Research on the pathogenic mechanism of FLNBI1542T-induced Scheuermann′s kyphosis in mice

English Keywords:Scheuermann′s kyphosis  Cartilage  Extracellular matrix  FLNB  Jun  SERPINH1

English Abstract:

  【Abstract】 Objectives: To investigate the pathogenic mechanism underlying FLNBI1542T-driven Scheuermann′s kyphosis(SK). Methods: A Han Chinese SK family underwent standardized clinical evaluation, including spinal X-ray, CT 3D reconstruction, and MRI inspection. Candidate pathogenic variants were identified by whole-exome sequencing and subsequently confirmed by Sanger sequencing, with pathogenicity assessed according to American College of Medical Genetics and Genomics(ACMG) guidelines. A FLNBI1542T knock-in mouse model was generated using CRISPR-Cas9. Phenotypic evaluation included gross observation, micro-CT imaging, and histological staining(HE, Masson, Safranin O/Fast Green) was performed to determine whether the model recapitulated SK-specific cartilaginous endplate defects and local kyphosis. Single-cell RNA sequencing(scRNA-seq) integrated with bioinformatics analysis was performed to delineate the key pathogenic chondrocyte subpopulations and their regulatory networks during endplate pathogenesis. Pathogenic chondrocyte subsets were isolated by fluorescence-activated cell sorting(FACS). Their molecular profiles were analyzed using immunohistochemistry(IHC) and co-immunoprecipitation(Co-IP) to assess the effect of FLNBI1542T on c-JUN. Quantitative PCR(qPCR) and dual-luciferase reporter assays were used to validate the transcriptional activation of the ECM homeostasis regulator Serpinh1 by Jun, with downstream effects confirmed by IHC and Western blot. Results: Through whole-exome sequencing of the proband, a heterozygous FLNB c.4625T>C (p.Ile1542Thr) mutation was identified, which co-segregated with the patient′s phenotype and was computationally predicted to impair protein stability. Using CRISPR-Cas9 technology, a FLNBI1542T mouse model was generated, which during growth and development exhibited imaging and histopathological features similar to those of human SK patients, including progressive thoracolumbar kyphosis, reduced vertebral volume and length, delayed ossification, and endplate depression. Single-cell RNA sequencing analysis revealed that the mutation primarily targeted and impaired the function of the chondroblast subpopulation within the cartilaginous endplate, leading to significant downregulation of gene expression programs related to "cartilage development" and "extracellular matrix organization" in this cell subset. Molecular mechanism studies demonstrated that the FLNBI1542T mutation reduced both the protein level and transcriptional activity of the transcription factor c-Jun. c-Jun could directly bind to and activate the promoter of the chaperone gene Serpinh1. The inhibition of Jun function caused by the FLNBI1542T mutation resulted in a marked decrease in SERPINH1 expression. Consequently, the expression of downstream key client proteins of SERPINH1, including the collagens COL2A1, COL9A3, and COL11A2, which were essential for maintaining ECM homeostasis, was also reduced, thereby compromising the integrity of the cartilaginous endplate ECM. Functional rescue experiments confirmed that overexpression of Jun reversed the downregulation of SERPINH1 and its downstream collagen proteins induced by the FLNBI1542T mutation. Conclusions: The FLNBI1542T mutation drives SK by impairing Jun-mediated transcriptional activation, which inhibits SERPINH1 expression and ultimately disrupts ECM collagen homeostasis, leading to cartilaginous endplate defects and kyphosis.