Abstract Pathogenic variants in RUBCN , encoding the Run domain Beclin-1 interacting and cysteine-rich domain-containing protein (Rubicon) have been implicated in autosomal recessive spinocerebellar ataxia 15 (SCAR15). However, the molecular mechanisms underlying disease pathogenesis remain poorly understood. Here, we report 18 individuals from 15 unrelated families harbouring biallelic RUBCN variants, who present with an aggressive neurodevelopmental disorder variably characterized by seizures, developmental delay, intellectual disability and movement abnormalities that cause regression, progressive brain atrophy and neurodegenerative features. Through functional characterization, we demonstrate that a subset of disease-associated putative truncating variants disrupt autophagy regulation. In Caenorhabditis elegans models, loss-of-function RUBCN variants result in an increased autophagic flux and impaired neuronal function, recapitulating key features in humans. Correspondingly, cellular assays reveal that nonsense and frameshift RUBCN variants lead to defective autophagy inhibition, underscoring a crucial role for RUBCN as a key negative autophagy regulator. Molecular dynamics simulations rank the eleven missense variants by structural effect, with p.Arg813Trp alone altering the target protein at both the local and the regional level and lying within the RAB7A-binding module that the truncating alleles remove altogether. Our findings establish and expand the RUBCN -related disorders as a clinically and molecularly distinct subset of autophagy-related diseases. By delineating both the genetic landscape and cellular consequences of Rubicon dysfunction, this study enhances our understanding of autophagy-related neurodevelopmental disorders and provides a foundation for future therapeutic investigations.

PURPOSE:XPO1 functions in key cellular processes, including nucleo-cytoplasmic export and mitosis. The gene is deleted in a subset of patients with the 2p15p16.1 microdeletion syndrome; however, no monogenic XPO1-related disorder has been described to date. METHODS:We collected clinical data of individuals with de novo XPO1 variants through online matchmaking. We used Drosophila to study XPO1 function in development and habituation learning. RESULTS:A total of 22 individuals met the criteria to be included in the main study cohort. Of these, half have putative loss-of-function variants, and half have coding variants (10 missense and 1 in-frame deletion variant). We found an overlapping phenotype, consistent with a monogenic neurodevelopmental disorder. We demonstrate XPO1 functions in development by ubiquitous and neuron-specific knockdown in Drosophila. GABAergic neuron specific knockdown flies demonstrated impaired habituation. CONCLUSION:Our results establish XPO1 as a novel dominant monogenic neurodevelopmental disorder gene and demonstrate a central role for XPO1 in development.

Pathogenic variants in ATP-dependent chromatin remodeling proteins are a recurrent cause of neurodevelopmental disorders (NDDs). The NURF complex consists of BPTF and either the SMARCA5 or SMARCA1 ISWI-chromatin remodeling enzyme. Pathogenic variants in BPTF and SMARCA5 have been previously implicated in NDDs. Here, we describe 35 individuals from 26 families with de novo or maternally inherited variants in the X-linked SMARCA1 gene. This SMARCA1-related NDD is associated with a spectrum of involvement, including mild to severe ID/DD, delayed or regressive speech development, ASD features, facial dysmorphisms, and other variable features. Individuals carrying SMARCA1 truncating variants exhibit a mildly unique genome-wide DNA methylation profile and a high penetrance of macrocephaly. Genetic dissection of the NURF complex using Smarca1, Smarca5, and Bptf single and double mouse knockouts reveals the importance of NURF composition and dosage for proper forebrain development. We propose that genetic alterations affecting different NURF components, including SMARCA1, result in a NDD with a broad clinical spectrum.

PURPOSE:A homozygous loss-of-function (LoF) variant in POC5 was previously described in an individual with retinitis pigmentosa. We identified POC5 variants in 12 probands with a syndromic phenotype. We aim to define the phenotype spectrum and molecular mechanism associated with biallelic POC5 LoF variants. METHODS:We studied a cohort of 12 families with bi-allelic LoF POC5 variants and performed detailed phenotype analysis. POC5 localization studies were performed in 3 proband-derived fibroblast cell lines. RESULTS:Detailed phenotyping of probands with POC5 variants expands the phenotype spectrum beyond ocular manifestations. This syndrome causes not only rod-cone dystrophy but also diabetes mellitus with severe insulin resistance and partial lipodystrophy, kidney disease, and muscle cramps. The POC5 protein plays an essential role during cell cycle and cilium formation. Interestingly, POC5 localization studies in 3 proband-derived fibroblast cell lines show aberrant localization suggesting a ciliary defect. The phenotypes of the 12 families in this study fit well within the ciliopathy phenotype spectrum, except for lipodystrophy, which is not common in ciliopathies. CONCLUSION:We describe a multiorgan syndrome caused by bi-allelic LoF variants in POC5. This underscores the pleiotropic effects of POC5 variants and highlights the significance of adipose tissue and metabolic dysfunction in ciliopathies.

Cilia are essential organelles, and variants in genes governing ciliary function result in ciliopathic diseases. The Ciliogenesis and PLANar polarity Effectors (CPLANE) protein complex is essential for ciliogenesis, and all but one subunit of the CPLANE complex have been implicated in human ciliopathy. Here, we identify three families in which variants in the remaining CPLANE subunit CPLANE2/RSG1 also cause ciliopathy. These patients display cleft palate, tongue lobulations and polydactyly, phenotypes characteristic of Oral-Facial-Digital Syndrome. We further show that these alleles disrupt two vital steps of ciliogenesis, basal body docking and recruitment of intraflagellar transport proteins. Moreover, APMS reveals that Rsg1 binds CPLANE and the transition zone protein Fam92 in a GTP-dependent manner. Finally, we show that CPLANE is generally required for normal transition zone architecture. Our work demonstrates that CPLANE2/RSG1 is a causative gene for human ciliopathy and also sheds new light on the mechanisms of ciliary transition zone assembly.

Alternative splicing is highly prevalent in the brain where it orchestrates key processes such as neurogenesis and synaptogenesis, both essential for the nervous system's complexity and plasticity. Dysregulation of splicing has increasingly been linked to neurodevelopmental disorders. Here, we describe unrelated individuals carrying de novo, likely deleterious heterozygous variants in Splicing Factor 1 (SF1), all presenting with neurodevelopmental disorders of variable severity, frequently accompanied by autistic traits and other non-specific features. SF1 is a core component of pre-mRNA processing, facilitating early spliceosome assembly at the 3' splice site and regulating alternative splicing. We conducted functional studies in neural progenitor cells, which showed that SF1 downregulation alters gene expression and alternative splicing programs, particularly in genes involved in neuronal differentiation, synaptic transmission, and axonal guidance, processes fundamental to brain development. Together, these findings establish SF1 dysfunction as an additional spliceosomopathy contributing to neurodevelopmental disorders and underscore its essential role in human neurodevelopment and disease.

Zinc and RING finger 3 (ZNRF3) is a negative-feedback regulator of Wnt/(3-catenin signaling, which plays an important role in human brain development. Although somatically frequently mutated in cancer, germline variants in ZNRF3 have not been established as causative for neurodevelopmental disorders (NDDs). We identified 12 individuals with ZNRF3 variants and various phenotypes via GeneMatcher/Decipher and evaluated genotype-phenotype correlation. We performed structural modeling and representative deleterious and control variants were assessed using in vitro transcriptional reporter assays with and without Wnt-ligand Wnt3a and/or Wnt-potentiator R-spondin (RSPO). Eight individuals harbored de novo missense variants and presented with NDD. We found missense variants associated with macrocephalic NDD to cluster in the RING ligase domain. Structural modeling predicted disruption of the ubiquitin ligase function likely compromising Wnt receptor turnover. Accordingly, the functional assays showed enhanced Wnt/(3-catenin signaling for these variants in a dominant negative manner. Contrarily, an individual with microcephalic NDD harbored a missense variant in the RSPO-binding domain predicted to disrupt binding affinity to RSPO and showed attenuated Wnt/(3-catenin signaling in the same assays. Additionally, four individuals harbored de novo truncating or de novo or inherited large in-frame deletion variants with non-NDD phenotypes, including heart, adrenal, or nephrotic problems. In contrast to NDD-associated missense variants, the effects on Wnt/(3-catenin signaling were comparable between the truncating variant and the empty vector and between benign variants and the wild type. In summary, we provide evidence for mirror brain size phenotypes caused by distinct pathomechanisms in Wnt/(3-catenin signaling through protein domain-specific deleterious ZNRF3 germline missense variants.

BACKGROUND AND OBJECTIVES:Heterozygous variants in RAR-related orphan receptor B (RORB) have recently been associated with susceptibility to idiopathic generalized epilepsy. However, few reports have been published so far describing pathogenic variants of this gene in patients with epilepsy and intellectual disability (ID). In this study, we aimed to delineate the epilepsy phenotype associated with RORB pathogenic variants and to provide arguments in favor of the pathogenicity of variants.METHODS:Through an international collaboration, we analyzed seizure characteristics, EEG data, and genotypes of a cohort of patients with heterozygous variants in RORB. To gain insight into disease mechanisms, we performed ex vivo cortical electroporation in mouse embryos of 5 selected variants, 2 truncating and 3 missense, and evaluated on expression and quantified changes in axonal morphology.RESULTS:We identified 35 patients (17 male, median age 10 years, range 2.5-23 years) carrying 32 different heterozygous variants in RORB, including 28 single-nucleotide variants or small insertions/deletions (12 missense, 12 frameshift or nonsense, 2 splice-site variants, and 2 in-frame deletions), and 4 microdeletions; de novo in 18 patients and inherited in 10. Seizures were reported in 31/35 (89%) patients, with a median age at onset of 3 years (range 4 months-12 years). Absence seizures occurred in 25 patients with epilepsy (81%). Nineteen patients experienced a single seizure type: absences, myoclonic absences, or absences with eyelid myoclonia and focal seizures. Nine patients had absence seizures combined with other generalized seizure types. One patient had presented with absences associated with photosensitive occipital seizures. Three other patients had generalized tonic-clonic seizures without absences. ID of variable degree was observed in 85% of the patients. Expression studies in cultured neurons showed shorter axons for the 5 tested variants, both truncating and missense variants, supporting an impaired protein function.DISCUSSION:In most patients, the phenotype of the RORB-related disorder associates absence seizures with mild-to-moderate ID. In silico and in vitro evaluation of the variants in our cohort, including axonal morphogenetic experiments in cultured neurons, supports their pathogenicity, showing a hypomorphic effect.

Large-scale next-generation sequencing (NGS) germline testing is technically feasible today, but variant interpretation represents a major bottleneck in analysis workflows. This includes extensive variant prioritization, annotation, and time-consuming evidence curation. The scale of the interpretation problem is massive, and variants of uncertain significance (VUSs) are a challenge to personalized medicine. This challenge is further compounded by the complexity and heterogeneity of the standards used to describe genetic variants and the associated phenotypes when searching for relevant information to support clinical decision making. To address this, all five Swiss academic institutions for Medical Genetics joined forces with the Swiss Institute of Bioinformatics (SIB) to create SwissGenVar as a user-friendly nationwide repository and sharing platform for genetic variant data generated during routine diagnostic procedures and research sequencing projects. Its aim is to provide a protected environment for expert evidence sharing about individual variants to harmonize and upscale their significance interpretation at the clinical grade according to international standards. To corroborate the clinical assessment, the variant-related data will be combined with consented high-quality clinical information. Broader visibility will be achieved by interfacing with international databases, thus supporting global initiatives in personalized healthcare.

Zinc and RING finger 3 (ZNRF3) is a negative-feedback regulator of Wnt/β-catenin signaling, which plays an important role in human brain development. Although somatically frequently mutated in cancer, germline variants in ZNRF3 have not been established as causative for neurodevelopmental disorders (NDDs). We identified 12 individuals with ZNRF3 variants and various phenotypes via GeneMatcher/Decipher and evaluated genotype-phenotype correlation. We performed structural modeling and representative deleterious and control variants were assessed using in vitro transcriptional reporter assays with and without Wnt-ligand Wnt3a and/or Wnt-potentiator R-spondin (RSPO). Eight individuals harbored de novo missense variants and presented with NDD. We found missense variants associated with macrocephalic NDD to cluster in the RING ligase domain. Structural modeling predicted disruption of the ubiquitin ligase function likely compromising Wnt receptor turnover. Accordingly, the functional assays showed enhanced Wnt/β-catenin signaling for these variants in a dominant negative manner. Contrarily, an individual with microcephalic NDD harbored a missense variant in the RSPO-binding domain predicted to disrupt binding affinity to RSPO and showed attenuated Wnt/β-catenin signaling in the same assays. Additionally, four individuals harbored de novo truncating or de novo or inherited large in-frame deletion variants with non-NDD phenotypes, including heart, adrenal, or nephrotic problems. In contrast to NDD-associated missense variants, the effects on Wnt/β-catenin signaling were comparable between the truncating variant and the empty vector and between benign variants and the wild type. In summary, we provide evidence for mirror brain size phenotypes caused by distinct pathomechanisms in Wnt/β-catenin signaling through protein domain-specific deleterious ZNRF3 germline missense variants.

BackgroundAs the technology of next generation sequencing rapidly develops and costs are constantly reduced, the clinical availability of whole genome sequencing (WGS) increases. Thereby, it remains unclear what exact advantage WGS offers in comparison to whole exome sequencing (WES) for the diagnosis of genetic diseases using current technologies. MethodsTrio-WGS was conducted for 20 patients with developmental or epileptic encephalopathies who remained undiagnosed after WES and chromosomal microarray analysis. ResultsA diagnosis was reached for four patients (20%). However, retrospectively all pathogenic variants could have been detected in a WES analysis conducted with today's methods and knowledge. ConclusionThe additional diagnostic yield of WGS versus WES is currently largely explained by new scientific insights and the general technological progress. Nevertheless, it is noteworthy that whole genome sequencing has greater potential for the analysis of small copy number and copy number neutral variants not seen with WES as well as variants in noncoding regions, especially as potentially more knowledge of the function of noncoding regions arises. We, therefore, conclude that even though today the added value of WGS versus WES seems to be limited, it may increase substantially in the future.

Next generation sequencing (NGS) can detect carrier status for rare recessive disorders, informing couples about their reproductive risk. The recent ACMG recommendations support offering NGS-based carrier screening (NGS-CS) in an ethnic and population-neutral manner for all genes that have a carrier frequency >1/200 (based on GnomAD). To evaluate current challenges for NGS-CS, we focused on the ciliopathies, a well-studied group of rare recessive disorders. We analyzed 118 ciliopathy genes by whole exome sequencing in ~400 healthy local individuals and ~1000 individuals from the UK1958-birth cohort. We found 20% of healthy individuals (1% of couples) to be carriers of reportable variants in a ciliopathy gene, while 50% (4% of couples) carry variants of uncertain significance (VUS). This large proportion of VUS is partly explained by the limited utility of the ACMG/AMP variant-interpretation criteria in healthy individuals, where phenotypic match or segregation criteria cannot be used. Most missense variants are thus classified as VUS and not reported, which reduces the negative predictive value of the screening test. We show how gene-specific variation patterns and structural protein information can help prioritize variants most likely to be disease-causing, for (future) functional assays. Even when considering only strictly pathogenic variants, the observed carrier frequency is substantially higher than expected based on estimated disease prevalence, challenging the 1/200 carrier frequency cut-off proposed for choice of genes to screen. Given the challenges linked to variant interpretation in healthy individuals and the uncertainties about true carrier frequencies, genetic counseling must clearly disclose these limitations of NGS-CS.

WDR5 is a broadly studied, highly conserved key protein involved in a wide array of biological functions. Among these functions, WDR5 is a part of several protein complexes that affect gene regulation via post-translational modification of histones. We collected data from 11 unrelated individuals with six different rare de novo germline missense variants in WDR5; one identical variant was found in five individuals and another variant in two individuals. All individuals had neurodevelopmental disorders including speech/language delays (n = 11), in-tellectual disability (n = 9), epilepsy (n = 7), and autism spectrum disorder (n = 4). Additional phenotypic features included abnormal growth parameters (n = 7), heart anomalies (n = 2), and hearing loss (n = 2). Three-dimensional protein structures indicate that all the residues affected by these variants are located at the surface of one side of the WDR5 protein. It is predicted that five out of the six amino acid substitutions disrupt interactions of WDR5 with RbBP5 and/or KMT2A/C, as part of the COMPASS (complex proteins associated with Set1) family complexes. Our experimental approaches in Drosophila melanogaster and human cell lines show normal protein expression, localization, and protein-protein interactions for all tested variants. These results, together with the clustering of variants in a specific region of WDR5 and the absence of truncating variants so far, suggest that dominant-negative or gain-of-function mechanisms might be at play. All in all, we define a neurodevelopmental disorder associated with missense variants in WDR5 and a broad range of features. This finding highlights the important role of genes encoding COMPASS family proteins in neurodevelopmental disorders.