Fgfr2c(C342Y/+)基因突变导致颅缝早闭症Crouzon综合征的分子机制研究

Crouzon syndrome is one of the frequent congenital deformities with patients exhibit premature fusion of multiple cranial sutures resulting in craniosynostosis during early childhood. The C342Y gain-of-function substitution in the Fgfr2 gene is a commonly reported mutation for this condition. A previous microarray study in human identified a number of novel genes incluing RBP4, GPC3 and C1QTNF3 were downregulated during this process and were similarly expressed in the Fgfr2c(C342Y/+) Crouzon mouse model. This strongly suggest they are downstream effector of FGF2 signaling. In this study, the onset of cranial suture fusion in Fgfr2c(C342Y/+) mice will be assessed from E16.5 to P10 using microCT and histology. Expression levels and localization of target genes with osteogenic markers of coronal suture-derived cell cultures from wildtype and Fgfr2c(C342Y/+) mice during osteogenesis will be determined using Realtime PCR and immunocytochemistry respectively. The metabolic and proliferation rates of coronal suture-derived and parietal bone-derived cell cultures will also be assessed by MTS and DNA assay. We will further indentify levels of target gene expression by both FGF2 stimulation and FGFR2 inhibition in these cells. Levels of FGF2 signaling proteins expression will then be detacted after overexpression and siRNA interference of target gene(s) using protein microarray. Confocal microscopy and chromatin immunoprecipitation assay will be undertaken to confirm target gene effectors of FGF2 signaling. We believe that the detailed study of target gene function through FGF pathway could lead to the understanding of Fgfr2c(C342Y/+) mutation mechanism and will provide a solid scientific basis for the subsequent development of adjunctive therapy of craniosynostosis.

颅缝早闭症Crouzon综合征是常见的先天性颅颌面畸形之一，由FFGR2突变所致，确切分子机制不清。Fgfr2c（C342Y/+）小鼠为针对该突变的动物模型，尚未有利用其开展基因分子调控机制的研究报道。课题组前期在患者颅缝组织基因芯片研究中发现RBP4、GPC3、C1QTNF3等新致病基因，并在Fgfr2c（C342Y/+）小鼠颅缝组织中验证，结果提示新基因可能处于FGFR2下游通路。申请者设想：上述新基因是否通过FGFR2信号通路调控颅缝细胞增殖与分化失衡，从而导致颅缝早闭？基于此，本课题首先研究新基因在小鼠颅缝细胞中的表达定位；通过激活、抑制FGFR2信号通路，探讨FGFR2调控新基因的作用机制；通过新基因过表达及沉默，双向检测主要通路蛋白的表达改变，并验证新基因与通路蛋白的相互作用。旨在初步阐明新基因通过FGFR2信号通路的作用模式，为进一步开展针对新致病基因的靶向治疗提供理论依据。

颅缝早闭症Crouzon综合征是常见的先天性颅颌面畸形之一，由FFGR2突变所致，确切分子机制不清，严重影响患儿的正常发育、生理功能。因而，研究Crouzon 综合征颅缝早闭的发病机制具有十分重要的社会意义。Fgfr2c（C342Y/+）小鼠为针对该突变的动物模型，尚未有利用其开展基因分子调控机制的研究报道。本项目在患者颅缝组织基因芯片研究中发现RBP4、GPC3等新致病基因，并在Fgfr2c（C342Y/+）小鼠颅缝组织、颅缝细胞中进行表达研究验证，证实致病基因表达与前期人颅缝组织Microarray研究结果一致，结果提示新基因可能处于FGFR2下游通路。通过慢病毒介导的过表达系统，构建稳定表达的FGFR2野生型和突变型的成骨细胞系，比较成骨细胞功能差异，并进一步探讨其涉及的差异表达蛋白和相关信号通路。结果显示：通过靶向基因干扰和（或）过表达RBP4和GPC3基因，成骨细胞功能发生显著变化：过表达RBP4能诱导颅缝细胞的增殖，但成骨矿化能力与正常细胞相比并无明显差异，分析可能与转染效率较低及低浓度视黄酸对RBP4功能的掩盖作用有关，因此无法得出RBP4在颅缝闭合中未产生调控的结论。GPC3过表达可明显诱导细胞增殖、抑制细胞凋亡，同时成骨细胞的分化能力明显增强；相反，靶向干扰GPC3基因可获得相反的结果。蛋白组学研究发现，突变型与野生型成骨细胞存在128个差异表达蛋白，其涉及到的关键性信号通路包括细胞周期和PI3K-Akt pathway。上述研究内容旨在初步阐明新基因通过FGFR2信号通路的作用模式，为深入研究FGFR2介导的颅缝早闭过程的分子机制奠定基础。