CTNNB1 Syndrome is a severe neurodevelopmental disorder caused by haploinsufficiency of the CTNNB1 gene, which encodes β-catenin, a central regulatory protein of the canonical Wnt signaling and nervous systems development. Today affected patients experience global developmental delays (GDD), intellectual disability, and progressive motor impairments, with no disease-modifying treatment available. We developed and evaluated the first gene therapy targeting CTNNB1 Syndrome based on recombinant adeno-associated virus (rAAV). Six therapeutic cassettes were engineered and screened in patient-derived cortical brain organoids, identifying the lead candidate that restores β-catenin function with downstream pathway normalization. The selected vector was evaluated in a mouse model recapitulating motor and behavioral symptoms of the CTNNB1 Syndrome patients. To do so, juvenile CTNNB1 Syndrome mice received escalating unilateral intracerebroventricular doses of the vector. Mice treated with the highest dose showed improved locomotor and anxiety-related behaviors, with no adverse effects over eight months. Drug material for toxicology and biodistribution studies was manufactured under current Good Manufacturing Practice (cGMP) conditions. Safety, biodistribution and tolerability were further assessed in wild-type mice and non-human primates (NHPs), of which some mice toxicology and biodistribution analyses were conducted under Good Laboratory Practice (GLP) and in compliance with the European Medicines Agency (EMA) regulatory requirements. Following infusion of animals with the drug candidate, no treatment-related clinical, biochemical or histopathological adverse findings were observed and the No Observed Adverse Effect Level (NOAEL) was established at the highest vector dose. These findings support the preclinical safety and therapeutic efficacy in AAV9-mediated β-catenin gene augmentation therapy, paving the way for a clinical trial in Europe, already initiated in December 2025.

Children with autism spectrum disorder (ASD) exhibit gut mucosal immune alterations, but the co-regulatory architecture linking stool immune proteins and cytokines within the same cohort remains unstudied. In 115 children (74 ASD, 41 controls; age 5-18 years), seven stool immune proteins (IgA subclasses, α1-antitrypsin and calprotectin subunits) were quantified by UHPLC-MS/MS and ten by Luminex-chemokines eotaxin/CCL11 and IL-8/CXCL8 plus eight cytokines-each normalised to total protein. Age-adjusted partial Spearman correlations were computed for all 70 protein-cytokine pairs per stratum, using Benjamini-Hochberg correction, bootstrap confidence intervals and Fisher r-to-z tests. No pair survived FDR correction in any stratum. IgA1 and IL-1β/TP correlated positively across all strata (full cohort ρ = 0.409, 95% CI 0.131-0.634, n = 62; controls 0.583; ASD 0.210), with no significant between-group difference (Fisher z = 1.70, p = 0.090). Multiple imputation attenuated this to ρ = 0.265 (95% CI 0.055-0.452). An inverse trend between IL-1β/TP and CARS score (ρ = -0.336, n = 41) did not survive correction (p-FDR = 0.576). This power-limited, hypothesis-generating study identifies an exploratory IgA1-IL-1β mucosal axis present across groups, with no confirmed between-group difference. Adequately powered multi-centre studies are required.

Autism spectrum disorder (ASD) is a neurodevelopmental condition associated with metabolic and environmental factors. We investigated associations between urinary tryptophan-pathway metabolites and essential/toxic trace elements in children with ASD and healthy controls. In a cross-sectional cohort of 216 children (149 ASD, 67 controls), urinary tryptophan metabolites were quantified by LC-MS/MS and normalized to creatinine. Trace elements were assessed by ICP-MS. Matching yielded 1:1 (n = 57/57) and 1:2 (n = 30/60) age- and sex-matched subsets. Correlations (Pearson or Spearman, FDR-adjusted) and group comparisons were performed; autism severity (CARS) was analyzed within ASD. Creatinine-normalized tryptamine, 5-hydroxyindoleacetic acid, and N-acetyltryptophan showed moderate, positive correlations with essential elements (Mg, Zn, Se; r ≈ 0.5-0.7; N-acetyltryptophan and IAA correlated modestly with toxic elements (Tl, Cs; r ≈ 0.3-0.4). Group differences in individual metabolites and elements were modest; however, the composite toxic element index was significantly lower in ASD (P = .002). CARS scores did not show robust, FDR-corrected associations. Essential trace elements are closely linked to tryptophan metabolism, suggesting cofactor-dependent modulation in ASD. N-acetyltryptophan may serve as a sensor for specific toxic elements. Intervention studies are warranted to clarify causality.

Background: Immune dysregulation and gut dysbiosis are increasingly implicated in autism spectrum disorder (ASD), but compartment-specific intestinal cytokine profiles remain poorly defined. Aim: To characterize stool cytokine profiles and pro-/anti-inflammatory balance in children with ASD across development. Methods: We analyzed stool samples from 283 children (109 controls, 104 mild ASD, 70 severe ASD; age 0.9–21.5 years) recruited at a tertiary centre. Nine cytokines (IFN-γ, IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17, TNF-α) were measured using a Luminex multi-plex assay; IL-15 was excluded due to >70% missing values. Group comparisons used Mann–Whitney U tests, with age stratification at the cohort median (≤9.5 vs. >9.5 years). A composite pro-/anti-inflammatory ratio (IL-1α + IL-1β + IL-6 + IL-8 + IL-17 + TNF-α + IFN-γ divided by IL-4 + IL-10) was calculated. Results: In the overall cohort, stool IL-8 and IL-4 were significantly decreased in ASD versus controls (IL-8: median 0.36 vs. 0.49 ng/L, p = 0.0041; IL-4: 0.28 vs. 0.30 ng/L, p = 0.0316), with a graded reduction from controls to mild and severe ASD. Age-stratified analysis revealed that IL-8 reduction was confined to younger children (≤9.5 years; p = 0.0025) and absent in older children, while IL-1β was significantly reduced in younger ASD children and tended to reverse in older ASD children. The pro-/anti-inflammatory ratio was markedly elevated in severe ASD (median 491 vs. 209 in controls; p = 0.059), particularly in older children. Stool IL-8 and IL-1β correlated inversely with CARS scores within the ASD group. Conclusions: Children with ASD show decreased stool IL-8 and IL-4 and a shift toward a pro-inflammatory cytokine balance, with the strongest alterations during early childhood. These findings are consistent with the hypothesis of a developmental window of intestinal immune dysregulation in ASD, with stool IL-8 as the primary FDR-corrected finding. The present cross-sectional data do not establish causality and independent replication is required before clinical conclusions are drawn.

Objectives SATB2-associated syndrome (SAS) is a rare genetic condition characterized by developmental delay and typical features. Currently, an evidence-based clinical practice guideline (CPG) is being developed by European Reference Network ITHACA in close collaboration with the patient community. To ensure that the guideline addresses the most pressing concerns of affected individuals, families, and clinicians, while remaining feasible to produce, a prioritization process was carried out. Study Design and Setting The prioritization process aimed to minimize the relevant clinical questions to a maximum of 12, based on criteria that were defined beforehand and based on input from a large patient community. The prioritization process included a SAS community-wide survey that collected all patient-relevant topics, a prioritization round using a tool that helps to calculate the items that were most voted on, and a final consensus round with the guideline core group. Results In the first round, a total of 376 topics was collected based on input from over 20 families. These were combined and refined into 48 clinical topics. A total of 269 valid responders filled in their prioritization on these topics in an online survey. Of these respondents, 234 identified as representative/family/carer, 30 identified as clinician, and 5 individuals identified as both. Rather than prioritizing a subset of 12 topics, that each would be answered with a systematic review, the core group decided on a final set of 22 questions and only 1 systematic review. Conclusion Despite using a rigorous, community-driven process with input from many parents, carers, and global clinical experts, the guideline core group could not agree on a final set of 12 clinical topics. The group concluded that completeness and clinical usability of the guideline should take precedence over adhering to 12 prioritized clinical topics that could each be answered with a systematic search in the literature. We concluded that completeness and usability, vs methodological rigor, are competing interests in CPG development. This methodological issue is a pressing matter in the field of rare disease CPG development, and possibly also beyond the context of rare diseases, for which no clear solution currently exists. Plain Language Summary SATB2-associated syndrome (SAS) is a rare genetic condition that causes developmental delays and other characteristic features. A new clinical practice guideline (CPG) for SAS is currently being developed by the European Reference Network ITHACA, together with families, caregivers, and health-care professionals. To make sure the guideline focuses on the most important needs of the SAS community, a structured process was used to decide which clinical topics should be included. First, families across the SAS community were asked to share their concerns. This resulted in 376 topics, which were combined into 48 broader clinical themes. Next, 269 people completed an online survey to rank these topics. Most respondents were family members or caregivers, and some were clinicians. Finally, the guideline development team reviewed the results and discussed which topics should be prioritized. Although the original goal was to select 12 questions to answer through systematic reviews, the team decided that limiting the guideline to only 12 topics would leave out important issues. Instead, they agreed on 22 key questions, which would be addressed with only 1 systematic review. The CPG recommendations would then be more complete but supported with less systematically searched evidence. This experience highlights an ongoing challenge in rare disease guideline development: balancing completeness and practicality with strict methodological standards.

Background: Autism spectrum disorder (ASD) arises from complex gene–environment interactions. While trace element abnormalities have been studied, associations with autism severity remain inconsistent. Ratios indicating detoxification balance, rather than single toxic elements, may better reflect severity. Objective: To examine the relationships between urinary trace element levels, detoxification-related element ratios, and autism severity measured by the Childhood Autism Rating Scale (CARS). Methods: In a cross-sectional study of 168 participants (103 ASD, 65 controls), thirty urinary trace elements were quantified by ICP-MS. ASD patients were stratified by CARS into subthreshold ASD (n = 29), mild–moderate ASD (n = 36), and severe ASD (n = 38). Analyses included Mann–Whitney U, Kruskal–Wallis, and Spearman correlation tests, focusing on Li/Pb, Cu/Pb, and Cr/Pb ratios. Results: Individual elements showed weak associations with CARS; lead correlated positively (ρ = 0.209, p = 0.035) and lithium inversely (ρ = −0.194, p = 0.051). In contrast, element ratios showed stronger links: Li/Pb (ρ = −0.349, p = 0.0003), Cu/Pb (ρ = −0.320, p = 0.0011), and Cr/Pb (ρ = −0.209, p = 0.035). Severe ASD exhibited modest 90th-percentile elevations for toxic elements but high heterogeneity. Conclusions: Single-element levels showed limited associations with ASD severity. Element ratios, particularly Li/Pb, showed stronger statistical associations than individual elements in this cross-sectional dataset; however, these findings should be interpreted as candidate correlates rather than causal or clinically validated biomarkers.

Objective:Primary headaches are a prevalent health concern among children and adolescents, often affecting their quality of life. Emerging evidence suggests that hydration status may play a role in primary headache occurrence and severity. This study aimed to evaluate the impact of hydration on headache characteristics in children and adolescents with primary headaches. Methods:A total of 60 children and adolescents diagnosed with primary headaches were enrolled from the outpatient Headache Clinic of the Department of Paediatric Neurology at the University Children's Hospital, University Medical Centre Ljubljana, Slovenia, between May and September 2023. Participants received personalised hydration recommendations based on established Slovenian dietary guidelines and guidance on appropriate fluid choices. The primary outcome was headache frequency, intensity, and duration, measured at baseline and after the four-month intervention using a headache questionnaire. Fluid intake was assessed using a Food Frequency Questionnaire, while hydration status was evaluated in a sub-group (n=28) using bioelectrical impedance analysis. Results:Following the four-month intervention, participants demonstrated a significant increase in daily fluid intake from 5-8 glasses/day (one glass=200 mL) to 8-10 glasses/day (p<0.001), which was associated with a reduction in primary headache frequency (1-2 times/week to 3 times/month; p<0.001) and headache intensity (7/10-5/10; p<0.001). However, no significant effect was observed on headache duration (3 hours/headache to 2 hours/headache; p=0.19). Following the intervention, hydration status improved, as evidenced by significant increases in median total body water and extracellular water (p<0.001 and p=0.007, respectively), while the total body water to extracellular water ratio remained unchanged. Conclusions:This study provides novel evidence supporting adequate fluid intake and hydration as a non-pharmacological intervention for reducing primary headache frequency and intensity in paediatric populations.

BACKGROUND:Congenital myasthenic syndromes (CMS) caused by pathogenic variants in CHRND, encoding the δ-subunit of the nicotinic acetylcholine receptor (AChR), are rare, and data on genotype-phenotype correlations and long-term outcomes are limited. METHODS:We performed a retrospective, multicenter study of nine patients with genetically confirmed CHRND-related CMS from specialized neuromuscular centers. Clinical, electrophysiological, genetic, and therapeutic data were systematically collected. All diagnoses were established by exome sequencing during routine clinical work-up. RESULTS:Eight patients were compound heterozygous and one was homozygous for pathogenic CHRND variants, including nonsense, missense, splice-site variants, and one microdeletion. Disease onset ranged from the neonatal period (n = 7) to adolescence (n = 2). Three patients were followed longitudinally for 22-43 years. Ocular involvement, particularly ptosis and ophthalmoparesis, was present in all patients. Generalized fatigable weakness was common, whereas bulbar and respiratory involvement occurred in a subset and reflected overall disease severity. Genotypes including a null allele or a homozygous missense variant tended to be associated with more severe phenotypes, while compound heterozygous missense variants were linked to a broader and generally milder spectrum, sometimes limited to ocular symptoms. Long-term outcomes ranged from minimal symptoms under therapy to severe motor impairment with respiratory insufficiency, highlighting substantial interindividual variability. CONCLUSIONS:This study expands the phenotypic and genotypic spectrum of CHRND-related CMS and underscores the critical role of genotype in determining disease severity. Comprehensive genetic testing, longitudinal phenotyping, and genotype-informed management are essential for optimal diagnosis and care in this rare disorder.

PURPOSE:To describe an atypical presentation of COQ2-related retinopathy in identical twins with nephropathy, mimicking intermediate uveitis with cystoid macular oedema (CMO). METHODS:Retrospective case report. RESULTS:A 33-year-old man presented with bilateral vision worsening and suspected intermediate uveitis. Examination revealed vitreous cells, CMO, retinal microangiopathy, and severely abnormal electrooculography (EOG) with only borderline full-field electroretinography (ERG) changes. CMO worsened with topical corticosteroids but improved bilaterally after a single unilateral intravitreal bevacizumab injection, suggesting a systemic therapeutic effect. His identical twin exhibited very similar retinal and systemic findings. Whole-exome sequencing identified a homozygous likely pathogenic COQ2 variant (c.683A > G), confirming primary coenzyme Q10 (CoQ10) deficiency type 1. Both twins also had nephropathy consistent with focal segmental glomerulosclerosis (FSGS) but no neurological involvement. CONCLUSIONS:This report expands the phenotypic spectrum of COQ2-related retinopathy, characterized by retinal microangiopathy, CMO, and primary retinal pigment epithelium (RPE) dysfunction with preserved rod function, in contrast to the typical retinitis pigmentosa-like phenotype. Recognition of this presentation is critical, as early CoQ10 supplementation may stabilize disease progression and prevent systemic complications. Genetic testing should be considered in young patients with CMO resembling intermediate uveitis, particularly when associated with nephropathy.

Introduction:Onasemnogene abeparvovec is one of the three disease-modifying therapies available that can significantly improve the outcome of patients with 5q-spinal muscular atrophy. Therapy-induced thrombotic microangiopathy is an ultra-rare, but potentially life-threatening condition of not yet clearly defined aetiology. Case Presentation:A case of a 2-year-old patient with 5q-spinal muscular atrophy, who developed thrombotic microangiopathy after gene replacement therapy with onasemnogene abeparvovec, is described. This severe adverse event was promptly recognized and successfully treated with the complement C5 inhibitor. Conclusion:Thrombotic microangiopathy is an ultra-rare, but potentially life-threatening condition that can occur after onasemnogene abeparvovec therapy. Anticipation of these serious adverse events, its prompt recognition and treatment is crucial for a better outcome.

Introduction Some parents initiate treatment with non-prescription, home-made (unlabeled and unregulated) cannabis products for their children with refractory epilepsy without consulting their physicians. This study aimed to analyze the composition of these products in a pediatric cohort. Methods Home-made cannabis samples were analyzed for cannabinoid content, cannabidiol (CBD):Δ9-tetrahydrocannabinol (THC) ratio, and potential contaminants. Results Sixty home-made cannabis samples from 31 patients were analyzed. The most frequently detected cannabinoids were CBD, THC, and their acidic forms. The CBD:THC ratio varied significantly, with the most common ratio ranging from 1:1 to 10:1 (25.0%). Approximately 11.7% of the samples contained mainly CBD, 38.3% contained more THC than CBD, and 18.3% contained primarily THC with undetectable CBD levels. Conclusions Health professionals should strongly discourage the use of unregulated home-made cannabis products, as undisclosed and highly variable cannabinoid content with often elevated THC levels and potential contaminants present serious health risks for children with refractory epilepsy. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study did not receive any funding. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This observational questionnaire-based study was performed in accordance with the Declaration of Helsinki. This human study was approved by The National Medical Ethics Committee of the Republic of Slovenia - approval: 0120-131/2021/14. The results of the study can be found at https://repozitorij.uni-lj.si/IzpisGradiva.php?id=137168 (in Slovene). Parents were also invited to provide a sample of the substance they have used for the analysis and those who provided a verbal informed consent, participated in this analysis. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

CTNNB1 neurodevelopmental syndrome is a rare disorder caused by de novo heterozygous variants in the CTNNB1 gene encoding β-catenin. This study aimed to characterize genetic variants in individuals with CTNNB1 neurodevelopmental syndrome, systematically assess the spectrum of clinical phenotypes using standardized measures, and explore potential genotype-phenotype correlations. In this cross-sectional cohort study, individuals diagnosed with CTNNB1 neurodevelopmental syndrome underwent structured interviews using standardized scales to evaluate motor skills, speech, communication, feeding abilities, visual function, neurodevelopment, and psychopathology. Genetic variants were analyzed, and, in a subset of cases, the impact of β-catenin variants on the Wnt/β-catenin signaling pathway was assessed. Across the 127 included participants (mean age, 70 months; range, 7-242 months) from 20 countries, we identified 88 different variants of the CTNNB1 gene, 87 of which were predicted to lead to loss of CTNNB1 function. Functional assays demonstrated reduced Wnt signaling activity, including 11 variants that also exhibited a dominant-negative effect. One missense variant demonstrated a gain-of-function effect. Dominant-negative variants were not clearly associated with a distinct phenotype; however, those with missense variants presented a milder phenotype, including earlier achievement of independent walking, fewer motor impairments, better conceptual and social skills, improved communication, and fewer feeding difficulties. This study describes the genetic, functional, and phenotypic characteristics in individuals with CTNNB1 neurodevelopmental syndrome. Further investigation into the genotypic and phenotypic characteristics of this syndrome and their interrelationships is essential to deepen our understanding of the disorder and inform the development of targeted therapies.

AimsTo assess the prevalence of endocrine/metabolic disorders among pediatric Duchenne muscular dystrophy (DMD) patients and identify individuals who would benefit from the new corticosteroid treatment available, vamorolone.MethodsA national pediatric cohort of DMD patients followed at University Children’s Hospital, University Medical Center Ljubljana in June 2025 was included in the study. The presence of endocrine/metabolic disorders was determined by clinical examination, laboratory data, and imaging at the last annual multidisciplinary evaluation.ResultsA total of 21 patients (average age 11.1, range 4.6-16.7 yrs) were included in the study. Two patients were corticosteroid-naive, the rest treated with deflazacort (average treatment duration 5.4, range 1.1-11.3 yrs). At the time of final follow-up, 90% were diagnosed with an endocrine and/or metabolic disorder. Short stature was present in 67% of patients, a decline in height standard deviation score (SDS) since the initiation of corticosteroid therapy was observed, -2.37 SDS on average. Based on body composition data (fat % SDS), 57% were classified as obese. Lipid abnormalities were detected in 76% of patients. Insulin resistance (determined by HOMA-IR) was increased in 9.5% of patients; type 2 diabetes was not detected.ConclusionOur findings demonstrate a high prevalence of endocrine and metabolic disturbances among patients with DMD. In light of emerging evidence on the potential benefits of vamorolone - particularly regarding bone health and growth - we identified a subgroup of patients most likely to benefit from its use. We propose that ambulatory, non-corticosteroid naive patients with pathological fractures, markedly reduced bone density, short stature, or significant growth deceleration could be considered for the therapeutic transition. Accordingly, we established national clinical criteria to guide individualized therapeutic transitions, aimed at optimizing clinical outcomes and ensuring efficient allocation of healthcare resources.

The CTNNB1 Connect & Cure and CTNNB1 Foundation, alongside Asociación CTNNB1, CTNNB1 Italia, Association CTNNB1 France, and researchers and clinicians globally are dedicated to finding effective treatments and cures for CTNNB1 syndrome. The syndrome is also characterized by progressive spasticity, which can in some cases cause loss of already achieved motor milestones. Since 2019, they have brought together researchers from different fields and invested in various research efforts to advance the search for treatment solutions for patients with CTNNB1 syndrome. Simons Searchlight serves as an important platform by remotely collecting high-quality, standardized data on the natural history of the disease and making it available to researchers around the world. Conducting genotype–phenotype correlation study and biochemically characterizing the mutations were critical to understand the effects of the patients’ mutations and related molecular function to symptoms. Several induced pluripotent stem cells were generated from patient cells, and preclinical mouse models have provided new insights into the molecular downstream effects of CTNNB1 haploinsufficiency. Multiple therapeutic approaches are in the developing, including small molecule treatments, RNA- and DNA-based therapies, AAV9 gene replacement therapy, which entered the manufacturing phase in November 2023. In this article, we summarize the journey of the CTNNB1 community and its organizations, highlight ongoing and future research projects, and outline the available research resources. The vision for the CTNNB1 community is that in the future several therapeutic options will be available that can be customized to every CTNNB1 patient’s needs.