Investigation of the Effect of Peptide p5 Targeting CDK5-p25 Hyperactivity on Munc18-1 (P67) Regulating Neuronal Exocytosis Using Molecular Simulations.

Munc18-1 is an SM (sec1/munc-like) family protein involved in vesicle fusion and neuronal exocytosis. Munc18-1 is known to regulate the exocytosis process by binding with closed- and open-state conformations of Syntaxin1, a protein belonging to the SNARE family established to be central to the exocytosis process. Our previous work studied peptide p5 as a promising drug candidate for CDK5-p25 complex, an Alzheimer's disease (AD) pathological target. Experimental in vivo and in vitro studies suggest that Munc18-1 promotes p5 to selectively inhibit the CDK5-p25 complex without affecting the endogenous CDK5 activity, a characteristic of remarkable therapeutic implications. In this paper, we identify several binding modes of p5 with Munc18-1 that could potentially affect the Munc18-1 binding with SNARE proteins and lead to off-target effects on neuronal communication using molecular dynamics simulations. Recent studies indicate that disruption of Munc18-1 function not only disrupts neurotransmitter release but also results in neurodegeneration, exhibiting clinical resemblance to other neurodegenerative conditions such as AD, causing diagnostic and treatment challenges. We characterize such interactions between p5 and Munc18-1, define the corresponding pharmacophores, and provide guidance for the in vitro validation of our findings to improve therapeutic efficacy and safety of p5.

A degranulation assay using Vγ9Vδ2 T cells for the rapid diagnosis of familial hemophagocytic syndromes.

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immune disorder characterized by uncontrolled lymphocyte and macrophage activation and a subsequent cytokine storm. The timely initiation of immunosuppressive treatment is crucial for survival. Methods: Here, we harnessed Vγ9Vδ2 T cell degranulation to develop a novel functional assay for the diagnosis of HLH. We compared the novel assay with the conventional natural killer (NK) cell stimulation method in terms of efficiency, specificity, and reliability. Our analysis involved 213 samples from 182 individuals, including 23 samples from 12 patients with degranulation deficiency (10 individuals with UNC13D deficiency, 1 with STXBP2 deficiency, and 1 with RAB27A deficiency). Results: While both tests exhibited 100% sensitivity, the Vγ9Vδ2 T cell degranulation assay showed a superior specificity of 86.2% (n=70) compared to the NK cell degranulation assay, which achieved 78.9% specificity (n=213). The Vγ9Vδ2 T cell degranulation assay offered simpler technical requirements and reduced labor intensity, leading to decreased susceptibility to errors with faster processing times. Discussion: This efficiency stemmed from the sole requirement of dissolving (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) powder, contrasting with the intricate maintenance of K562 cells necessary for the NK cell degranulation assay. With its diminished susceptibility to errors, we anticipate that the assay will require fewer repetitions of analysis, rendering it particularly well-suited for testing infants. Conclusion: The Vγ9Vδ2 T cell degranulation assay is a user-friendly, efficient diagnostic tool for HLH. It offers greater specificity, reliability, and practicality than established methods. We believe that our present findings will facilitate the prompt, accurate diagnosis of HLH and thus enable rapid treatment and better patient outcomes.

α-Synuclein pathology in Drosophila melanogaster is exacerbated by haploinsufficiency of Rop: connecting STXBP1 encephalopathy with α-synucleinopathies.

Syntaxin-binding protein 1 (STXBP1) is a presynaptic protein that plays important roles in synaptic vesicle docking and fusion. STXBP1 haploinsufficiency causes STXBP1 encephalopathy (STXBP1-E), which encompasses neurological disturbances including epilepsy, neurodevelopmental disorders, and movement disorders. Most patients with STXBP1-E present with regression and movement disorders in adulthood, highlighting the importance of a deeper understanding of the neurodegenerative aspects of STXBP1-E. An in vitro study proposed an interesting new role of STXBP1 as a molecular chaperone for α-Synuclein (αSyn), a key molecule in the pathogenesis of neurodegenerative disorders. However, no studies have shown αSyn pathology in model organisms or patients with STXBP1-E. In this study, we used Drosophila models to examine the effects of STXBP1 haploinsufficiency on αSyn-induced neurotoxicity in vivo. We demonstrated that haploinsufficiency of Ras opposite (Rop), the Drosophila ortholog of STXBP1, exacerbates compound eye degeneration, locomotor dysfunction, and dopaminergic neurodegeneration in αSyn-expressing flies. This phenotypic aggravation was associated with a significant increase in detergent-insoluble αSyn levels in the head. Furthermore, we tested whether trehalose, which has neuroprotective effects in various models of neurodegenerative disorders, mitigates αSyn-induced neurotoxicity exacerbated by Rop haploinsufficiency. In flies expressing αSyn and carrying a heterozygous Rop null variant, trehalose supplementation effectively alleviates neuronal phenotypes, accompanied by a decrease in detergent-insoluble αSyn in the head. In conclusion, this study revealed that Rop haploinsufficiency exacerbates αSyn-induced neurotoxicity by altering the αSyn aggregation propensity. This study not only contributes to understanding the mechanisms of neurodegeneration in STXBP1-E patients, but also provides new insights into the pathogenesis of α-synucleinopathies.

Differential SNARE chaperoning by Munc13-1 and Munc18-1 dictates fusion pore fate at the release site.

The regulated release of chemical messengers is crucial for cell-to-cell communication; abnormalities in which impact coordinated human body function. During vesicular secretion, multiple SNARE complexes assemble at the release site, leading to fusion pore opening. How membrane fusion regulators act on heterogeneous SNARE populations to assemble fusion pores in a timely and synchronized manner, is unknown. Here, we demonstrate the role of SNARE chaperones Munc13-1 and Munc18-1 in rescuing individual nascent fusion pores from their diacylglycerol lipid-mediated inhibitory states. At the onset of membrane fusion, Munc13-1 clusters multiple SNARE complexes at the release site and synchronizes release events, while Munc18-1 stoichiometrically interacts with trans-SNARE complexes to enhance N- to C-terminal zippering. When both Munc proteins are present simultaneously, they differentially access dynamic trans-SNARE complexes to regulate pore properties. Overall, Munc proteins' direct action on fusion pore assembly indicates their role in controlling quantal size during vesicular secretion.

Reduced Protein Stability of 11 Pathogenic Missense STXBP1/MUNC18-1 Variants and Improved Disease Prediction.

BACKGROUND: Pathogenic variants in STXBP1/MUNC18-1 cause severe encephalopathies that are among the most common in genetic neurodevelopmental disorders. Different molecular disease mechanisms have been proposed, and pathogenicity prediction is limited. In this study, we aimed to define a generalized disease concept for STXBP1-related disorders and improve prediction. METHODS: A cohort of 11 disease-associated and 5 neutral variants (detected in healthy individuals) were tested in 3 cell-free assays and in heterologous cells and primary neurons. Protein aggregation was tested using gel filtration and Triton X-100 insolubility. PRESR (predicting STXBP1-related disorder), a machine learning algorithm that uses both sequence- and 3-dimensional structure-based features, was developed to improve pathogenicity prediction using 231 known disease-associated variants and comparison to our experimental data. RESULTS: Disease-associated variants, but none of the neutral variants, produced reduced protein levels. Cell-free assays demonstrated directly that disease-associated variants have reduced thermostability, with most variants denaturing around body temperature. In addition, most disease-associated variants impaired SNARE-mediated membrane fusion in a reconstituted assay. Aggregation/insolubility was observed for none of the variants in vitro or in neurons. PRESR outperformed existing tools substantially: Matthews correlation coefficient = 0.71 versus <0.55. CONCLUSIONS: These data establish intrinsic protein instability as the generalizable, primary cause for STXBP1-related disorders and show that protein-specific ortholog and 3-dimensional information improve disease prediction. PRESR is a publicly available diagnostic tool.

SM protein Sly1 and a SNARE Habc domain promote membrane fusion through multiple mechanisms.

SM proteins including Sly1 are essential cofactors of SNARE-mediated membrane fusion. Using SNARE and Sly1 mutants and chemically defined in vitro assays, we separate and assess proposed mechanisms through which Sly1 augments fusion: (i) opening the closed conformation of the Qa-SNARE Sed5; (ii) close-range tethering of vesicles to target organelles, mediated by the Sly1-specific regulatory loop; and (iii) nucleation of productive trans-SNARE complexes. We show that all three mechanisms are important and operate in parallel, and that close-range tethering promotes trans-complex assembly when cis-SNARE assembly is a competing process. Further, we demonstrate that the autoinhibitory N-terminal Habc domain of Sed5 has at least two positive activities: it is needed for correct Sed5 localization, and it directly promotes Sly1-dependent fusion. "Split Sed5," with Habc presented solely as a soluble fragment, can function both in vitro and in vivo. Habc appears to facilitate events leading to lipid mixing rather than promoting opening or stability of the fusion pore.

SNARE chaperone Sly1 directly mediates close-range vesicle tethering.

The essential Golgi protein Sly1 is a member of the Sec1/mammalian Unc-18 (SM) family of SNARE chaperones. Sly1 was originally identified through remarkable gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory but also acts as a positive effector. An amphipathic lipid packing sensor (ALPS)-like helix within the loop directly binds high-curvature membranes. Membrane binding is required for relief of Sly1 autoinhibition and also allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 mutation bypasses requirements for diverse tethering factors but loses this ability if the tethering activity is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to initiate trans-SNARE complex assembly and fusion in the early secretory pathway.

Reduced synaptic depression in human neurons carrying homozygous disease-causing STXBP1 variant L446F.

MUNC18-1 is an essential protein of the regulated secretion machinery. De novo, heterozygous mutations in STXBP1, the human gene encoding this protein, lead to a severe neurodevelopmental disorder. Here, we describe the electrophysiological characteristics of a unique case of STXBP1-related disorder caused by a homozygous mutation (L446F). We engineered this mutation in induced pluripotent stem cells from a healthy donor (STXBP1LF/LF) to establish isogenic cell models. We performed morphological and electrophysiological analyses on single neurons grown on glial micro-islands. Human STXBP1LF/LF neurons displayed normal morphology and normal basal synaptic transmission but increased paired-pulse ratios and charge released, and reduced synaptic depression compared to control neurons. Immunostainings revealed normal expression levels but impaired recognition by a mutation-specific MUNC18-1 antibody. The electrophysiological gain-of-function phenotype is in line with earlier overexpression studies in Stxbp1 null mouse neurons, with some potentially human-specific features. Therefore, the present study highlights important differences between mouse and human neurons critical for the translatability of pre-clinical studies.

Accelerating therapeutic development and clinical trial readiness for STXBP1 and SYNGAP1 disorders.

Gene-targeted therapies for genetic neurodevelopmental disorders (NDDs) are becoming a reality. The Center for Epilepsy and Neurodevelopmental Disorders (ENDD) is currently focused on the development of therapeutics for STXBP1 and SYNGAP1 disorders. Here we review the known clinical features of these disorders, highlight the biological role of STXBP1 and SYNGAP1, and discuss our current understanding of pathogenic mechanisms and therapeutic development. Finally, we provide our perspective as scientists and parents of children with NDDs, and comment on the current challenges for both clinical and basic science endeavors.

Munc18c accelerates SNARE-dependent membrane fusion in the presence of regulatory proteins α-SNAP and NSF.

Intracellular vesicle fusion is driven by the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and their cofactors, including Sec1/Munc18 (SM), α-SNAP, and NSF. α-SNAP and NSF play multiple layers of regulatory roles in the SNARE assembly, disassembling the cis-SNARE complex and the prefusion SNARE complex. How SM proteins coupled with NSF and α-SNAP regulate SNARE-dependent membrane fusion remains incompletely understood. Munc18c, an SM protein involved in the exocytosis of the glucose transporter GLUT4, binds and activates target (t-) SNAREs to accelerate the fusion reaction through a SNARE-like peptide (SLP). Here, using an in vitro reconstituted system, we discovered that α-SNAP blocks the GLUT4 SNAREs-mediated membrane fusion. Munc18c interacts with t-SNAREs to displace α-SNAP, which overcomes the fusion inhibition. Furthermore, Munc18c shields the trans-SNARE complex from NSF/α-SNAP-mediated disassembly and accelerates SNARE-dependent fusion kinetics in the presence of NSF and α-SNAP. The SLP in domain 3a is indispensable in Munc18c-assisted resistance to NSF and α-SNAP. Together, our findings demonstrate that Munc18c protects the prefusion SNARE complex from α-SNAP and NSF, promoting SNARE-dependent membrane fusion through its SLP.

GABAergic/Glycinergic and Glutamatergic Neurons Mediate Distinct Neurodevelopmental Phenotypes of STXBP1 Encephalopathy.

An increasing number of pathogenic variants in presynaptic proteins involved in the synaptic vesicle cycle are being discovered in neurodevelopmental disorders. The clinical features of these synaptic vesicle cycle disorders are diverse, but the most prevalent phenotypes include intellectual disability, epilepsy, movement disorders, cerebral visual impairment, and psychiatric symptoms ( Verhage and Sørensen, 2020; Bonnycastle et al., 2021; John et al., 2021; Melland et al., 2021). Among this growing list of synaptic vesicle cycle disorders, the most frequent is STXBP1 encephalopathy caused by de novo heterozygous pathogenic variants in syntaxin-binding protein 1 (STXBP1, also known as MUNC18-1; Verhage and Sørensen, 2020; John et al., 2021). STXBP1 is an essential protein for presynaptic neurotransmitter release. Its haploinsufficiency is the main disease mechanism and impairs both excitatory and inhibitory neurotransmitter release. However, the disease pathogenesis and cellular origins of the broad spectrum of neurological phenotypes are poorly understood. Here we generate cell type-specific Stxbp1 haploinsufficient male and female mice and show that Stxbp1 haploinsufficiency in GABAergic/glycinergic neurons causes developmental delay, epilepsy, and motor, cognitive, and psychiatric deficits, recapitulating majority of the phenotypes observed in the constitutive Stxbp1 haploinsufficient mice and STXBP1 encephalopathy. In contrast, Stxbp1 haploinsufficiency in glutamatergic neurons results in a small subset of cognitive and seizure phenotypes distinct from those caused by Stxbp1 haploinsufficiency in GABAergic/glycinergic neurons. Thus, the contrasting roles of excitatory and inhibitory signaling reveal GABAergic/glycinergic dysfunction as a key disease mechanism of STXBP1 encephalopathy and suggest the possibility to selectively modulate disease phenotypes by targeting specific neurotransmitter systems.

The DDHD2-STXBP1 interaction mediates long-term memory via generation of saturated free fatty acids.

The phospholipid and free fatty acid (FFA) composition of neuronal membranes plays a crucial role in learning and memory, but the mechanisms through which neuronal activity affects the brain's lipid landscape remain largely unexplored. The levels of saturated FFAs, particularly of myristic acid (C14:0), strongly increase during neuronal stimulation and memory acquisition, suggesting the involvement of phospholipase A1 (PLA1) activity in synaptic plasticity. Here, we show that genetic ablation of the PLA1 isoform DDHD2 in mice dramatically reduces saturated FFA responses to memory acquisition across the brain. Furthermore, DDHD2 loss also decreases memory performance in reward-based learning and spatial memory models prior to the development of neuromuscular deficits that mirror human spastic paraplegia. Via pulldown-mass spectrometry analyses, we find that DDHD2 binds to the key synaptic protein STXBP1. Using STXBP1/2 knockout neurosecretory cells and a haploinsufficient STXBP1+/- mouse model of human early infantile encephalopathy associated with intellectual disability and motor dysfunction, we show that STXBP1 controls targeting of DDHD2 to the plasma membrane and generation of saturated FFAs in the brain. These findings suggest key roles for DDHD2 and STXBP1 in lipid metabolism and in the processes of synaptic plasticity, learning, and memory.

Disease-linked mutations in Munc18-1 deplete synaptic Doc2.

Heterozygous de novo mutations in the neuronal protein Munc18-1/STXBP1 cause syndromic neurological symptoms, including severe epilepsy, intellectual disability, developmental delay, ataxia and tremor, summarized as STXBP1 encephalopathies. Although haploinsufficiency is the prevailing disease mechanism, it remains unclear how the reduction in Munc18-1 levels causes synaptic dysfunction in disease as well as how haploinsufficiency alone can account for the significant heterogeneity among patients in terms of the presence, onset and severity of different symptoms. Using biochemical and cell biological readouts on mouse brains, cultured mouse neurons and heterologous cells, we found that the synaptic Munc18-1 interactors Doc2A and Doc2B are unstable in the absence of Munc18-1 and aggregate in the presence of disease-causing Munc18-1 mutants. In haploinsufficiency-mimicking heterozygous knockout neurons, we found a reduction in Doc2A/B levels that is further aggravated by the presence of the disease-causing Munc18-1 mutation G544D as well as an impairment in Doc2A/B synaptic targeting in both genotypes. We also demonstrated that overexpression of Doc2A/B partially rescues synaptic dysfunction in heterozygous knockout neurons but not heterozygous knockout neurons expressing G544D Munc18-1. Our data demonstrate that STXBP1 encephalopathies are not only characterized by the dysfunction of Munc18-1 but also by the dysfunction of the Munc18-1 binding partners Doc2A and Doc2B, and that this dysfunction is exacerbated by the presence of a Munc18-1 missense mutant. These findings may offer a novel explanation for the significant heterogeneity in symptoms observed among STXBP1 encephalopathy patients.

Early life seizures and epileptic spasms in STXBP1-related disorders.

OBJECTIVE: Individuals with disease-causing variants in STXBP1 frequently have epilepsy onset in the first year of life with a variety of seizure types, including epileptic spasms. However, the impact of early onset seizures and antiseizure medication (ASM) on the risk of developing epileptic spasms and impact on their trajectory are poorly understood, limiting informed and anticipatory treatment, as well as trial design. METHODS: We retrospectively reconstructed seizure and medication histories in weekly intervals for individuals with STXBP1 developmental and epileptic encephalopathy (DEE) with epilepsy onset in the first year of life and quantitatively analyzed longitudinal seizure histories and medication response. RESULTS: We included 61 individuals with early onset seizures, 29 of whom had epileptic spasms. Individuals with neonatal seizures were likely to have continued seizures after the neonatal period (25/26). The risk of developing epileptic spasms was not increased in individuals with neonatal seizures or early infantile seizures (21/41 vs. 8/16, odds ratio [OR] = 1, 95% confidence interval [CI] = .3-3.9, p = 1). We did not find any ASM associated with the development of epileptic spasms following prior seizures. Individuals with prior seizures (n = 16/21, 76%) had a higher risk of developing refractory epileptic spasms (n = 5/8, 63%, OR = 1.9, 95% CI = .2-14.6, p = .6). Individuals with refractory epileptic spasms had a later onset of epileptic spasms (n = 20, median = 20 weeks) compared to individuals with nonrefractory epileptic spasms (n = 8, median = 13 weeks, p = .08). SIGNIFICANCE: We provide a comprehensive assessment of early onset seizures in STXBP1-DEE and show that the risk of epileptic spasms is not increased following a prior history of early life seizures, nor by certain ASMs. Our study provides baseline information for targeted treatment and prognostication in early life seizures in STXBP1-DEE.

Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2.

Munc18-interacting proteins (Mints) are multidomain adaptors that regulate neuronal membrane trafficking, signaling, and neurotransmission. Mint1 and Mint2 are highly expressed in the brain with overlapping roles in the regulation of synaptic vesicle fusion required for neurotransmitter release by interacting with the essential synaptic protein Munc18-1. Here, we have used AlphaFold2 to identify and then validate the mechanisms that underpin both the specific interactions of neuronal Mint proteins with Munc18-1 as well as their wider interactome. We found that a short acidic α-helical motif within Mint1 and Mint2 is necessary and sufficient for specific binding to Munc18-1 and binds a conserved surface on Munc18-1 domain3b. In Munc18-1/2 double knockout neurosecretory cells, mutation of the Mint-binding site reduces the ability of Munc18-1 to rescue exocytosis, and although Munc18-1 can interact with Mint and Sx1a (Syntaxin1a) proteins simultaneously in vitro, we find that they have mutually reduced affinities, suggesting an allosteric coupling between the proteins. Using AlphaFold2 to then examine the entire cellular network of putative Mint interactors provides a structural model for their assembly with a variety of known and novel regulatory and cargo proteins including ADP-ribosylation factor (ARF3/ARF4) small GTPases and the AP3 clathrin adaptor complex. Validation of Mint1 interaction with a new predicted binder TJAP1 (tight junction-associated protein 1) provides experimental support that AlphaFold2 can correctly predict interactions across such large-scale datasets. Overall, our data provide insights into the diversity of interactions mediated by the Mint family and show that Mints may help facilitate a key trigger point in SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) complex assembly and vesicle fusion.

Sec1 regulates intestinal mucosal immunity in a mouse model of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a common immune-mediated condition with its molecular pathogenesis remaining to be fully elucidated. This study aimed to deepen our understanding of the role of FUT2 in human IBD, by studying a new surrogate gene Sec1, a neighboring gene of Fut2 and Fut1 that co-encodes the α 1,2 fucosyltransferase in mice. CRISPR/Cas9 was used to prepare Sec1 knockout (Sec1-/-) mice. IBD was induced in mice using 3% w/v dextran sulphate sodium. Small interfering RNA (siRNA) was employed to silence Sec1 in murine colon cancer cell lines CT26.WT and CMT93. IBD-related symptoms, colonic immune responses, proliferation and apoptosis of colon epithelial cells were assessed respectively to determine the role of Sec1 in mouse IBD. Impact of Sec1 on the expression of death receptor 5 (DR5) and other apoptosis-associated proteins were determined. Sec1 knockout was found to be associated with deterioration of IBD in mice and elevated immune responses in the colonic mucosa. Silencing Sec1 in CT26.WT and CMT93 cells led to greater secretion of inflammatory cytokines IL-1ß, IL-6 and TNF-α. Cell counting kit 8 (CCK8) assay, flow cytometry and TUNEL detection suggested that Sec1 expression promoted the proliferation of colon epithelial cells, inhibited cell apoptosis, reduced cell arrest in G0/G1 phase and facilitated repair of inflammatory injury. Over-expression of DR5 and several apoptosis-related effector proteins was noticed in Sec1-/- mice and Sec1-silenced CT26.WT and CMT93 cells, supporting a suppressive role of Sec1 in cell apoptosis. Our results depicted important regulatory roles of Sec1 in mouse IBD, further reflecting the importance of FUT2 in the pathogenesis of human IBD.

Microcircuit failure in STXBP1 encephalopathy leads to hyperexcitability.

De novo mutations in STXBP1 are among the most prevalent causes of neurodevelopmental disorders and lead to haploinsufficiency, cortical hyperexcitability, epilepsy, and other symptoms in people with mutations. Given that Munc18-1, the protein encoded by STXBP1, is essential for excitatory and inhibitory synaptic transmission, it is currently not understood why mutations cause hyperexcitability. We find that overall inhibition in canonical feedforward microcircuits is defective in a P15-22 mouse model for Stxbp1 haploinsufficiency. Unexpectedly, we find that inhibitory synapses formed by parvalbumin-positive interneurons were largely unaffected. Instead, excitatory synapses fail to recruit inhibitory interneurons. Modeling confirms that defects in the recruitment of inhibitory neurons cause hyperexcitation. CX516, an ampakine that enhances excitatory synapses, restores interneuron recruitment and prevents hyperexcitability. These findings establish deficits in excitatory synapses in microcircuits as a key underlying mechanism for cortical hyperexcitability in a mouse model of Stxbp1 disorder and identify compounds enhancing excitation as a direction for therapy.

A Case of Fetal Familial Hemophagocytic Lymphohistiocytosis Type 5 caused by STXBP2 Gene Mutation.

BACKGROUND: Familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) is a rare hyper-inflammatory syndrome caused by mutations in STXBP2. Most cases present at 2 - 6 months of age, and FHL-5 is extremely rare in neonates. METHODS: Appropriate laboratory tests, abdominal ultrasonography and whole exome sequencing were carried out. Respiratory support, antibiotics, and transfusion of blood products were done. RESULTS: Laboratory tests revealed metabolic acidosis, thrombocytopenia, mild anemia, and low fibrinogen level. Blood culture, metagenomics, and TORCH screening were negative. Liver and spleen enlargements were confirmed by abdominal ultrasonography. Whole exome sequencing identified a homozygous mutation in STXBP2 c. 1432del G (p. V478Sfs*5). The heterozygous STXBP2 mutation was identified in the paternal grandfather, maternal grandfather, and parents. CONCLUSIONS: Here we report a case with a novel homozygous deletion in exon 16 of STXBP2, which caused the earliest reported case of FHL-5 in a neonate. Our results identify a new pathogenic variant for the early identification and clinical consultation of FHL-5.

STXBP1-Related Disorders: Clinical Presentation, Molecular Function, Treatment, and Future Directions.

In recent years, the affordability and availability of genetic testing have led to its increased use in clinical care. The increased frequency of testing has led to STXBP1 variants being identified as one of the more common variants associated with neurological disorders. In this review, we aim to summarize the common clinical phenotypes associated with STXBP1 pathogenic variants, provide an overview of their known natural history, and discuss current research into the genotype to phenotype correlation. We will also provide an overview of the suspected normal function of the STXBP1-encoded Munc18-1 protein, animal models, and experimental techniques that have been developed to study its function and use this information to try to explain the diverse phenotypes associated with STXBP1-related disorders. Finally, we will explore current therapies for STXBP1 disorders, including an overview of treatment goals for STXBP1-related disorders, a discussion of the current evidence for therapies, and future directions of personalized medications for STXBP1-related disorders.

Delineating clinical and developmental outcomes in STXBP1-related disorders.

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.