EXT2基因C339Y新突变致遗传性多发性骨软骨瘤的分子发病机制研究

Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder most frequently caused by the EXT gene family mutations. Malignant transformation of the exostoses to a chondrosarcoma or an osteosarcoma occurs in about 5% of the HME patients. This project is focus on the novel EXT2 gene mutation, i.e. C339Y, which we previously identified in three unrelated Chinese pedigrees with HME. Real time RT-PCR, pulse-chase, immunofluorescent assay and immunoprecipitation will be used to detect the mutant EXT2 expression and the interaction between EXT1 and EXT2. To explore the influence of EXT2 C339Y on heparan sulfate (HS) synthesis, the content and length of HS will be tested by RPIP-HPLC. The glycotransferase activity of EXT2 and EXT1/EXT2 complex will be analyzed by using corresponding enzymatic reactions. Through this study, we will illustrate the molecular mechanisms of EXT2 C339Y mutation in HME. This study will also provide experimental basis for the further study of malignant transformation of HME so as to make it possible to take intervention to reduce the malignant risk. What's more, this study will make sense to the HS biosynthesis enzyme targeted pharmaceutical research.

遗传性多发性骨软骨瘤（HME）是常见的常染色体显性遗传性骨骼异常性疾病，EXT基因家族与其发生相关，约5％的HME患者会恶变为软骨肉瘤。本项目拟对本课题组前期在三个独立HME家系中发现的EXT2基因新突变C339Y进行功能研究。通过实时定量RT-PCR、同位素代谢示踪、激光共聚焦、免疫沉淀等技术，探讨EXT2 C339Y突变对EXT2蛋白合成、代谢、稳定性以及EXT2与EXT1相互作用的影响；通过RPIP-HPLC技术分析硫酸乙酰肝素（HS）链的含量和长度，探讨EXT2 C339Y突变对HS合成的影响；利用酶促反应探讨突变对EXT2和EXT1/EXT2糖基转移酶活性的影响。本研究将阐明EXT2 C339Y突变对HS合成的影响及其导致HME的分子机制，将为进一步研究肿瘤的恶变机制提供实验基础，有利于对疾病加以干预降低恶变风险，同时对以HS生物合成中的酶为治疗靶点的药物研发也具有一定意义。

遗传性多发性骨软骨瘤（HME）为常染色体显性遗传性骨骼发育疾病，目前主要由EXT1、EXT2、EXT3及EXT-like家族基因突变造成。本项目前期在三个HME家系中同时发现了EXT2 C339Y突变，该突变在ExAc数据库及1000基因组数据库中均未见报道。体外实验揭示C339Y突变不影响EXT2蛋白质的表达水平，EXT2与EXT1的结合及硫酸乙酰肝素(HS)的表达，但是，我们发现EXT2 C339Y突变体与野生型相比其糖胺聚糖（GAGs）的分泌量显著增加，而细胞内则明显降低，该现象提示C339Y突变可通过影响GAGs细胞分布进而促进软骨细胞的增殖分化，在HME发病过程中发挥重要的作用。