Neurodevelopmental disorders (NDDs) affect 2-4% of the population, are predominantly genetic and remain unsolved in ~50% of individuals. We show that rare biallelic variants in RNU2-2 are enriched and over-transmitted in individuals with unresolved NDDs. We define a recessive RNU2-2 syndrome, delineate its unique genetic architecture and show that it manifests clinically as a severe developmental and epileptic encephalopathy. We find that candidate biallelic variants are significantly correlated with reduced U2-2 abundance, implicating compromised transcript stability as a probable pathomechanism. We identify a decreased ratio of U2-2 to its paralog U2-1 as a potential diagnostic biomarker for this condition. We show that the recessive RNU2-2 syndrome is genetically, clinically and mechanistically distinct from the dominant RNU2-2 disorder. Within our cohort, the recessive RNU2-2 syndrome emerges as by far the most frequent recessive NDD, greatly disproportionate to the small genomic footprint of this non-protein-coding gene.

Ceramides are essential skin lipids for maintaining the mammalian skin permeability barrier, which protects against external stimuli. The precursor of epidermal ceramides, glucosylceramides (GlcCer), is synthesized within granular keratinocytes while its precise cellular transport mechanisms remain poorly characterized. Here, we identified 3 pathogenic variants in the GLTP gene, which encodes glycolipid transfer protein, in 5 unrelated families with nonsyndromic epidermal differentiation disorder presenting with generalized skin scaling. The biallelic GLTPvariants resulted in loss of competent GLTP expression. CRISPR/Cas9-generated Gltp-knockout mice exhibited lethal barrier defects, partially recapitulating the clinical features of our patients. We demonstrated that GLTP facilitated GlcCer transport in differentiated keratinocytes, with its deficiency causing impaired GlcCer trafficking and consequent aberrant retention in lysosomes, thereby disrupting lysosome function. The lysosomal dysfunction impaired autophagy flux, resulting in delayed keratinocyte terminal differentiation, which is expected to compromise the skin barrier integrity and ultimately lead to abnormal scaling. Pharmacological inhibition of GlcCer synthesis effectively rescued both autophagy and keratinocyte differentiation defects. Our findings establish GLTP as a novel underlying gene for nonsyndromic epidermal differentiation disorders and unravel its essential role in maintaining skin homeostasis during terminal differentiation by mediating epidermal GlcCer transport.

PIGC encodes a protein essential for the biosynthesis of glycophosphatidylinositol-anchored proteins (GPI-APs). So far, three families with biallelic PIGC variants have been reported to exhibit developmental delay/intellectual disability and seizures. Our aim was to further elucidate the clinical and biomolecular characteristics of PIGC pathogenic or likely pathogenic variants. We established a cohort of 18 previously unreported probands. Clinical data were collected, and causative variants were identified though genome/exome sequencing. Variants were modelled in silico using AlphaFold2. Flow cytometry was performed to analyze the cell-surface expression of GPI-APs. The probands displayed a severe neurodevelopmental disorder characterized by developmental and cognitive impairment, early-onset and treatment-resistant seizures, and premature death affecting 10 out of 18 individuals (median age of 40 months, ranging from 40 days to 7 years). Additional features included brain imaging abnormalities (14/15), hypotonia (15/18), and skeletal anomalies (5/17). One patient exhibited mildly elevated alkaline phosphatase levels. All harbored biallelic PIGC variants, with 14 out of 18 of those being homozygous variants. Analysis of samples derived from probands and cellular models showed reduced cell surface levels of GPI-APs. This study confirms the association of PIGC biallelic variants with refractory seizures, severe developmental and cognitive impairments, and highlights their association with childhood-onset mortality. Additionally, it shows that dysfunctional PIGC results in defective biosynthesis of GPI-AP.

Genetic diagnosis of rare diseases requires accurate identification and interpretation of genomic variants. Clinical and molecular scientists from 37 expert centers across Europe created the Solve-Rare Diseases Consortium (Solve-RD) resource, encompassing clinical, pedigree and genomic rare-disease data (94.5% exomes, 5.5% genomes), and performed systematic reanalysis for 6,447 individuals (3,592 male, 2,855 female) with previously undiagnosed rare diseases from 6,004 families. We established a collaborative, two-level expert review infrastructure that allowed a genetic diagnosis in 506 (8.4%) families. Of 552 disease-causing variants identified, 464 (84.1%) were single-nucleotide variants or short insertions/deletions. These variants were either located in recently published novel disease genes (n = 67), recently reclassified in ClinVar (n = 187) or reclassified by consensus expert decision within Solve-RD (n = 210). Bespoke bioinformatics analyses identified the remaining 15.9% of causative variants (n = 88). Ad hoc expert review, parallel to the systematic reanalysis, diagnosed 249 (4.1%) additional families for an overall diagnostic yield of 12.6%. The infrastructure and collaborative networks set up by Solve-RD can serve as a blueprint for future further scalable international efforts. The resource is open to the global rare-disease community, allowing phenotype, variant and gene queries, as well as genome-wide discoveries.

Neurodevelopmental disorders (NDDs) affect 2-4% of the population, are predominantly genetic, and remain unsolved in ~50% of individuals. We show that rare biallelic variants in RNU2-2 are enriched and over-transmitted in individuals with unresolved NDDs. We define a novel recessive RNU2-2 syndrome, delineate its unique genetic architecture and show that clinically it manifests as a severe developmental epileptic encephalopathy. We find that candidate biallelic variants are significantly correlated with reduced U2-2 abundance, implicating compromised transcript stability as likely pathomechanism. We identify decreased ratio of U2-2 to its paralog U2-1 as a potential diagnostic biomarker for this condition. We show that the recessive RNU2-2 syndrome is genetically, clinically, and mechanistically distinct from the dominant RNU2-2 disorder. Within our cohort, the recessive RNU2-2 syndrome emerges as by far the most frequent recessive NDD, greatly disproportionate to the small genomic footprint of this non-protein coding gene. ### Competing Interest Statement L.M. has received personal consultancy fees from Mendelian Ltd., a rare disease digital health company, outside of the submitted work. No other authors have any conflicts to declare. ### Funding Statement A.J. and S.B. acknowledge the support of SolveRD. The SolveRD project has received funding from the European Unions Horizon 2020 research and innovation program under grant agreement 779257. This study has been delivered through the National Institute for Health and Care Research (NIHR) Manchester Biomedical Research Centre (NIHR203308). A.B. is supported by a Wellcome PhD Training Fellowship for Clinicians and the 4Ward North PhD Programme for Health Professionals (223521/Z/21/Z). This research was made possible through access to data in the National Genomic Research Library, which is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The National Genomic Research Library holds data provided by patients and collected by the NHS as part of their care and data collected as part of their participation in research. The National Genomic Research Library is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. This research has been conducted using the UK Biobank Resource under Application Number 90952. The Australian Undiagnosed Diseases Network (UDN-Aus) acknowledges financial support from the Australian Governments Medical Research Future Fund (2007567), Australian Genomics and The Centre for Population Genomics (Garvan Institute of Medical Research and Murdoch Childrens Research Institute); funded in part by a Medical Research Future Fund (MRFF) Genomics Health Futures Mission grant (2008820). ### 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 research was performed under the ethical approvals given by the South Manchester National Health Service (NHS) Research Ethics Committee (REC; 11/H1003/3/AM02 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 Genomic and phenotypic data are available for the 100KGP and individuals who have had WGS through the Genomic Medicine Service in the NGRL. Access to the NGRL may be granted following application via https://www.genomicsengland.co.uk/research/academic/join-research-network, which gives access to the secure GERE. Genomic data used pertain to participants in 100KGP in the Main Programme v.18 and the GMS data v.4. SolveRD data are accessible by application through the RD-CONNECT platform. All data presented in this paper, pertaining to 100kGP participants, were requested for the Airlock transfer through GERE. The paper was submitted for approval by the Genomics England Publication Committee on 25th August 2025 and was approved on 27th August 2025. Access to the Australian Centre for Population Genomics dataset can be requested through contact with the authors. The GRCh38 human genome reference assembly can be accessed at https://www.ncbi.nlm.nih.gov/datasets/genome/GCF\_000001405.26/. The GENCODE v.32 comprehensive annotations were accessed within the GERE but can be downloaded from https://www.gencodegenes.org/human/release\_32.html. The gnomADv4 genotype VCF files were accessed within the GERE but can also be downloaded from https://gnomad.broadinstitute.org/.

Background: Why some individuals experience severe neuropathy following infection is unknown. Nucleocytoplasmic trafficking (NCT) is an essential process in nucleated cells, and its disruption has been implicated in many neurodegenerative conditions including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Methods: We performed genomic and clinical studies in 24 individuals from 12 families with acute onset axonal neuropathy. Genetic variants were characterized by thermal stability and enzymatic assays using recombinantly expressed protein. Protein localization was determined in patient fibroblasts using immunofluorescence following heat or oxidative stress. A humanized Drosophila model was generated to determine the effect of stress on in vivo function. Results: We identified deleterious biallelic variants in human RCC1, encoding a GTP exchange factor essential in maintaining Ran GTPase-dependent NCT function. Clinical presentations ranged from a rapidly progressive, fatal axonal neuropathy with encephalopathy to a mild motor neuropathy resulting in impaired walking. In most patients (n=22/24), neurological presentation was secondary to infection, resulting in prior diagnosis of Guillain-Barre syndrome (GBS) in 13. The efficiency of cellular Ran GDP-GTP exchange and the thermal stability of Rcc1 protein was reduced by disease-associated variants. Heat shock or oxidative stress revealed defects in Ran nuclear localization, impaired NCT, and TDP-43 mislocalization in patient fibroblasts. Disease associated variants were unable to rescue the thermosensitive phenotype of a rcc1 deficient hamster cell line. RCC1 Drosophila models revealed a fatal intolerance to oxidative stress. Conclusion: We describe a novel autosomal recessive acute onset axonal neuropathy triggered by infection caused by biallelic RCC1 variants, which mimics GBS and has important mechanistic overlap with ALS. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement We acknowledge grant support from: the Wellcome Trust ; the Manchester NIHR BRC (NIHR203308); LifeArc Pathfinder award; and NIHR Doctoral Fellow, 301748). This research was made possible through access to data in the National Genomic Research Library, which is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The National Genomic Research Library holds data provided by patients and collected by the NHS as part of their care and data collected as part of their participation in research. The National Genomic Research Library is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. Further acknowledgements of funding are provided in the supplementary appendix. A.B. is supported by a Wellcome PhD Training Fellowship for Clinicians and the 4Ward North PhD Programme for Health Professionals (223521/Z/21/Z). P.L. is supported by Ministry of Health of the Czech Republic, grant. no: NW24-04-00349. R.H. is supported by the Wellcome Discovery Award (226653/Z/22/Z), the Medical Research Council (UK) (MR/V009346/1), the Addenbrookes Charitable Trust (G100142), the Hereditary Neuropathy Foundation, the Stoneygate Trust, the Lily Foundation, Ataxia UK, Action for AT, the Muscular Dystrophy UK, the LifeArc Centre to Treat Mitochondrial Diseases (LAC-TreatMito) and the UKRI/Horizon Europe Guarantee MSCA Doctoral Network Programme (Project 101120256: MMM). This research was supported by the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. H.L. receives support from the Canadian Institutes of Health Research (CIHR) for Foundation Grant FDN-167281 (Precision Health for Neuromuscular Diseases), Transnational Team Grant ERT-174211 (ProDGNE) and Network Grant OR2-189333 (NMD4C), from the Canada Foundation for Innovation (CFI-JELF 38412), the Canada Research Chairs program (Canada Research Chair in Neuromuscular Genomics and Health, 950-232279), the European Commission (Grant # 101080249) and the Canada Research Coordinating Committee New Frontiers in Research Fund (NFRFG-2022-00033) for SIMPATHIC, and from the Government of Canada Canada First Research Excellence Fund (CFREF) for the Brain-Heart Interconnectome (CFREF-2022-00007). RWT is funded by the Wellcome Centre for Mitochondrial Research (203105/Z/16/Z), the Mitochondrial Disease Patient Cohort (UK) (G0800674), the Medical Research Council (MR/W019027/1), the Lily Foundation, the Pathological Society, the UK NIHR Biomedical Research Centre for Ageing and Age-related disease award to the Newcastle upon Tyne Foundation Hospitals NHS Trust, LifeArc and the UK NHS Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children. R.D.S.P. is funded by The Lily Foundation, Muscular Dystrophy UK (MDUK), and a seedcorn award from the Rosetrees Trust and Stoneygate Foundation. R.D.S.P. is supported by a Medical Research Council (UK) Transition Support award (MR/X02363X/1), Medical Research Council (UK) award MC\_PC\_21046 to establish a National Mouse Genetics Network Mitochondria Cluster (MitoCluster), and the LifeArc Centre to Treat Mitochondrial Diseases (LAC-TreatMito). ### 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: Written informed consent was obtained from all persons in the study (or from their parents or guardians) in accordance with the Declaration of Helsinki protocols, and our experimental protocols were approved by the NHS institutional review board (IRAS 64321). 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

Accurately determining the sample size (“N”) of a dataset is a key consideration for experimental design. Misidentification of sample size can lead to pseudoreplication, a process of artificially inflating the number of experimental replicates which systematically underestimates variability, overestimates effect sizes and invalidates statistical tests performed on the data. While many journals have adopted stringent requirements with regard to statistical reporting over the last decade, it remains unknown whether such efforts have had a meaningful impact on statistical rigour. Here, we evaluated the prevalence of this type of statistical error among neuroscience studies involving animal models of Fragile-X Syndrome (FXS) and those using animal models of neurological disorders at large published between 2001 and 2024. We found that pseudoreplication was present in the majority of publication, increasing over time despite marked improvements in statistical reporting over the last decade. This trend generalised beyond the FXS literature to rodent studies of neurological disorders at large between 2012 and 2024, suggesting that pseudoreplication remains a widespread issue in the literature. The scope of this study was limited to rodent-model studies of neurological disorders which had the potential for being pseudoreplicated, by allowing repeat observations from individual animals. We did not consider reviews or articles whose experimental design could not allow for pseudoreplication, for example studies which reported only behavioural results, or studies which did not use inferential statistics. These observations identify an urgent need for better standards in experimental design and increased vigilance for this type of error during peer review. While reporting standards have significantly improved over the past two decades, this alone has not been enough to curb the prevalence of pseudoreplication. We offer suggestions for how this can be remedied as well as quantifying the severity of this particular type of statistical error. Although the examined literature concerns a specific neuroscience-related area of research, the implications of pseudoreplication apply to all fields of empirical research.

BACKGROUND:The reasons why some individuals have severe neuropathy following an infection are not known. Through the agnostic screening of children with acute axonal neuropathy after an infection, we identified several families with biallelic variants in RCC1. We aimed to describe the clinical phenotype of these patients, and the molecular and cellular pathology associated with the genetic variants identified in these families. METHODS:For this case series, we identified children affected by a severe, acute-onset axonal neuropathy following infection through an international research consortium of paediatric neurologists and clinical geneticists from nine countries (Canada, Cyprus, Czechia, Germany, Iran, Saudi Arabia, Slovakia, Türkiye, and the UK). Clinical assessments included nerve conduction studies and neuroimaging. We did exome or genome sequencing in DNA samples from all patients. We characterised the proteins encoded by the genetic variants by use of thermal stability and enzymatic assays, using recombinantly expressed proteins. We assessed cellular protein transport under heat or oxidative stress by use of immunofluorescence in primary fibroblasts, obtained from patients. We generated a humanised Drosophila knock-in model to assess the effects of stress on the in vivo function of RCC1. FINDINGS:Between Nov 2, 2011, and July 10, 2024, we identified 24 individuals from 12 families who had severe, acute-onset axonal neuropathy following infection (13 female and 11 male patients, with a mean age at diagnosis of 1 year 10 months [SD 2·27]). Eight biallelic missense variants in RCC1 were identified in affected individuals with autosomal recessive inheritance. Patients had variable phenotypes, ranging from rapidly progressive fatal axonal neuropathy to mild motor neuropathy with impaired walking. Neurological presentation was often secondary to an infection, resulting in initial misdiagnoses of Guillain-Barré syndrome in several patients. 15 children had disease recurrence. The disease was fatal in 15 patients. The RCC1 variants in these patients code for proteins that alter GDP-to-GTP exchange activity and have reduced thermal stability in vitro. In primary fibroblasts, heat shock or oxidative stress revealed defects in Ran nuclear localisation and impaired nucleocytoplasmic transport. A Drosophila model of the disease revealed a fatal intolerance to oxidative stress. INTERPRETATION:We describe an autosomal recessive, acute-onset paediatric axonal neuropathy, seemingly triggered by infection, that affects individuals with biallelic RCC1 variants. In these children, the disease can mimic Guillain-Barré syndrome. The pathological mechanisms underlying this novel axonal neuropathy might overlap with those of amyotrophic lateral sclerosis. Cellular studies indicate that RCC1 variants affect nucleocytoplasmic transport, which is crucial for healthy axonal function. Future studies should be directed at pre-symptomatic treatment by exploring ways to maintain nucleocytoplasmic transport. FUNDING:National Institute for Health and Care Research, LifeArc, and Wellcome Trust.

Kabuki syndrome type 1 (KS1) is a neurodevelopmental disorder caused by loss-of-function variants in KMT2D which encodes a H3K4 methyltransferase. The mechanisms underlying neurodevelopmental problems in KS1 are still largely unknown. Here, we track the epigenome and transcriptome across three stages of neuronal differentiation using patient-derived induced pluripotent stem cells (iPSCs) to gain insights into the disease mechanism of KS1. In KS1 iPSCs we detected significantly lower levels of functional KMT2D transcript and KMT2D protein, and lower global H3K4me1, H3K4me2 levels and modest reduction in H3K4me3. We identify loss of thousands of H3K4me1 peaks in iPSCs, neuronal progenitors (NPs) and early cortical neurons (CNs) in KS1. We show that the number of lost peaks increase as differentiation progresses. We also identify hundreds of differentially expressed genes (DEGs) in iPSCs, NPs and CNs in KS1. In contrast with the epigenomic changes, the number of DEGs decrease as differentiation progresses. Our analysis reveals significant enrichment of differentially downregulated genes in areas containing putative enhancer regions with H3K4me1 loss. We also identify a set of distinct transcription factor binding sites in differentially methylated regions and a set of DEGs related to KS1 phenotypes. We find that genes regulated by SUZ12, a subunit of Polycomb Repressive complex 2, are over-represented in KS1 DEGs at early stages of differentiation. In conclusion, we present a disease-relevant human cellular model for KS1 that provides mechanistic insights for the disorder and could be used for high throughput drug screening for KS1.

R-loops are DNA-RNA hybrid structures that may promote mutagenesis. However, their contribution to human Mendelian disorders is unexplored. Here we show excess de novo variants in genomic regions that form R-loops (henceforth, 'R-loop regions') and demonstrate enrichment of R-loop region variants (RRVs) in ribozyme, snoRNA and snRNA genes, specifically in rare disease cohorts. Using this insight, we report neurodevelopmental disorders (NDDs) caused by rare variants in two major spliceosomal RNA encoding genes, RNU2-2 and RNU5B-1. These, along with the recently described RNU4-2-related ReNU syndrome, provide a genetic explanation for a substantial proportion of individuals with NDDs.

Mutations in SYNGAP1 are a common genetic cause of intellectual disability (ID) and a risk factor for autism. SYNGAP1 encodes a synaptic GTPase-activating protein (GAP) that has both signaling and scaffolding roles. Most pathogenic variants of SYNGAP1 are predicted to result in haploinsufficiency. However, some affected individuals carry missense mutations in its calcium/lipid binding (C2) and GAP domains, suggesting that many clinical features result from loss of functions carried out by these domains. To test this hypothesis, we targeted the exons encoding the C2 and GAP domains of SYNGAP. Rats heterozygous for this deletion exhibit reduced exploration and fear extinction, altered social investigation, and spontaneous seizures—key phenotypes shared with Syngap heterozygous null rats. Together, these findings indicate that the reduction of SYNGAP C2/GAP domain function is a main feature of SYNGAP haploinsufficiency. This rat model provides an important system for the study of ID, autism, and epilepsy.

PURPOSE:Valproic acid or valproate is an effective antiepileptic drug; however, embryonic exposure to valproate can result in a teratogenic disorder referred to as fetal valproate syndrome (OMIM #609442). Currently there are no diagnostic biomarkers for the condition. This study aims to define an episignature biomarker for teratogenic antenatal exposure to valproate. METHODS:DNA extracted from peripheral blood of individuals with teratogenic antenatal exposure to valproate was processed using DNA methylation microarrays. Subsequently, methylation profiling and construction of support vector machine classifiers were performed in R. RESULTS:Genomic DNA methylation analysis was applied, and a distinct DNA methylation profile was identified in the majority of affected individuals. This profile was used to develop a diagnostic episignature classifier. The valproate exposure episignature exhibited high sensitivity and specificity relative to a large reference data set of unaffected controls and individuals with a wide spectrum of syndromic disorders with episignatures. Gene set enrichment analysis demonstrated an enrichment for terms associated with cell adhesion, including significant overrepresentation of the cadherin superfamily. CONCLUSION:This study provides evidence of a robust peripheral blood-based diagnostic epigenetic biomarker for a prenatal teratogenic disorder.

Detection of structural variants (SVs) is currently biased toward those that alter copy number. The relative contribution of inversions toward genetic disease is unclear. In this study, we analyzed genome sequencing data for 33,924 families with rare disease from the 100,000 Genomes Project. From a database hosting >500 million SVs, we focused on 351 genes where haploinsufficiency is a confirmed disease mechanism and identified 47 ultra-rare rearrangements that included an inversion (24 bp to 36.4 Mb, 20/47 de novo). Validation utilized a number of orthogonal approaches, including retrospective exome analysis. RNA-seq data supported the respective diagnoses for six participants. Phenotypic blending was apparent in four probands. Diagnostic odysseys were a common theme (>50 years for one individual), and targeted analysis for the specific gene had already been performed for 30% of these individuals but with no findings. We provide formal confirmation of a European founder origin for an intragenic MSH2 inversion. For two individuals with complex SVs involving the MECP2 mutational hotspot, ambiguous SV structures were resolved using long-read sequencing, influencing clinical interpretation. A de novo inversion of HOXD11-13 was uncovered in a family with Kantaputra-type mesomelic dysplasia. Lastly, a complex translocation disrupting APC and involving nine rearranged segments confirmed a clinical diagnosis for three family members and resolved a conundrum for a sibling with a single polyp. Overall, inversions play a small but notable role in rare disease, likely explaining the etiology in around 1/750 families across heterogeneous clinical cohorts.

The homologous genes GTPBP1 and GTPBP2 encode GTP-binding proteins 1 and 2, which are involved in ribosomal homeostasis. Pathogenic variants in GTPBP2 were recently shown to be an ultra-rare cause of neurodegenerative or neurodevelopmental disorders (NDDs). Until now, no human phenotype has been linked to GTPBP1. Here, we describe individuals carrying bi-allelic GTPBP1 variants that display an identical phenotype with GTPBP2 and characterize the overall spectrum of GTP-binding protein (1/2)-related disorders. In this study, 20 individuals from 16 families with distinct NDDs and syndromic facial features were investigated by whole-exome (WES) or whole-genome (WGS) sequencing. To assess the functional impact of the identified genetic variants, semi-quantitative PCR, western blot, and ribosome profiling assays were performed in fibroblasts from affected individuals. We also investigated the effect of reducing expression of CG2017, an ortholog of human GTPBP1/2, in the fruit fly Drosophila melanogaster. Individuals with bi-allelic GTPBP1 or GTPBP2 variants presented with microcephaly, profound neurodevelopmental impairment, pathognomonic craniofacial features, and ectodermal defects. Abnormal vision and/or hearing, progressive spasticity, choreoathetoid movements, refractory epilepsy, and brain atrophy were part of the core phenotype of this syndrome. Cell line studies identified a loss-of-function (LoF) impact of the disease-associated variants but no significant abnormalities on ribosome profiling. Reduced expression of CG2017 isoforms was associated with locomotor impairment in Drosophila. In conclusion, bi-allelic GTPBP1 and GTPBP2 LoF variants cause an identical, distinct neurodevelopmental syndrome. Mutant CG2017 knockout flies display motor impairment, highlighting the conserved role for GTP-binding proteins in CNS development across species.

Pathogenic variants in multiple genes on the X chromosome have been implicated in syndromic and non-syndromic intellectual disability disorders. ZFX on Xp22.11 encodes a transcription factor that has been linked to diverse processes including oncogenesis and development, but germline variants have not been characterized in association with disease. Here, we present clinical and molecular characterization of 18 individuals with germline ZFX variants. Exome or genome sequencing revealed 11 variants in 18 subjects (14 males and 4 females) from 16 unrelated families. Four missense variants were identified in 11 subjects, with seven truncation variants in the remaining individuals. Clinical findings included developmental delay/intellectual disability, behavioral abnormalities, hypotonia, and congenital anomalies. Overlapping and recurrent facial features were identified in all subjects, including thickening and medial broadening of eyebrows, variations in the shape of the face, external eye abnormalities, smooth and/or long philtrum, and ear abnormalities. Hyperparathyroidism was found in four families with missense variants, and enrichment of different tumor types was observed. In molecular studies, DNA-binding domain variants elicited differential expression of a small set of target genes relative to wild-type ZFX in cultured cells, suggesting a gain or loss of transcriptional activity. Additionally, a zebrafish model of ZFX loss displayed an altered behavioral phenotype, providing additional evidence for the functional significance of ZFX. Our clinical and experimental data support that variants in ZFX are associated with an X-linked intellectual disability syndrome characterized by a recurrent facial gestalt, neurocognitive and behavioral abnormalities, and an increased risk for congenital anomalies and hyperparathyroidism.

BACKGROUND:Desmosomes are complex cell junction structures that connect intermediate filaments providing strong cell-to-cell adhesion in tissues exposed to mechanical stress.OBJECTIVES:To identify causal variants in individuals with woolly hair and skin fragility of unknown genetic cause.METHODS:This research was conducted using whole-genome sequencing, whole-exome sequencing, clinical phenotyping, haplotype analysis, single-cell RNA sequencing data analysis, immunofluorescence microscopy and transmission electron microscopy.RESULTS:We identified homozygous predicted loss-of-function tuftelin-1 (TUFT1) variants in nine individuals, from three families, with woolly hair and skin fragility. One donor splice-site variant, c.60+1G>A, was present in two families, while a frameshift variant, p.Gln189Asnfs*49, was found in the third family. Haplotype analysis showed the c.60+1G>A substitution to be a founder variant in the Irish population that likely arose approximately 20 generations ago. Human and mouse single-cell RNA sequencing data showed TUFT1 expression to be enriched in the hair dermal sheath and keratinocytes. TUFT1 expression was highly correlated with genes encoding desmosomal components implicated in diseases with phenotypes that overlap with the cohort presented here. Immunofluorescence showed tuftelin-1 to be mainly localized to the peripheral cell membranes of keratinocytes in normal skin. Skin samples from individuals with TUFT1 variants showed markedly reduced immunoreactivity for tuftelin-1, with a loss of the keratinocyte cell membrane labelling. Light microscopy revealed keratinocyte adhesion, mild hyperkeratosis and areas of superficial peeling. Transmission electron microscopy showed panepidermal acantholysis with widening of intercellular spaces throughout the epidermis and desmosomal detachment through the inner plaques.CONCLUSIONS:Biallelic loss-of-function TUFT1 variants cause a new autosomal recessive skin/hair disorder characterized by woolly hair texture and early-onset skin fragility. Tuftelin-1 has a role in desmosomal integrity and function.