Pathogenic rewiring of the three-dimensional (3D) genome architecture is increasingly being identified as the cause of genetic diseases, but recognizing the cis-regulatory effects of structural variation remains a challenge. The Xq27.1 region contains a quasi-palindrome identified as a pleiotropic hotspot for disease-causing interchromosomal insertions. In a large Danish family affected by X-linked recessive complex spastic paraplegia, we identified the segregation of a 149-kb interchromosomal insertion at Xq27.1 originating from 4q24. To understand the disease mechanism, we generated induced pluripotent stem cells (iPSCs) from affected individuals. Using CRISPR perturbation and neural differentiation experiments combined with high-throughput chromatin conformation capture (Hi-C) and transcriptomic analyses, we identify a 3D regulatory rewiring of SOX3 and transcriptional dysregulation of SOX3 targets in iPSC-derived neurons. Consistent with regulatory partitioning of the SOX3 topologically associating domain (TAD) in affected individuals, our experiments show that upstream cis-regulatory elements have a reduced ability to activate SOX3 expression and that the observed dysregulation depends on CTCF-binding sites within the insertion. This work provides mechanistic evidence that a position effect at the SOX3 locus can cause hereditary spastic paraplegia.

The circadian system synchronizes physiology, improving the adaptation to daily environmental changes. In mammals, the central pacemaker, in the suprachiasmatic nuclei (SCN) of the hypothalamus, coordinates "wake" functions by inducing the circadian release of glucocorticoids (GCs). GCs entrain the clocks of a wide variety of tissues through GC receptor (GR) activation, however, the influence of GCs on the SCN is unclear and seems to depend on the maturity of the circuit. During the perinatal period, the mouse SCN express GR and respond directly to GCs while the adult SCN express low GR and have been traditionally considered resistant to GCs. To understand the change of sensitivity to GCs we followed the developmental trajectory of the mouse SCN, and found that while GR is expressed in all SCN cells early in life, it remains expressed mainly in astrocytes in the adult. Using a model of prenatal exposure to GCs, we found that offspring from treated mothers, adapt slower to shifted light-dark cycle and shows reduced expression of GR in SCN astrocytes. The adult SCN astrocytes can indeed sense and respond to GCs with rapid astrocytic Ca2+ events that propagate across neighboring cells, an effect that is prevented by the specific inhibition of astrocyte-astrocyte communication. Our findings provide a conceptual advance on how the mouse clock develops and on the influence that GCs have on the SCN. This might be relevant to understand how circadian synchrony is restored in conditions of temporal misalignment, such as jet lag.

Marfan syndrome (MFS) is a rare connective tissue disorder characterized by involvement of the cardiovascular, ocular, and musculoskeletal systems. Pathogenic variants in FBN1 cause most of the MFS cases; however, intellectual disability (ID) is rarely observed. A non-consanguineous Pakistani family with four affected individuals was recruited. Physical examinations, echocardiography, and doppler ultrasound were performed as part of the clinical assessment. Exome sequencing was conducted on the index patient, and Sanger sequencing was performed for the entire family. ID was the primary symptom in all the affected individuals. A detailed examination showed that all affected individuals and their affected mother were tall, had long limbs, craniofacial abnormalities, and exhibited low IQ, aggressive, and hyperactive behaviors. Heart defects, such as atrial septal defects and pulmonary hypertension, were observed in one affected individual and her mother. Genetic analysis identified two rare missense variants in FBN1, c.1552G>A (p.Gly518Arg) and c.3046A>G (p.Thr1016Ala), both predicted to be deleterious. The p.Gly518Arg variant is predicted to be likely pathogenic, while the p.Thr1016Ala variant is of uncertain significance. Notably, these variants were found in two affected individuals in a compound heterozygous state, correlating with more severe symptoms. Each variant alone, seen in the two patients, is associated with milder symptoms, indicating incomplete penetrance. In conclusion, this study identified rare heterozygous missense variants in FBN1 , suggesting a potential connection between neurodevelopmental outcomes and variants in FBN1 . However, further research is needed to clarify the role of FBN1 in ID.

Short-read sequencing (SRS) methods have improved the detection of small genetic variants but remain limited in highly homologous genomic regions, such as segmental duplications with gene-pseudogene pairs. These paralogous regions often require complex, locus-specific assays for accurate analysis. Long-read genome sequencing (lrGS) technologies, such as PacBio HiFi sequencing, can span these regions but still face challenges in variant calling due to alignment ambiguities. Here, we evaluated PacBio HiFi lrGS combined with Paraphase, a dedicated haplotype-based variant caller, in 86 individuals with 125 known clinically relevant variants across 11 paralogous loci. Standard HiFi variant callers detected 95/125 variants, while the remaining 30 variants were only identified by Paraphase. Together, the standard variant callers and Paraphase detected all known variants, including single-nucleotide variants (SNVs), insertions or deletions (InDels), copy-number variants (CNVs), structural variants (SVs), and gene conversions. In addition, lrGS allowed for accurate phasing and gene-pseudogene copy-number detection. We demonstrate that PacBio HiFi lrGS, particularly when integrated with Paraphase, enables comprehensive variant detection in previously difficult-to-assess genomic regions. These results also suggest that lrGS is ready for a wider implementation, possibly as a first-tier diagnostic approach for individuals with suspected variants in these paralogous regions. Ideally, clinical adoption is guided by prospectively designed clinical utility studies, alongside evaluation of sensitivity, specificity, and cost-effectiveness.

Abstract Viral infections have long been proposed as environmental contributors to neurodegenerative diseases, including Parkinson’s disease (PD), yet the molecular mechanisms linking infection and neurodegeneration are not well defined. Neuroinflammation and disruption of central nervous system (CNS) homeostasis have emerged as potential mediators. In this study, we used severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, as a model pathogen to investigate convergent molecular pathways between viral infection and PD. Single-nucleus RNA sequencing (snRNA-seq) was performed on post-mortem striatal tissue from 14 individuals stratified into four groups: COVID-19 only (COVID-19), PD only (PD), comorbid PD with COVID-19 (PD/COVID-19), and controls (Control). The PD/COVID-19 group exhibited an expanded astrocytic population and a pronounced interferon-associated molecular signature characterized by increased expression of canonical interferon-stimulated genes, including IFI44L (average log2FC= 3.9; adjusted p=2.3 x 10 −373 ) , IFI44 (average log2FC=2.9; adjusted p=8.0 x 10 −266 ) , ISG15 (average log2FC=3.1; adjusted p=1.2 x 10 −197 ), and RSAD2 (average log2FC= 3.5; adjusted p=8.6 x 10 −111 ). Pathway analyses demonstrated activation of innate immune and antiviral signaling pathways, particularly within microglia and astrocytes, including interferon signaling, pattern-recognition receptor pathways, and complement-associated responses. In parallel, genes involved in lipid metabolism, cholesterol homeostasis, synaptic maintenance, and neuronal signaling were reduced across disease groups. Proteomic analyses independently confirmed enrichment of antiviral and interferon-associated pathways and identified convergent suppression of sterol, cholesterol, and lipid metabolic processes. Our findings identify a convergent molecular signature linking PD and COVID-19, pronounced in comorbid individuals and characterized by interferon-driven innate immune activation, glial inflammatory responses, and dysregulation of lipid metabolic homeostasis. Collectively, the data support a model in which severe viral infection amplifies biological pathways already implicated in PD pathogenesis.

Spatial maps of gene expression in whole human embryos across several weeks of early development offer insights into the molecular programs that drive organogenesis. Spatial maps of gene expression in whole human embryos across several weeks of early development offer insights into the molecular programs that drive organogenesis.

Abstract Purpose Periodic reanalysis of genome sequencing data can yield additional diagnoses as knowledge evolves, yet manual reanalysis is labour-intensive. We compared automated and manual reanalysis approaches in rare disease genomics. Methods We reanalyzed 377 rare disease cases: 158 with pathogenic or likely pathogenic (P/LP) findings, 49 with variants of uncertain significance (VUS) findings, and 170 had no findings. Manual reanalysis used standard diagnostic workflow for all cases without prior P/LP diagnoses (219 cases). An automated pipeline using Talos was benchmarked on the 158 P/LP cases before application to the 219-case reanalysis cohort. The mean reanalysis interval was 660 days. Results Manual reanalysis identified three additional P/LP cases and two newly classified as VUS, increasing P/LP cases from 158 (41.9%) to 161 (42.7%). Talos recovered all three P/LP findings but only identified one of the two new VUS findings. Benchmarking showed 80.0% singleton concordance and 75.2% (82.8% proband-only) trio concordance, with an output of approximately three variants per case. Conclusion Reanalysis at 1.8 years yields modest but clinically meaningful gain. Automated reanalysis closely approximates manual performance while reducing hands-on effort, supporting scalable reanalysis in routine genomic care.

We developed a human Bone-on-a-Chip (BOAC) model that integrates autologous primary immune and bone cells on native human bone substrates within a xeno-free, perfused environment. In contrast to existing models, which focus primarily on hematopoietic or stromal compartments, our system recapitulates key aspects of functional bone remodeling and enables the maintenance of mature immune cells (up to 42 days), with preserved functional responsiveness at defined time points during culture. Dynamic flow and sequential cell seeding facilitated osteoclast-mediated resorption, osteoblast-driven matrix formation, and the maintenance of donor-specific immune profiles over extended culture periods. The balance between bone cell activity and immune cell persistence was further optimized by controlled temperature modulation. The BOAC model preserves key features of bone and bone marrow physiology, including extracellular matrix formation, soluble factor signaling, and cellular heterogeneity. The bone scaffold provides a physiologically relevant 3D architecture derived from decellularized human trabecular bone. Single-nucleus RNA sequencing confirmed the presence of major donor-specific immune and bone cell populations. This 3D human in vitro system provides a robust platform for translational research and personalized medicine.

Purpose:In uveal melanoma (UM), coexistence of the fatal monosomy 3 with the benign gain of chromosome 6p occurs rarely. The spatial organization of chromosomes can be influenced by the nucleoli, which become larger under hyperglycemia. We therefore hypothesized that hyperglycemia may be responsible for chromosome-specific aberrations in UM and analyzed its effect on nucleolar organization, chromosome territories, and missegregation rates in vitro. Methods:UM cell lines 92.1 and OMM2.5, UM cells from the primary tumors of two patients, and Tenon fibroblasts from a control were incubated in normo- or hyperglycemic medium (with 5.5 or 25 mM glucose, respectively) for one day, followed by the mitotic arrest with Nocodazole for 18-24 hours and recovery in fresh medium. Co-detection of proteins with the centromeres of chromosomes 3 and 6 was performed by two-dimensional immunofluorescent in situ hybridization. Results:In the UM cells undergoing interphase, hyperglycemia promoted the dislocation of chromosome 3 toward the center along with nucleolar growth. During prometaphase, the mean angle between the centromeres of chromosome 3 was reduced below 90° under hyperglycemia (P = 0.02). During the later mitotic phases, hyperglycemia resulted in a 3.8-fold increase in the missegregation rate of chromosome 3 in UM cells (P < 0.001), whereas chromosome 6 rather than 3 was more prone to missegregation in the normoglycemic UM cells and hyperglycemic Tenon fibroblasts. Conclusions:Hyperglycemia can favor chromosome-specific aneuploidies by altering chromosome territories in a cell-type dependent manner. Prevention of hyperglycemia may be a simple therapeutic approach to impede the generation of monosomy 3 in UM.

Genetic factors are fundamental in the etiology of thoracic aortic aneurysm and dissection (TAAD), but the genetic cause is detected in only about 30% of cases. To define unreported TAAD-associated sequence variants, exome and gene panel sequencing was performed in 323 patients. We identified heterozygous CDKL1 variants [c.427T>C p.(Cys143Arg), c.617C>T p.(Ser206Leu), and c.404C>T p.(Thr135Met)] in 6 patients from 3 families with TAAD spectrum disorders. CDKL1 encodes a protein kinase involved in ciliary biology. Amino acid substitutions were predicted to affect CDKL1 catalytic activity or protein binding properties. CDKL1 was expressed in vascular smooth muscle cells in normal and diseased human aortic wall tissue. Cdkl1 knockdown and transient knockout in zebrafish resulted in intersomitic vessel (ISV) malformations and aortic dilation. Coinjection of human CDKL1wild-type RNA, but not CDKL1Cys143Arg and CDKL1Ser206Leu RNA, rescued ISV malformations. All variants affected CDKL1 kinase function and profiling data, and altered protein-protein binding properties, particularly with ciliary transport molecules. Expression of CDKL1 variants in heterologous cells interfered with cilia formation and length, CDKL1 localization, and p38 MAPK and Vegf signaling. Our data suggest a role of CDKL1 variants in the pathogenesis of TAAD spectrum disorders. The association between primary cilia dysregulation and TAAD expands our knowledge of the underlying molecular pathophysiology.

The spatial organization of the genome within the nucleus — also known as genome architecture or 3D genome — is important to the regulation of gene expression. Disruption of the 3D genome, for example, by structural variation, can contribute to disease, including developmental disorders and cancer. Structural variants can rearrange higher-order chromatin structures, such as topologically associating domains, and disrupt interactions between cis-regulatory elements, which can lead to altered gene expression, a phenomenon known as position effects. New experimental and computational approaches are revealing the effect of structural variants on the 3D genome and gene expression and can help interpret their pathogenic potential, which has important implications for patients. Here, we review mechanisms of disease caused by position effects owing to disruptions of genome architecture, and more specifically topologically associating domains, as well as their consequences and clinical impact. Disruption of the 3D genome caused by structural variation contributes to developmental disorders and cancer. The authors review the causes and molecular and clinical consequences of position effects arising from disruptions to the genome architecture.

The major spliceosome contains five small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) essential for splicing. Variants in RNU4-2, encoding U4, cause a neurodevelopmental disorder called ReNU syndrome. We investigated de novo variants in 50 snRNA-encoding genes in a French cohort of 23,649 individuals with rare disorders and gathered additional cases through international collaborations. Altogether, we identified 145 previously unreported probands with (likely) pathogenic variants in RNU4-2 and 21 individuals with de novo and/or recurrent variants in RNU5B-1 and RNU5A-1, encoding U5. Pathogenic variants typically arose de novo on the maternal allele and cluster in regions critical for splicing. RNU4-2 variants mainly localize to two structures, the stem III and T-loop/quasi-pseudoknot, which position the U6 ACAGAGA box for 5' splice site recognition and associate with different phenotypic severity. RNU4-2 variants result in specific defects in alternative 5' splice site usage and methylation patterns (episignatures) that correlate with variant location and clinical severity. This study establishes RNU5B-1 as a neurodevelopmental disorder gene, suggests RNU5A-1 as a strong candidate and highlights the role of de novo variants in snRNAs.

BACKGROUND:Androgen signalling through the androgen receptor (AR) is crucial for male genital development. Disruptions in this pathway are associated with androgen insensitivity syndrome (AIS), which is typically caused by mutations in the AR gene, although the underlying genetic mechanisms remain unknown in many cases. To better understand androgen-dependent transcriptional changes in human genital tissue, we performed transcriptomic profiling of foreskin- and scrotum-derived human genital skin fibroblasts (GSFs) treated with dihydrotestosterone. RESULTS:Differential gene expression analysis revealed 409 and 260 reproducibly up-regulated genes in foreskin- and scrotum-derived GSFs, respectively. GSFs from individuals with complete androgen insensitivity syndrome, carrying inactivating mutations in the AR gene, showed no reproducible androgen response. Androgen response element motif scanning confirmed direct AR binding in key up-regulated genes, including AOX1, APOD, FKBP5, and FAM107A. Gene ontology analysis revealed enrichment in pathways related to neuronal, muscle, cardiovascular, and sex development. CONCLUSION:Identifying new AR target genes broadens the current understanding of androgen signalling and aids in better understanding the aetiology of AIS, and other androgen-related conditions.