatal or early infantile seizures that resolved by 7 months of life, or a shorter duration of seizures in infancy (Fig. 4C). Particularly, individuals who achieved seizure freedom within a few months following onset showed a highly predictable seizure trajectory. This subgroup accounted for ∼50% of individuals with epilepsy. We were also able to forecast future refractory seizures in individuals who had focal seizures, infantile spasms after 6 months and ongoing seizures at 12 months of life; however, this subgroup represented only 2% of the cohort (Supplementary Table 3).
Conversely, seizure characteristics indicative of an atypical, unpredictable course later in life included refractory epilepsy throughout the first year of life, especially between 7 to 12 months, and longer periods of consecutive months with seizures, indicating a more severe epilepsy subgroup within STXBP1. In contrast to the high-fidelity subgroup, 60% of the low-fidelity subgroup had ongoing epilepsy at 12 months of life. Furthermore, while there was no notable difference in the proportion of individuals with infantile spasms between subgroups (n = 9/27 in high-fidelity, n = 11/25 in low-fidelity subgroup), the longitudinal distribution of seizure frequencies after 6 months of life suggests that the differentiating factor was determined by ongoing and intractable spasms and seizures during this period. There was no significant difference in the variant spectrum between the high-fidelity (n = 28 PTV/del, n = 25 missense) and low-fidelity subgroups (n = 13 PTV/del, n = 9 missense, n = 3 in-frame indel).
Treatment effects in STXBP1 vary over time and align with existing guidelines
We assessed existing treatment strategies and characterized the relative effectiveness of ASMs in our cohort. We reconstructed ASM histories across 307 total patient-years in a subgroup of 61 individuals with a total of 20 unique ASMs in addition to the ketogenic diet. The most frequently prescribed medications were levetiracetam (n = 45, 99.5 patient-years), phenobarbital (n = 37, 23.2 patient-years), topiramate (n = 24, 31.3 patient-years), adrenocorticotropic hormone (ACTH) (n = 21, 2.25 patient-years) and vigabatrin (n = 20, 22.6 patient-years). The treatment landscape was dynamic across the age span, with phenobarbital, ACTH and levetiracetam commonly used for treating neonatal and early infantile seizures, vigabatrin and levetiracetam in early childhood and clobazam and the ketogenic diet preferred as treatment strategies later in life (Fig. 5A).

Characterization of treatment strategies and medication response in STXBP1-related disorders. (A) Anti-seizure medication (ASM) landscape across 61 individuals with STXBP1, showing the relative density of ASMs used in at least 10 individuals across the age span. (B) Characterization of medication response over time following treatment initiation, showing the reduction in seizure frequencies on a monthly basis for seizure more broadly (top) and spasms (bottom). (C) Relative effectiveness of ASM for seizures vary across short-term and long-term treatment response, with phenobarbital as a superior ASM in reducing seizure frequency while clobazam and the ketogenic diet were effective in maintaining seizure freedom. Only medications used in at least 10 individuals are shown. ACTH = adrenocorticotropic hormone.
We found that the use of any ASM or combination of ASMs was able to reduce overall seizure burden by ∼50% within 5 months following initiation. This effect was seen to a greater extent for treating spasms, in which 50% of individuals with spasms had offset within 3 months following ASM initiation (Fig. 5B). When assessing seizure burden across epilepsy syndromes, we found a difference in medication response; individuals with Lennox-Gastaut syndrome (LGS) and developmental and epileptic encephalopathies (DEEs) (n = 15) were significantly less likely to experience a reduction in seizure frequencies when compared to individuals with West syndrome and individuals with other early onset epileptic encephalopathies (EOEE) (n = 22) (Supplementary Fig. 7). This points to differences in disease severity within STXBP1-related disorders.
We then analysed the relative efficacy for both short-term and long-term response (Fig. 5C). Phenobarbital was 2-fold more likely to reduce overall seizures [n = 37, odds ratio (OR) 2.11, 95% CI 1.49–2.95], primarily driven by the offset of neonatal seizures in a subgroup of 21 individuals by 4 months of life. When accounting for periods of seizure freedom, we found that clobazam (n = 14, OR 2.71, 95% CI 2.09–3.55) and the ketogenic diet (n = 10, OR 1.47, 95% CI 1.07–2.03) were superior treatment strategies. The medication response in clobazam and ketogenic diet was seen to a greater degree when assessing for maintaining seizure freedom only, and topiramate emerged as another ASM with relative efficacy compared to other medication choices (n = 22, OR 1.26, 95% CI 1.00–1.59). Accordingly, we derived a framework for quantifying longitudinal response to ASMs in STXBP1-related disorders, providing a foundation upon which the efficacy of future therapeutic and treatment strategies can be evaluated.
Virtual clinical trials demonstrate an age-dependent distribution of treatment success
Given the heterogeneity of seizure trajectories in STXBP1-related epilepsy, we identified optimal time windows during which a treatment effect would have the highest probability of being detected in a clinical trial. Virtual clinical trials using seizure frequency as the primary outcome resulted in wide range of trial success probabilities across the age span. We developed a measure for the probability of success: the OFTS, defined as the proportion of 1000 simulated trials in which a significant difference (P < 0.05) was detected. We were able to achieve the highest OFTS (range 0.87 to >0.95) in early childhood between 8 months and 3.5 years of life (Fig. 6A), with a wide overall range of probabilities from 0 to >0.95. While we found a second window between 7 and 10 years of life, this effect was driven by repeated sampling of a limited number of individuals with refractory seizures during this period. When stratified by seizure type (Fig. 6B–D), we found an optimal window starting at 3 months and a second window after 7 months for individuals with intractable spasms, 6 to 18 months and 2 to 3.5 years for focal-onset seizures, and two periods spanning from 8 months to just prior to 2 years and 2 to 3.5 years for tonic seizures. This demonstrates an age and seizure type-dependent landscape for optimal treatment windows.

Virtual clinical trial framework for STXBP1-related disorders. (A) Virtual clinical trial using seizure frequency as the primary outcome, highlighting optimal age windows in green. The observed frequency of trial success (OFTS) is defined as the proportion of trials out of 1000 virtual trials in which a significant effect can be detected if the trial were to be started at the respective month. A trial duration of 6 months versus 12 months and the targeted seizure reduction ranging from 10% to 50% was selected based on the widest distribution of OFTS across the age span. (B) Virtual trials for targeting infantile spasms demonstrate an early window for optimal intervention starting at 3 months, and a second window is observed after 7 months for individuals with intractable spasms. (C) Virtual trials for targeting focal-onset seizures show an optimal window of 6 to 18 months and 2 to 3.5 years, although the OFTS was more variable. (D) Virtual trials for targeting tonic seizure, showing broad windows between 8 months to just prior to 2 years and 2 to 3.5 years.
Discussion
Disease-causing variants in STXBP1 lead to a heterogenous group of clinical presentations with varying disease severity and a wide range of outcomes. Understanding the natural history of STXBP1-related disorders, including long-term epilepsy trajectories and developmental end points, is critical to the development of clinical trials and delivery of novel therapeutic strategies. In our study of 162 individuals with STXBP1-related disorders, we found a heterogeneous pattern of seizures, especially in the first year of life and into early childhood, alongside high baseline variability in developmental domains including gross motor, fine motor and communication abilities. Notably, we characterized epilepsy and development using validated outcome scales across the age span, demonstrating a dynamic disease trajectory that sets the groundwork for future prospective natural history studies.
Seizures in STXBP1
Seizure onset is within the second month of life in 50% of individuals and in the first year of life in >80%. In contrast to other common genetic epilepsies, such as Dravet syndrome,38,39 the overall pattern of seizures in STXBP1 is highly variable. While the first 3 months of life are characterized by the highest seizure burden with focal-onset seizures followed by infantile spasms as the most frequent types, the dynamic risk of different seizure types had not previously been delineated.1,17,40 In our study, we found a heterogeneous interplay of seizure onset and offset in the first year of life into early childhood, providing precise insight for counselling of families in the clinical setting. The recognition of early infantile seizures in STXBP1 may lead to earlier molecular diagnoses, which has important implications in prognosis and targeted care including early intervention for epilepsy management.
As previously recognized,3 we found that epilepsy histories diverge between individuals with missense variants compared to PTV and deletions. Individuals with PTV/del had increased risk of infantile spasms, while individuals with missense variants were more likely to have intractable seizures later in life. We also found that individuals with p.Arg292Cys/His/Pro were at greater risk for neonatal seizures, while individuals with p.Arg551Cys/His were more likely to have ongoing seizures after 12 months of life. This adds to previously unrecognized clinical signatures associated with recurrent variants in STXBP1.3,12,14 The identification and recognition of natural histories specific to underlying aetiologies will be necessary in tailored prognostic care and in moving towards genotype-guided trials in the future.
Developmental outcomes
With increased clinical genetic testing, an increasing number of individuals are recognized to have developmental delay and intellectual disability without epilepsy within STXBP1-related disorders.1,5,8 As initial cohort studies tend to include more severely affected individuals, interpretation of outcomes can be variable when considering selection bias.1,17,40 In contrast, 30% of our cohort did not have seizures, pointing to the need to delineate developmental end points as an additional outcome measure complementary to epilepsy end points. However, unlike for well studied neurological conditions such as cerebral palsy, disease-specific outcomes using validated measures for STXBP1 and most rare neurogenetic conditions currently do not exist.
We found that development is variable in the first 5 years of life, with observed improvement in gross and fine motor development and acquisition of milestones during this window. This underscores the importance of intervention in early childhood to maximize development and achieve better long-term outcomes. Our study shows that most individuals with STXBP1-related disorders attain gross motor milestones including independent ambulation in more than 45% of individuals and reach GMFM-66-IS scores ranging from 50–75 after 5 years of life, providing granular context to existing findings on independent ambulation.40 The wide range of outcomes suggest that gross motor development can be considered a primary end point as a comprehensive and modifiable measure.
Delineation of PDMS-2 percentiles in STXBP1 demonstrated an overall age equivalent of less than 14 months in fine motor development. However, the assessment of fine motor function in children with STXBP1 may be difficult due to the need to follow directions, which may be limited by language and cognitive deficits.41,42 As families of children with STXBP1 have reported difficulties with activities of daily living as a major concern,18 additional research to validate reliable fine motor function measures is necessary as potential treatments for STXBP1 are developed in the future.
In addition, challenges in language and communication may be more prominent in STXBP1-related disorders compared to other genetic epilepsies. Families of children with STXBP1 have identified expressive and receptive communication challenges as major symptoms that affect their lives.18 Our study demonstrates that the milestones with lowest frequencies achieved across the cohort are primarily regarding differences in language abilities, including the ability to use words or sentences in <25% of individuals. This aligns with previously published studies, in which verbal communication was identified as the domain with the greatest delay and impairment in up to 70% of individuals with STXBP1.17,40 However, we found that almost all individuals assessed use non-verbal means of communication, highlighting that expressive communication is not limited to verbal communication. Additional longitudinal assessments of language function using more comprehensive scales such as the Observer-Reported Communication Ability (ORCA) measure will be critical to examine the development of language skills in children with STXBP1.43,44
Furthermore, current challenges in trials are the switching of developmental scales across the age span and floor effects, when existing scales cannot capture and differentiate the range of outcomes at the lower extremes.45 The use of raw scores rather than standard scores and testing children outside the intended age range for outcome measures may be necessary to accurately measure children with STXBP1.41 The development of disease-specific outcomes measures has been completed with some neurogenetic conditions such as CDKL5 deficiency disorder and Aicardi Goutières syndrome to avoid floor effects, which could mask potential changes as the result of an intervention.46,47
Through validated outcome measures, we then demonstrate differences in developmental trajectories between subgroups stratified by markers of seizure severity. We found that individuals with epilepsy and spasms had greater delay in gross motor development than individuals with neurodevelopmental phenotypes and other seizure types, respectively. Furthermore, individuals with an earlier seizure onset were less likely to achieve independent ambulation and had lower verbal communication abilities, most prominently seen in individuals with neonatal onset.17,40 These findings point to the need for future prospective studies to further distinguish the full range of outcomes within subgroups in STXBP1.
Epilepsy variability and virtual clinical trials
Seizures in STXBP1 are heterogeneous and subgroups can be stratified based on the variability or ‘unpredictability’ of seizures across the age span. Through a longitudinal seizure frequency forecasting model based on interrogation of epilepsy histories in the first year of life, we show that seizures can be forecasted better than chance in 87% of individuals. We quantified the degree of variability and identified two subgroups, namely individuals who had predictable versus unpredictable seizures later in life. The subgroup with high predictability included individuals with early infantile seizures that resolved within several months, which accounted for up to 50% of individuals with epilepsy. Conversely, individuals whose seizures diverged from forecasted trajectories constituted up to one-third of individuals with STXBP1-related disorders, representing a high proportion of individuals with variable outcomes. Markers of epilepsy histories in the first year of life indicative of highly unpredictable seizure histories later in life included refractory epilepsy throughout the first year with ongoing seizures at 12 months. Understanding the clinical factors associated with specific trajectories is critical for clinical decision-making, enabling personalized risk stratification and monitoring disease progression over time.24,33,40,48–50 Furthermore, longitudinal forecasting facilitates identification of atypical presentations, which allow for variant prioritization in functional studies in addition to providing a prognostic baseline score for inclusion and exclusion of robust subgroups for future trials.
Seizure frequency is an objective measure that has thus far been used as the primary outcome when interpreting change from baseline in clinical trials in the epilepsies51,52; however, the overall variability of seizures in a given patient cohort can decrease statistical power when evaluating treatment effect. Accordingly, we modelled virtual clinical trials using a synthetic control approach, leveraging real world data captured from routine clinical care to analyse the probability of trial success across the age span.49,53 We demonstrate that the most optimal age range for detecting a significant treatment effect is between 8 months and 3.5 years of life in STXBP1-related disorders. The probability of success when targeting focal-onset seizures was more variable, and trials for spasms and tonic seizures revealed varying windows. In summary, we provide insight into the design of efficient and cost-effective trials in the future. However, understanding the generalizability of this approach across genetic epilepsies will remain critical in the future as the methodology of virtual clinical trials in rare disorders has not yet been validated.
Treatment efficacy
ASMs are the primary treatment strategy for epilepsy management; however, whether certain ASMs are effective has largely relied on provider experience. Our study demonstrates that a systematic approach to capture ASM response without a priori hypotheses from medical record data can be used as a guiding framework for characterizing the efficacy of current treatment strategies in STXBP1. We show that phenobarbital is effective for short-term treatment of neonatal and early infantile seizures, while clobazam and the ketogenic diet are effective for long-term management. In contrast to prior reports, we did not find a superior effect of levetiracetam in seizure reduction in STXBP1, even when assessing a subset of individuals with refractory epilepsy.54 Stratification of treatment response across the age span, seizure types and between subgroups will provide granular insight into subtypes in STXBP1 that will have the greatest prospect of benefit in a future trial.
Limitations
Our study presents findings on the natural history of STXBP1-related disorders, leveraging existing data captured from clinical care through a retrospective, cross-sectional study. As our cohort represents a primarily paediatric cohort with the median age at last encounter of 9 years, future studies will benefit from understanding longitudinal epilepsy and developmental outcomes in adulthood.40 To this end, the outcomes for early-to-late childhood in our study likely include over-representation of a more severely affected subgroup for whom data were available. Accordingly, follow-up studies of the younger population in which the age at genetic diagnosis was earlier will be critical, including a standardized framework to capture the full range of seizure and developmental outcomes across the age span. Additionally, while our study demonstrated that clinical heterogeneity is associated with markers of disease severity, such as a greater degree of developmental delay in individuals with epilepsy and more variable seizures trajectories in individuals with refractory epilepsy early in life, we were not able to assess the impact of genomic background and variation contributing to the wide range of outcomes.17 To this end, biomarkers including EEG signatures, will serve as critical objective measurements of underlying disease pathophysiology and enable stratification of subtypes, prognostication of trajectories and treatment response in future studies.55 Lastly, families of individuals with STXBP1 have identified clinical features including gastrointestinal and respiratory symptoms that have been under-represented in the existing literature and may serve as additional end points alongside measures for tremor and ataxia in STXBP1.18 The validation, harmonization and integration of real-world data from various healthcare resources and disease registries will remain critical in the future as these resources have only been used recently in the context of research studies. Accordingly, future prospective natural history studies may benefit from integration of a comprehensive range of symptom domains in affected individuals and expand upon the windows for which meaningful improvements can be measured.
Conclusion
The clinical heterogeneity of STXBP1-related disorders has historically posed challenges in delineating longitudinal trajectories and end points. Here, we established a baseline to understand the disease trajectory in STXBP1-related disorders, which is critical for tailored care. We identified treatment windows and outcome measures that will facilitate interpretation of efficacy for future therapeutic strategies and developed a more precise understanding of the current landscape of treatment strategies. This information provides a framework focused on quantifying clinical outcomes for assessing longitudinal phenotypes in STXBP1-related disorders.
Data availability
Primary data are available in the Supplementary material. All code is available at github.com/helbig-lab/STXBP1-NHS.
Acknowledgements
We would like to thank the individuals and families with STXBP1-related disorders who participated in our study.
Funding
I.H. was supported by The Hartwell Foundation through an Individual Biomedical Research Award. This work was also supported by the National Institute for Neurological Disorders and Stroke (R01 NS127830-01A1, R01 NS131512-01 and K02 NS112600), the Eunice Kennedy Shriver National Institute of Child Health and Human Development through the Intellectual and Developmental Disabilities Research Center (IDDRC) at Children’s Hospital of Philadelphia and the University of Pennsylvania (U54 HD086984), and by intramural funds of the Children’s Hospital of Philadelphia through the Epilepsy NeuroGenetics Initiative (ENGIN). Research reported in this publication was also supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR001878. This project was also supported in part by the Institute for Translational Medicine and Therapeutics’ (ITMAT) Transdisciplinary Program in Translational Medicine and Therapeutics at the Perelman School of Medicine of the University of Pennsylvania. The study also received support through the EuroEPINOMICS-Rare Epilepsy Syndrome (RES) Consortium, which provided the capacity for exome sequencing, by the German Research Foundation (HE5415/3-1 to I.H.) within the EuroEPINOMICS framework of the European Science Foundation, by the German Research Foundation (DFG; HE5415/5-1, HE5415/6-1 to I.H.) by the DFG/FNR INTER Research Unit FOR2715 (We4896/4-570 1, and He5415/7-1 to I.H.), and by the Genomics Research and Innovation Network (GRIN, grinnetwork.org).
Competing interests
The authors report no competing interests.
Supplementary material
Supplementary material is available at Brain online.