FLNBI1542T致小鼠休门氏后凸畸形的机制研究

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

中文关键词:  休门氏后凸畸形  软骨  细胞外基质  FLNB  Jun  SERPINH1

英文关键词:Scheuermann′s kyphosis  Cartilage  Extracellular matrix  FLNB  Jun  SERPINH1

中文摘要:

  【摘要】 目的：阐明细丝蛋白B基因（filamin B，FLNB）第1542位异亮氨酸（Ile/I）→苏氨酸（Thr/T）突变（FLNB p.Ile1542Thr，FLNBI1542T）在休门氏后凸畸形（Scheuermann′s kyphosis，SK）中的机制。方法：对一个汉族SK家系进行标准化临床评估，包括脊柱X线、CT三维重建及MRI检查。通过全外显子测序与Sanger测序筛选并验证致病性变异，依据美国医学遗传学与基因组学学会（American College of Medical Genetics and Genomics， ACMG）指南进行致病性评估。采用CRISPR-Cas9技术构建携带FLNBI1542T突变的小鼠模型，通过外观观察、显微CT成像及组织学染色（HE、Masson、Safranin O/Fast Green）对模型表型进行评估，以确认其是否重现SK特异的软骨终板缺陷与局部后凸畸形。利用单细胞RNA测序（scRNA-seq）结合生物信息学分析，解析软骨终板病理发育过程中关键的致病软骨细胞亚群及其靶分子调控网络。通过单细胞流式分选致病性软骨细胞亚群，并进一步利用免疫组化（immunohistochemistry，IHC）、免疫共沉淀（co-immunoprecipitation，Co-IP）验证FLNBI1542T对c-Jun蛋白表达水平的影响；利用定量聚合酶链式反应（quantitative polymerase chain reaction，qPCR）、双荧光素酶报告基因等实验分析c-Jun对细胞外基质（extracellular matrix，ECM）稳态关键分子Serpinh1的转录激活调控，并利用免疫组化、Western blot分析Jun对ECM稳态的调控作用。结果：通过对先证者全外显子测序，鉴定出一个杂合FLNB c.4625T>C（p.Ile1542Thr）突变，与患者表型共分离，且计算预测提示其破坏蛋白稳定性。利用CRISPR-Cas9技术构建的FLNBI1542T基因敲入小鼠，在生长发育过程中表现出与人SK患者相似的进行性胸腰段后凸、椎体体积与长度减小、骨化延迟及终板凹陷等影像学与组织病理学特征。单细胞转录组测序分析揭示，突变主要靶向并损害了终板软骨中的软骨形成细胞亚群的功能，导致该群细胞中与“软骨发育”和“细胞外基质组织”相关的基因表达程序显著下调。分子机制研究表明，FLNBI1542T突变削弱了转录因子c-Jun的蛋白水平及其转录活性。c-Jun可直接结合并激活分子伴侣Serpinh1的启动子，而FLNBI1542T突变导致的Jun功能抑制致使SERPINH1表达显著下降。SERPINH1的下游关键客户蛋白，包括COL2A1、COL9A3和COL11A2等维持ECM稳态的胶原蛋白，其表达也随之降低，从而破坏了软骨终板的ECM完整性。功能挽救实验证实，过表达Jun可逆转由FLNBI1542T突变引起的SERPINH1及其下游胶原蛋白的表达下调。结论：FLNBI1542T通过损害Jun的转录活性，抑制SERPINH1表达，从而破坏ECM胶原稳态，驱动SK的软骨终板缺陷与后凸畸形。

英文摘要:

  【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.