Generation of a P4hbY393C mouse model of cole-carpenter syndrome and therapeutic proof-of-concept.

Comprehensive characterization and translational implications of the GalnsR384C mouse model of Mucopolysaccharidosis IVA.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to progressive accumulation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S) and resulting in systemic skeletal dysplasia. Severe, early-onset disease is frequently associated with destabilizing structural missense variants, including p.R386C. To model a loss-of-function missense variant associated with severe MPS IVA, we generated a GalnsR384C knock-in mouse, the murine ortholog of the most common human variant, p.R386C. Biochemical, histological, and skeletal phenotypes were evaluated across multiple tissues, and bone microarchitecture was assessed using microcomputed tomography (micro-CT). Genomic and biochemical assays were performed to assess allelic integrity, and principal component analysis (PCA) was used to integrate biochemical and structural parameters. GalnsR384C mice exhibited significantly reduced GALNS activity and elevated KS levels across various tissues. Histological examination revealed considerable vacuolization in cartilage and cardiac valves, while micro-CT illustrated altered bone microarchitecture consistent with disrupted endochondral ossification. During allele validation, a secondary missense variant (p.R384Y) was identified and characterized as a comparative model that led to defective GALNS activity, substrate accumulation, and analogous skeletal and cardiovascular pathology. PCA demonstrated clear differentiation between WT and mutant groups, with considerable multivariate overlap observed between GalnsR384C and GalnsR384Y mice. In conclusion, GalnsR384C and GalnsR384Y mice recapitulate key biochemical, skeletal, and histopathological features of MPS IVA and provide well-characterized murine models of severe GALNS loss-of-function resulting from clinically relevant missense variants. Rigorous genomic validation underscores the importance of careful allele-level characterization during genome-editing-based model generation.

PIK3CA in-frame deletion-activated PI3K/AKT signaling pathway drives macrodactyly.

EXTL3 dysfunction identified as a driver of aberrant bone development in severe familial ankylosing spondylitis.

OBJECTIVES Pathological ossification leading to spinal ankylosis is a hallmark of ankylosing spondylitis (AS), yet its molecular mechanisms remain poorly understood. We investigated the genetic basis and functional consequences of severe familial AS to identify pathways driving structural progression. METHODS Genome-wide linkage analysis and whole-exome sequencing were performed in a multiplex family with severe AS. A heterozygous knock-in mouse carrying the identified EXTL3 variant was generated and characterized using microCT, histology, transcriptomics, primary osteoblast and chondrocyte cultures, and biochemical analyses of heparan sulfate (HS) metabolism. RESULTS A rare missense variant in EXTL3, a key enzyme involved in HS biosynthesis, was identified in affected family members. Extl3mut/+ mice developed sacroiliac structural abnormalities, osteoid accumulation and early joint bridging, together with impaired trabecular bone architecture. Mechanistically, the mutation accelerated chondrocyte hypertrophy while delaying osteoblast maturation and mineralization. These changes were associated with activation of Wnt signalling, increased HS accumulation, enhanced heparanase expression and altered glycosaminoglycan homeostasis. Collectively, these findings identify EXTL3-mediated HS dysregulation as a novel mechanism linking abnormal bone remodelling to pathological ossification. CONCLUSIONS Our study identifies EXTL3 as a genetic modifier of structural severity in AS and reveals a previously unrecognised role for HS metabolism in pathological ossification. These findings support a model in which HLA-B27 primarily confers disease susceptibility, whereas rare variants such as EXTL3 contribute to structural progression, highlighting HS-related pathways as potential therapeutic targets.

The abnormal C-terminus caused by DVL1 variants impacts Robinow syndrome phenotypes

Abstract Robinow Syndrome is a genetically heterogeneous, rare skeletal disorder characterized by craniofacial and limb defects. All 7 causative genes lie in the Wingless-related Integration site-1 (WNT) pathway. Here we study the pathogenesis of DVL1 (Dishevelled 1), the most commonly affected gene, where variants cause a frameshift that replaces the C terminus with a novel peptide. We compared phenotypes caused by DVL11519ΔT to the effects of wtDVL1 or DVL1 with a stop codon at position 1519. Misexpression of DVL11519ΔT in chicken embryos with an avian retrovirus, leads to increased width of the frontonasal mass similar to the facial phenotype in RS. Ultimately skeletogenesis is inhibited, which was verified in primary cultures of frontonasal mass mesenchyme. In luciferase assays carried out in facial mesenchyme, wtDVL1 activated canonical and JNK-PCP WNT signalling whereas the DVL11519* and the DVL11519ΔT variant had significantly lower signaling activity. These data confirm that the C-terminus plays an important role in WNT signal transduction and skeletogenesis. We also determined that there is mislocalization of the protein expressed from DVL11519ΔT in the nucleus while the other two constructs were expressed in the cytoplasm. Nuclear expression of DVL1 may alter transcription in RS. In complementary Drosophila experiments using a variety of readouts, only the DVL11519ΔT variant and not the 1519* impacted morphogenesis and signaling. This is the first study to show that the novel C-terminus of DVL1 is sufficient to interfere with the function of DVL1 protein expressed from the normal allele in heterozygous, autosomal dominant RS.

Clinical and zebrafish studies of truncating SF3B2-variants in craniofacial microsomia

NFIX missense variants that disrupt the β-hairpin loop result in a severe form of Malan syndrome in adolescence with rapidly evolving scoliosis and muscle wasting

In vitro functional studies support variant-specific disruption of DNA binding, providing a mechanistic basis of genotype-phenotype correlations and informing prognosis, clinical surveillance, and therapy development.