凝血因子无义突变引起重型血友病的分子机制及药物治疗

The hemophilia, which is caused by the mutation in the factor Ⅷ/Ⅸ (FⅧ/FⅨ)gene, is known as an X-linked recessive disease with high disability rate and mortality. At present, due to lacking of the eradicative therapy, the patients needed infusion the expensive blood coagulation factor in their lifetime. One of the most important molecular mechanisms in severe hemophilia is nonsense mutation, carrying premature termination codon(PTC),which lead to the production of nonfunctional truncated protein or the degradation of PTC-bearing mRNA.Because the percentage of nonsense mutation in hemophilia in our country is not clear, we try to use PCR and direct DNA sequencing to screen and identify the percentage of nonsence mutation in patients with hemophilia A and B. Furthermore,isolating and culturing the bone marrow mesenchyme stem cells of the patient's family members with nonsense mutation, induced bone marrow mesenchyme stem cells differentiate into hepatocyte-like cells and detected the FⅧ/FⅨ protein level and activities. Then, after treated with different concentration of chemical compound PTC124, testing the variations of FⅧ/FⅨ mRNA and protein quantities and activities,further research the mechanism and treatment of hemophilia.

血友病是凝血因子Ⅷ/Ⅸ基因突变引起的性连锁隐性遗传性出血性疾病，致残率及死亡率高，需终生输注凝血因子，药物治疗尚属空白。凝血因子基因中产生提前终止的无义密码子(PTC)，启动无义密码子介导的mRNA降解（NMD），导致凝血因子的表达质/量降低，引起重型血友病的发生。由于目前在我国血友病中无义突变的比例尚不明确,本课题拟采用PCR和直接测序进一步筛查血友病A/B患者所有外显子的无义突变，明确无义突变在血友病发病中的比例；分离携带无义突变家系成员骨髓间充质干细胞，诱导分化为肝样细胞，检测其FVIII/FIX的含量及活性，进行不同浓度非氨基糖苷类小分子化合物（PTC124）药物干预，检测药物干预前后FⅧ/FⅨ的 mRNA、蛋白表达量及活性变化，为血友病的发病机制和药物治疗奠定基础。

血友病由于凝血因子Ⅷ/Ⅸ基因突变引起凝血因子VIII/IX缺乏，分别导致血友病A和血友病B。其中一个最重要的导致重型血友病的分子机制为无义突变，使得凝血因子基因中产生提前终止的无义密码子(PTC)，启动无义密码子介导的mRNA降解（NMD），或导致无功能的截断型凝血因子的产生。首先对于山西省血友病中心223例血友病A及23例血友病B患者联合使用长距离PCR、双管多重PCR、直接测序法及多重探针链接技术（MLPA）进行致病基因的筛查。其中18例血友病A患者及2例血友病B患者由无义突变导致。其次，我们选择发生频率高的无义突变位点（FⅧ：W14X， R1696X，K1827X，R2307X；FⅨ：R29X，R116X，W194X，R333X）作为研究对象，构建相应的上述FⅧ、FⅨ无义突变体，进行不同浓度氨基糖苷类药物庆大霉素及非氨基糖苷类小分子化合物（PTC124）药物干预，检测药物干预前后FⅧ/FⅨ的 mRNA、蛋白表达量及活性变化。结果显示FⅧ和 FⅨ无义突变体的mRNA及蛋白水平在PTC124和庆大霉素干预后明显提高。PTC124和庆大霉素均具有促通读疗效。