新致病基因CTGF突变致家族性脊柱骨骺干骺端发育不良的发病机制研究

Spondyloepimetaphyseal dysplasia (SEMD) is the collective term for a clinically and genetically heterogeneous group of inherited human skeletal disorders, seriously affecting the health among adolescents. The clinical phenotypes of affected subjects are usually short-stature, abnormal joint motion, and early-onset osteoarthritis (OA). We previously collected a pedigree of SEMD with autosomal-dominant pattern of inheritance. Interestingly, further studies revealed that CTGF was a new disease-causing gene in the familial SEMD. Previous studies have shown that CTGF is a critical regulator implicated in the process of endochondral ossification, indicating the key role of CTGF in skeleton development. Moreover, emerging evidences suggest that CTGF is a promising regeneration factor that plays an important role in articular cartilage homeostasis. However, no human mendelian disorders were found to be linked to germline mutations in CTGF previously. In this study, we intend to use CTGF mutation knock-in mice model as well as OA mice model to clarify the role of CTGF in bone formation and in homeostasis of articular cartilage, especially its role in the development of OA, and to explore the potential application value of CTGF in OA treatment. This study is expected to elucidate the molecular mechanism of CTGF gene mutation in pathogenesis of SEMD, and to gain understanding of pathogenesis of OA, as well providing new insight for the development of therapy for OA.

脊柱骨骺干骺端发育不良是一组以身材矮小、先天性关节活动异常及早发性骨关节炎等为主要临床特征的遗传性骨病，严重影响青少年健康。我们前期临床诊断一个常显遗传方式的脊柱骨骺干骺端发育不良大家系，令人兴奋的是，我们通过进一步研究明确CTGF是该疾病的新致病基因。已发现CTGF是软骨内成骨过程关键因子，并且参与关节软骨稳态维持与损伤修复，但此前该基因突变导致人类疾病未见报道。因此，本项目拟在我们已构建成功的CTGF基因R21P点突变敲入小鼠模型基础上，建立骨关节炎小鼠模型，通过体内外实验明确CTGF在骨软骨发育及关节软骨稳态维持，尤其在骨关节炎发生发展中的关键作用，并探讨CTGF在骨关节炎干预中的潜在价值。本项目有望阐明CTGF基因突变致家族性脊柱骨骺干骺端发育不良的发病机制，并明确CTGF对骨关节炎的作用，为其治疗药物研发提供关键靶点。

按照本项目计划书要求，我们对所收集的常染色体显性遗传脊柱骨骺干骺端发育不良家系进行全面详致的临床表型分析，归纳总结表型特点，绘制疾病临床表型谱。通过体外细胞实验明确所鉴定到的CTGF基因点突变为功能丢失型突变，CTGF基因点突变降低CTGF蛋白细胞外分泌而不影响CTGF基因转录过程，同时该突变降低了CTGF对间充质干细胞成骨分化过程的促进作用，明确CTGF基因点突变致病机制。随后我们构建CTGFA基因缺陷斑马鱼模型，在研究中我们观察到CTGFA基因缺陷斑马鱼表现出明显的体轴弯曲与颅面部发育障碍，提示CTGFA基因在斑马鱼骨软骨发育过程中发挥重要作用。我们使用CRISPR /Cas9技术先后构建了CtgfR21P点突变敲入小鼠模型与Ctgffl/fl;Prx1Cre小鼠模型。首先，我们对前期已构建成功的CtgfR21P点突变敲入小鼠模型的不同天数的胚胎及不同周龄小鼠进行整体骨架染色、X线摄片、micro-CT检查、组织形态学分析等，均未能检测到CtgfR21P点突变敲入小鼠骨软骨异常表型，更无法模拟脊柱骨骺干骺端发育不良疾病表型。为此，我们进一步构建Ctgffl/fl;Prx1Cre小鼠模型，该小鼠模型特异性在胚胎肢芽和部分颅面间质细胞中敲除Ctgf基因。目前通过对该小鼠的表型分析，我们已观察到Ctgffl/fl;Prx1Cre小鼠存在骨量降低的重要表型，且部分模拟了脊柱骨骺干骺端发育不良疾病表型，进一步研究提示软骨细胞与成骨细胞分化障碍及破骨吸收增强共同参与Ctgffl/fl;Prx1Cre小鼠异常骨骼表型的发生。后续我们将在上述研究基础上，利用该模型小鼠开展深入的机制研究。.本项目协助培养博士1名（已毕业）；发表SCI论文3篇。本研究首次全面绘制常染色体显性遗传脊柱骨骺干骺端发育不良疾病临床表型谱，为该类罕见病的诊断、遗传咨询及产前诊断提供了临床重要依据。同时基本明确CTGF在间充质干细胞介导骨软骨发育过程的重要作用，也会后续深入的机制研究提供关键线索。