Genetic variants in RNU4-2, which is transcribed into the U4 small nuclear RNA component of the major spliceosome, were recently shown to cause ReNU syndrome, a prevalent dominant neurodevelopmental disorder (NDD). These variants almost exclusively arise de novo and cluster within 18 nucleotides of RNU4-2. Here we describe a new recessive NDD associated with homozygous and compound heterozygous variants in RNU4-2. We identify 38 individuals with biallelic variants outside the 18-nucleotide ReNU syndrome region that cluster within other functionally important elements of U4: Stem II, the k-turn and the Sm protein binding site. We characterize the clinical phenotype in 31 individuals, demonstrating that the recessive disorder is clinically distinct from ReNU syndrome and is associated with distinctive white matter abnormalities, including enlarged perivascular spaces. Finally, we find reduced RNU4-2 transcript levels in individuals with the recessive disorder, suggesting a loss-of-function disease mechanism that is distinct from the mechanism underlying ReNU syndrome. Together, these findings expand the genotypic and phenotypic spectrum of RNU4-2-associated NDDs.

A variety of genomic rearrangement mechanisms contribute to copy number variations at the 17p11.2 locus driven in part by its complex genomic architecture which is characterized by low copy repeats (LCRs) and other repetitive elements. These copy number variants are primarily mediated by nonallelic homologous recombination (NAHR) leading to recurrent tandem duplications and reciprocal deletions of the genomic interval mapping between the repeats. Two notable neurodevelopmental genomic disorders: Potocki-Lupski Syndrome (PTLS; MIM: 610883) and Smith-Magenis Syndrome (SMS; MIM: 182290) are driven by LCRs that undergo NAHR between the directly oriented repeats causing a duplication (PTLS) or deletion (SMS) encompassing the dosage-sensitive gene RAI1. We observed that other uncommon gains of varying sizes and extent at the 17p11.2 locus, which do not include the RAI1 gene, could be found in patients ascertained with a neurodevelopmental delay (NDD) phenotype. We ascertained 15 individuals from 11 families with copy number gains at the 17p11.2 locus not encompassing the driver gene-RAI1; such individuals manifested a broad spectrum of neurodevelopmental phenotypes. To validate our genomic findings, investigate DNA rearrangement mechanism(s), and refine our understanding at the breakpoint junctions, we performed a combination of high-resolution array CGH (n = 15), short-read whole-genome sequencing (sr-GS, n = 4), long-read GS (lr-GS; ONT; n = 4 and PacBio HiFi; n = 4), and breakpoint junctional analysis on this subset. Phenotypes in each individual were systematically studied. The phenotypes noted in these 15 individuals from 11 families primarily included developmental delay, intellectual disability, and behavioral problems. The genomic variations found in these 11 families included simple copy number gains (n = 7), higher order amplifications (n = 2), and complex genomic rearrangements (n = 2) at the 17p11.2 locus, surrounding the RAI1 gene and not encompassing it. Individuals from 4/11 families carried inherited variants. Identification of such rearrangement gains at the 17p11.2 locus that do not include the driver gene RAI1 and yet research subjects still exhibit neurodevelopmental phenotypes creates an opportunity to (i) dissect the gene(s) and genetic mechanisms that might contribute to phenotypic variability at the PTLS locus and (ii) uncover previously unrecognized genes or disease pathways and mechanisms.

Background: Genetic studies have disproportionately focused on populations of European ancestry, limiting the generalizability of allele-frequency references and genetic associations to underrepresented groups, including South American populations. This gap is particularly relevant for rare diseases and cancer, where accurate variant interpretation depends in part on appropriate population context. In addition, population-specific haplotype structure influence genome-wide association analyses and the portability of polygenic scores across ancestries. By focusing on the Chilean population, our work aims to bridge these gaps, providing more accurate reference data for genetic research and enhancing diagnostic and therapeutic strategies for underrepresented communities. Methods: We aggregated exomes from seven Chilean cohorts and processed samples using a standardized best practices workflow, including population-scale quality control and relatedness filtering. Aggregated variants were annotated with VEP (including LOFTEE, REVEL, AlphaMissense, EVE, and CADD) and clinically classified using InterVar. Population structure was assessed with PCA/admixture. We compared overlap and allele frequencies against external references, performed gene-level variant burden analysis, and constructed phased haplotypes for ancestry association using linear regression with Bonferroni correction. Finally, we developed a dashboard with R shiny framework to enable gene-wise exploration of annotated variants. Results: We present CHANGER (Chilean Aggregated National Genomics Resource), a comprehensive aggregation of 902 unrelated Chilean exomes designed to create a detailed reference of genetic variation in Chile. By incorporating data from multiple cohorts, we identified 774,110 unique genetic variants, with an average of 42,601 aggregated variants per individual. We identified 132,363 novel variants, of which 31,470 were common within our cohort (frequency > 1 %). We provide variant annotation and direct comparisons with other publicly available general population references. Beyond variant discovery, CHANGER supports gene-level burden scans (61 Missense-Damaging depleted genes; 2 Loss-of-Function enriched genes) and a haplotype resource for ancestry association (51,171 haplotypes), enabling downstream interpretation, improved imputation, and more powerful genome-wide association analyses in Chileans. Conclusions: CHANGER provides a valuable resource for genetic research and a reference for local variant interpretation. Importantly, CHANGER follows a high-quality methodological baseline and an open, FAIR-aligned infrastructure designed to grow, increasing its value for discovery, imputation, and equitable clinical interpretation over time.

ASTN1 encodes astrotactin 1, a neuronal-glial ligand in the developing brain that promotes neuronal migration along radial glia in brain structures with laminar organization, such as the cerebral cortex, hippocampus, and cerebellum. In mouse models, disruption of Astn1 results in neuronal migration deficits, a mild reduction in cerebellar volume, and balance and coordination deficits. In humans, bi-allelic ASTN1 variants have been identified in nine individuals with neurodevelopmental disorders (NDDs) with or without brain malformations. ASTN1 additionally interacts with astrotactin 2 (ASTN2) to implement neuronal migration; ASTN2 deletions associate with NDDs with reduced penetrance. Here, we describe eighteen individuals with NDDs from twelve unrelated families with bi-allelic, ultra-rare, predicted damaging variants in ASTN1 and one individual with heterozygous variants in both ASTN1 and ASTN2. We expand the clinical phenotypic descriptions of ASTN1-related NDDs, which range from mild to profound developmental delay or intellectual disability and can be associated with autism, attention-deficient hyperactivity disorder (ADHD), and epilepsy. Other recurrent abnormalities include dysmorphic facial features, hypotonia, spasticity, and ataxia. Additionally, we add to the neuroradiographic phenotype of this condition, which can be normal, mildly dysmorphic (a thin corpus callosum and cerebellar dysgenesis), or severely dysmorphic (polymicrogyria and lissencephaly). Remarkably, three genetic models of multilocus pathogenic variation (MPV), including tri-allelic, double heterozygous, and double homozygous due to distributive absence of heterozygosity (AOH), were observed. This ASTN1 allelic series characterizes the consequences of perturbations in radial-glia-guided neuronal migration in humans, the phenotypic spectrum of ASTN1-related NDDs, and the contribution of MPV to the genetic basis of NDDs.

CONTEXT:Current practice guidelines recommend considering a diagnosis of maturity-onset diabetes of the young (MODY) in patients with diabetes with an affected parent but without typical features of type 1 or type 2 diabetes. OBJECTIVE:To test if these criteria apply to a real-world cohort of racially/ethnically diverse children with diabetes. METHODS:We performed a retrospective case review of electronic medical records of youth diagnosed with MODY in a large academic pediatric hospital in Southwestern United States. Cases were ascertained based on the identification of a molecular diagnosis of MODY. RESULTS:We studied 50 genetically confirmed cases of MODY: 60% GCK-MODY (MODY2, n = 30), 16% HNF1A-MODY (MODY3, n = 8), 18% HNF1B-MODY (MODY5, n = 9), 4% HNF4A-MODY (MODY1, n = 2), and 2% with a dual molecular diagnosis resulting from variants in more than one MODY gene (n = 1). Race/ethnicity other than non-Hispanic White, lack of parental diabetes history, obesity/overweight at diagnosis, dyslipidemia and/or hypertension and acanthosis nigricans documented on physical examination were observed, respectively, in 52% (n = 26), 24% (n = 12), 22.2% (n = 11), 30% (n = 15), and 12% (n = 6) of MODY cases. Furthermore, islet antibody positivity was observed in 8% of the cases GCK-MODY (n = 4). The mean hemoglobin A1c at diabetes diagnosis was higher in patients with HNF4A-MODY (7.0%), HNF1A-MODY (6.7%), and HNF1B-MODY (6.7%), than in those with GCK-MODY (6.1%, P = 0.005). Four (13.3%) GCK-MODY patients had persistent proteinuria or neuropathy of undetermined cause. CONCLUSION:The current guidelines to consider a diagnosis of MODY may not apply to a racially/ethnically diverse US population of children with diabetes.

PURPOSE:RAPGEF2 encodes a guanine nucleotide exchange factor (GEF) that activates small GTPases and has not been linked to a Mendelian disorder. RAPGEF2 is highly intolerant to loss-of-function variants. We report 5 de novo heterozygous variants in RAPGEF2 in unrelated individuals with developmental delay, attention deficit hyperactivity disorder, epilepsy, dysmorphic features, or other manifestations. We used a Drosophila model to assess the functional impact of the identified human variants. METHODS:We generated a Kozak-GAL4 null allele of the Drosophila ortholog of RAPGEF2, PDZ-GEF, and used the allele to determine the gene expression pattern as well as the loss-of-function phenotypes. We expressed the reference and variant RAPGEF2 in PDZ-GEF mutant background to conduct "humanization" studies. RESULTS:Our experiments show that PDZ-GEF is expressed in the central nervous system. Loss of PDZ-GEF leads to severe locomotion defects, aberrant microtubular stability in motor neuron axons, and synaptic overgrowth at neuromuscular junctions in third instar larvae. Mutant animals are lethal at various developmental stages. Importantly, the neurodevelopmental phenotypes can be rescued by expression of the human RAPGEF2 reference cDNA but not by any of the variants. CONCLUSION:Our findings provide functional evidence that the tested RAPGEF2 variants are loss-of-function alleles and that the RAPGEF2 variants are associated with a neurodevelopmental disorder.

Rare diseases often remain unsolved because causal genetic changes can be complex and thus missed by standard sequencing or difficult to prioritize. Long-read sequencing can reveal structural variants, repeat expansions, DNA methylation and inherited haplotypes, but trio sequencing of an affected child and both parents remains costly. Here we show that phenotype-driven Trio-barcoded Oxford Nanopore Adaptive Sequencing (TBAS) enables cost-efficient long-read analysis of rare-disease trios on one flow cell. TBAS workflow uses clinical features to select broad disease-gene panels, barcodes all three family members and enriches these regions during sequencing rather than targeting a known causal locus. In benchmark regions, TBAS increased coverage and accurately detected small variants, structural variants, tandem repeat expansions, methylation and read-backed phasing, while reducing estimated sequencing consumable costs to 32.2% of conventional three-flow-cell trio long-read sequencing. Across 13 trios, TBAS recovered all five known diagnoses and prioritized candidates in five of eight unresolved cases. Here the authors present Phenotype-driven Trio-barcoded Adaptive Sequencing (TBAS) that enables cost-efficient long-read sequencing of rare-disease trios on a single flow cell, detecting diverse genetic variants while cutting costs to 32% of standard trio sequencing.

Nonsense-mediated decay (NMD) is a conserved RNA quality-control pathway that degrades transcripts containing premature termination codons. Because roughly a third of pathogenic variants in ClinVar can lead to truncated protein synthesis, predicting whether such transcripts undergo NMD is central to interpreting variant effects, yet the canonical 50-55 nucleotide rule explains only about half of observed outcome variability. Using paired whole-genome and RNA-sequencing from 10,306 individual samples in the Trans-Omics for Precision Medicine (TOPMed) program, we quantified NMD efficiency for 5,749 germline truncating variants via allele-specific expression and trained a gradient-boosting classifier, TrunCat, that distinguished NMD-sensitive from NMD-escape transcripts with ∼78% ROC-AUC (Receiver Operating Characteristic - Area Under the Curve). A reduced model using the ten features with the highest mean SHAP (SHapley Additive exPlanations) value as a measure of each feature's average contribution to predictions nearly matched this performance. Applied across large variant databases and a rare-disease cohort, the model produced NMD outcome predictions, with variants of uncertain significance showing higher predicted escape than pathogenic ones. This framework confirms the canonical rule, identifies non-canonical determinants, and offers a scalable resource for interpreting protein-truncating variants.

TUBB2B encodes a β-tubulin isotype essential for neuronal proliferation, migration, and organization during brain development. Pathogenic heterozygous variants in TUBB2B are associated with neurodevelopmental disorders including polymicrogyria and corpus callosum abnormalities. However, the phenotypic spectrum remains heterogeneous, most likely reflecting variant-specific effects on microtubule formation and stability. Homozygous TUBB2B variants are exceedingly rare, with one family reported to date. We describe five individuals in four families with rare TUBB2B variants. Four variants are described, including a previously reported de novo missense variant, c.292G>A (GenBank: NM_178012.5) (p.Gly98Arg), with potential phenotypic expansion including panhypopituitarism; a previously reported de novo missense variant, c.605T>C (GenBank: NM_178012.5) (p.Ile202Thr), showing interindividual heterogeneity; a de novo missense variant, c.43C>A (GenBank: NM_178012.5) (p.Gln15Lys) at a polyamination site critical for microtubule stability; and a homozygous missense variant within a region of absence of heterozygosity in two siblings from consanguineous parents, c.145G>A (GenBank: NM_178012.5) (p.Val49Ile). Both individuals also carry a pathogenic homozygous truncating ALKBH8 variant c.1675del (GenBank: NM_138775.3) (p.Arg559Alafs∗56), representing a potential dual molecular diagnosis driving clinical features reflective of contributions from both genes. These reports expand the clinical spectrum of TUBB2B-related tubulinopathies, illustrate phenotypic heterogeneity, and provide insights into disease mechanisms including effects at polyamination sites and rare recessive inheritance, underscoring the need for nuanced genotype-phenotype interpretation in diagnostic and counseling contexts.

Background Genome-wide distributions of Alu elements contribute to a broad range of structural variants (SVs) through Alu/Alu-mediated genomic rearrangement (AAMR). Yet, the prevalence and characteristics of AAMR on the human genome and its scale in generating pathogenic SVs remain poorly understood. Methods We established a disease-focused, AAMR-SV dataset and a control dataset to comprehensively delineate the genomic landscape of Alu mutagenesis. The disease-focused dataset included 407 published pathogenic AAMR-SV alleles in 115 known genes for Mendelian disorders or traits through a literature survey. A control dataset was collected from short-read genome sequencing analyses of 100 randomly selected, healthy individuals. Results AAMR favors the formation of copy number variant (CNV) less than 100 kb, including single-exon dropout and intragenic multi-exonic copy number variation. Genome-wide deletion length distribution from analyses of 526,806 deletion calls from 100 genomes reveals a high prevalence of AAMR in healthy individuals. Orthogonal experimental validations of these predicted AAMR events indicated their contributions mostly to non-coding CNVs. Conclusions Our study provides a comprehensive survey of Alu-related SV mutagenesis across global populations, analyzing their roles in reported pathogenic events and their prevalence among healthy individuals. It further documents AAMR-SVS responsible for a broad spectrum of Mendelian diseases and cancers.

Introduction and Objective: Insulin-deficient diabetes in the absence of islet autoantibodies (Ab) remains poorly understood, with unclear mechanisms and limited guidance for clinical management. We aimed to characterize this entity using data from the RADIANT cohort. Methods: We evaluated 237 RADIANT participants with non-secondary diabetes who initiated continuous insulin therapy within 1 yr of diagnosis and tested negative for GAD65, IA-2 and ZnT8 Ab. Participants who completed both genetic (e.g., type 1 diabetes genetic risk score-2, T1D GRS2) and metabolic testing (n=92) were stratified into those who tested Ab-negative <5 yrs since diabetes diagnosis (Group 1, n=47) or ≥5 yrs (Group 2, n=45). Each group was further stratified by maximum OGTT-stimulated C-peptide (A: <0.3, B: 0.3-0.7, or C: >0.7 nmol/L). Group 1 included 9, 6, and 32 and Group 2 included 14, 6, and 25 participants in strata A, B, and C, respectively. Results: At enrollment, mean ±SD age was 36.0 ±16.9 yrs and diabetes duration 10.3 ±13.1 yrs; 54% were female, 68.4% White, 8.9% Black, and 7.6% Asian. Diabetic ketoacidosis was reported in 34.2% of participants (23.6% at diagnosis), and personal or family histories of autoimmunity in 6.8% and 35.4%, respectively.The lowest C-peptide group had the longest diabetes duration (A: 2.9, B: 2.8, C: 1.3 yrs; p=0.011) and the lowest BMI at diagnosis (18.4, 20.9, 23.6; p=0.018) in Group 1; the lowest neck acanthosis severity (0.08, 0.50, 1.05; p=0.024) in Group 2; and the highest T1D-GRS2 percentile in both groups (Group 1: 0.84, 0.72, 0.51; p=0.017; Group 2: 0.86, 0.59, 0.40; p<0.001). Conclusion: The clinical phenotype of autoantibody-negative insulin-deficient diabetes includes subgroups with divergent genetic risk and beta-cell function that traditional criteria cannot distinguish. Incorporating C-peptide and genetic risk scores into evaluation may improve etiologic classification and guide treatment decisions in atypical diabetes. Disclosure K.R. Klein: Consultant; Current; Roche Pharmaceuticals, Novo Nordisk A/S. Advisory Panel; Current; vTv Therapeutics. Consultant; Current; Antag Therapeutics, Metsera. H. Parikh: None. A. Balasubramanyam: None. S.D. Gage: None. I. Hirsch: Research Support; Current; MannKind Corporation, Sequel Tech. Consultant; Current; Abbott Diabetes, Roche Diabetes Care, Hagar. C. Kirk: None. R.J. Kreienkamp: None. E.A. Kubota-Mishra: None. R. Naylor: None. L. Philipson: Research Support; Current; Novo Nordisk, Novo Nordisk Foundation. Research Support; Ended; Dompé. Research Support; Current; Vertex Pharmaceuticals Incorporated. Consultant; Current; Abbott. Research Support; Current; Zucara Therapeutics. Research Support; Ended; Diasome. Other - Data safety committee; Current; Cour. Consultant; Ended; Ono Pharmaceuticals. J.E. Posey: None. M. Tosur: None. M. Udler: Advisory Panel; Ended; Novo Nordisk. Research Support; Current; Novo Nordisk. C. Pihoker: None. M.J. Redondo: Advisory Panel; Current; Sanofi. Other - Data Safety Monitoring committee; Current; Lilly. Funding The RADIANT Study is funded by U54 DK118638 and U54 DK118612 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Purpose:Aicardi-Goutières syndrome (AGS) is a type I interferonopathy presently associated with nine genes. PTPN1 is a negative regulator of the interferon pathway previously associated with chronic inflammation and recently type 1 IFN autoinflammation. Methods:Genomic data from undiagnosed individuals with suspected AGS were interrogated for PTPN1 variants, and predicted loss-of-function (pLOF) and damaging missense variants in PTPN1 were sought in two additional academic databases as well as the All of Us database. Results:We identified 13 cases with ultra-rare heterozygous pLOF or highly damaging missense variants in PTPN1. Nine cases were identified in a cohort of 53 individuals (~ 17%) with clinical, imaging and persistent biochemical features of AGS. Median age of onset is 1.75 years (IQR 0.67), significantly later (p< 0.0001) than other AGS genotypes. Four additional cases were identified in academic datasets with variable clinical features suggestive of autoinflammation. Additionally, 49 individuals with ultra-rare, damaging PTPN1 variants were identified in the All of Us database, none had features suggestive of AGS, but autoimmunity was highly prevalent (~21.6%). Conclusion:Our data implicate PTPN1 as a cause of later-onset presentations of AGS within a broader spectrum of autoinflammatory phenotypes. Segregation and biobank data demonstrate reduced penetrance, with carriers being enriched for autoimmune disorders.

Purpose: This study aimed to apply cardiometabolic genetic testing in a community setting with a predominantly Hispanic population and assess feasibility and perspectives toward genetic testing. Methods: A genome-sequencing-based genetic panel for cardiometabolic disorders (177 genes related to monogenic conditions, 2 LPA risk alleles, 2 pharmacogenomic loci, and ancestryadjusted polygenic risk scores for type 2 diabetes and coronary artery disease) was deployed in community cardiology and endocrinology clinics in South Texas. A survey on perceptions toward genetic testing was administered after return of results. Results: Testing was completed for 776 patients (18-92 years old, 92% Hispanic). 26 patients (3.4%) were identified with a pathogenic or likely pathogenic variant in a monogenic disease gene, including 17 diagnostic and 9 secondary findings. Additionally, 291 (37.5%), 181 (23.3%), and 298 (38.4%) patients were identified with at least 1 LPA risk allele, pharmacogenomic finding, or elevated polygenic risk scores, respectively. Patients perceived the testing to be beneficial with few concerns. Conclusion: This study expanded cardiometabolic genetic testing to a predominantly Hispanic population in a community setting, providing actionable guidance for disease diagnosis, intervention, and preventive care with a favorable patient perception.

Idiopathic scoliosis (IS) is the most common form of spinal deformity with unclear pathogenesis. In this study, we first reanalyzed the loci associated with IS, drawing upon previous studies. Subsequently, we mapped these loci to candidate genes using either location-based or function-based strategies. To further substantiate our findings, we verified the enrichment of variants within these candidate genes across several large IS cohorts encompassing Chinese, East Asian, and European populations. Consequently, we identified variants in the EPHA4 gene as compelling candidates for IS. To confirm their pathogenicity, we generated zebrafish mutants of epha4a. Remarkably, the zebrafish epha4a mutants exhibited pronounced scoliosis during later stages of development, effectively recapitulating the IS phenotype. We observed that the epha4a mutants displayed defects in left-right coordination during locomotion, which arose from disorganized neural activation in these mutants. Our subsequent experiments indicated that the disruption of the central pattern generator (CPG) network, characterized by abnormal axon guidance of spinal cord interneurons, contributed to the disorganization observed in the mutants. Moreover, when knocked down efnb3b, the ligand for Epha4a, we observed similar CPG defects and disrupted left-right locomotion. These findings suggested that ephrin B3-Epha4 signaling is vital for the proper functioning of CPGs, and defects in this pathway could lead to scoliosis in zebrafish. Furthermore, we identified two cases of IS in NGEF, a downstream molecule in the EPHA4 pathway. Collectively, our data provide compelling evidence that neural patterning impairments and disruptions in CPGs may underlie the pathogenesis of IS.

Cytotoxic immune cells mediate precise attacks against diseased cells to maintain organismal health. Their operational unit of killing and host defense is lytic granules (LGs), which are specialized lysosomal-related organelles. Precision in cytotoxicity is achieved by converging the many LGs to the microtubule-organizing center (MTOC) and polarizing these to the diseased cell for secretion. We identify unappreciated intimate relationships between the Golgi, MTOC, and LGs after cytotoxic cell activation, as well as the trans-Golgin protein GCC2 on the LG surface. GCC2 serves to tether LGs to the Golgi following convergence, and both GCC2 and the Golgi are required for the persistence of convergence. GCC2 allows LGs to utilize the Golgi as a docking station preventing LG dispersion and innocent bystander killing in complex three-dimensional environments. We also identify GCC2 variants causing human natural killer cell deficiency, further emphasizing the importance of LG convergence and Golgi linkage in precision targeting for human immunity.

Purpose:While heterozygous de novo missense variants in the microtubule-binding GAR domain of Microtubule-actin cross-linking factor 1 (MACF1) cause Lissencephaly 9 with Complex Brainstem Malformations [MIM #618325], the phenotypic impact of variants outside this domain remains unclear. Methods:Through collaborative efforts, we assembled a cohort of 10 affected individuals from 8 unrelated families with either biallelic or monoallelic non-GAR domain MACF1 variants who exhibit partially overlapping yet unique phenotypic traits. Combined with previously reported cases, we analyzed genotype and phenotype data from 29 individuals using Human Phenotype Ontology (HPO)-based unsupervised hierarchical clustering. Results:Clustering revealed two distinct phenotypic signatures, suggesting domain-specific effects. Variants outside the GAR domain associate with broader neurodevelopmental phenotypes and variable craniofacial and skeletal expressivity. Additionally, enrichment analysis (p < 0.001) using OMIM HPO sets supported these findings. In contrast to the GAR domain's strong correlation with lissencephaly and brainstem malformations, biallelic non-GAR domain MACF1 variants were linked to diverse developmental anomalies. Conclusion:These results expand the phenotypic spectrum of MACF1-related disorders and highlight the relevance of domain-specific variant effects. Comprehensive genetic and phenotypic assessments are essential for understanding the role of MACF1 in development, informing diagnosis, and guiding future research on cytoskeletal regulation in neurodevelopment.

BackgroundPrevious studies in mouse,Xenopusand zebrafish embryos show strongtfap2eexpression in progenitor cells of neuronal and neural crest tissues suggesting its involvement in neural crest specification. However, the role of human transcription factor activator protein 2 (TFAP2E)in human embryonic central nervous system (CNS), orofacial and maxillofacial development is unknown.MethodsThrough a collaborative work, exome survey was performed in families with congenital CNS, orofacial and maxillofacial anomalies. Exome variant prioritisation promptedTFAP2Egene for functional analysis in zebrafish embryos. Embryonic morphology and development were assessed after antisense morpholino (MO) knockdown (KD), CRISPR/Cas9 knockout and overexpression oftfap2ein fluorescent zebrafish reporter lines using in vivo microscopy. Computational structural protein modelling of the identified human variants was performed.ResultsIn total, exome survey identified novel or ultra-rare heterozygous missense variants inTFAP2Ein seven individuals from five independent families with predominantly CNS, orofacial and maxillofacial anomalies. One variant was found de novo and another variant segregated in an affected multiplex family. Protein modelling of the identified variants indicated potential distortion of TFAP2E in the transactivation or dimerisation domain. MO KD and CRISPR/Cas9 knockout oftfap2ein zebrafish revealed hydrocephalus and a significant reduction of brain volume, consistent with a microencephaly phenotype. Furthermore, mRNA overexpression ofTFAP2Eindicates dosage-sensitive phenotype expression. In addition, zebrafish showed orofacial and maxillofacial anomalies followingtfap2eKD, recapitulating the human phenotype.ConclusionOur human genetic data and analysis of Tfap2e manipulation in zebrafish indicate a potential role ofTFAP2Ein human CNS, orofacial and maxillofacial anomalies.

SPOUT1/CENP-32 encodes a putative SPOUT RNA methyltransferase previously identified as a mitotic chromosome associated protein. SPOUT1/CENP-32 depletion leads to centrosome detachment from the spindle poles and chromosome misalignment. Aided by gene matching platforms, here we identify 28 individuals with neurodevelopmental delays from 21 families with bi-allelic variants in SPOUT1/CENP-32 detected by exome/genome sequencing. Zebrafish spout1/cenp-32 mutants show reduction in larval head size with concomitant apoptosis likely associated with altered cell cycle progression. In vivo complementation assays in zebrafish indicate that SPOUT1/CENP-32 missense variants identified in humans are pathogenic. Crystal structure analysis of SPOUT1/CENP-32 reveals that most disease-associated missense variants are located within the catalytic domain. Additionally, SPOUT1/CENP-32 recurrent missense variants show reduced methyltransferase activity in vitro and compromised centrosome tethering to the spindle poles in human cells. Thus, SPOUT1/CENP-32 pathogenic variants cause an autosomal recessive neurodevelopmental disorder: SpADMiSS ( SPOUT1 Associated Development delay Microcephaly Seizures Short stature) underpinned by mitotic spindle organization defects and consequent chromosome segregation errors.

Despite significant advances in genomic sequencing, the resolution of many rare disease cases is still hindered by variant detection limitations. Short reads struggle in homologous regions, and long reads remain costly and difficult to scale. Here, we present the first systematic evaluation of Illumina's Constellation sequencing technology for rare disease research. By fragmenting long DNA molecules directly on the flow cell surface, Constellation unlocks proximity information that enables long-range phasing and structural variant detection. Across 21 families, Constellation independently identified all known causative variants and resolved previously unsolved trios. It reliably resolved complex structural and copy number variants (e.g. impacting MECP2) and recovered haplotype phasing information across key disease impacting variants, all from low DNA input using existing Illumina infrastructure. These findings establish Constellation as a scalable, cost-efficient advance, closing critical diagnostic gaps and broadening access to long-range variant analysis in rare disease genomics.