CTLA-4在重症肌无力患者中的表达及其多态性导致的无效转录研究

CTLA-4 gene polymorphisms result in inefficient transcription in myasthenia gravis

目的 探讨重症肌无力(MG)患者细胞毒性T淋巴细胞相关抗原 -4 -(CTLA -4 )的表达状况及由CTLA -4基因启动区多态性导致的不同遗传易感性机制。方法 ELISA法测定MG患者血清中sCTLA 4的水平;限制性片段长度多态性分析用于检测启动区 1772、16 6 1位点的多态性;转录因子NF- 1和c -EBPβ结合位点通过染色质免疫沉淀实验(CHIP)得以验证。结果 MG患者血清sCTLA 4的表达水平与等位基因的突变相关,携带T→C -1 772 突变基因的患者可表达高水平的sCTLA -4。TC -1 772 基因型的频率在MG患者尤其是胸腺瘤亚组明显高于对照组,而G -1 6 6 1 等位基因和GG -1 6 6 1 基因型的频率在MG患者则显著降低。当 1772位点的等位基因是T而非C时,存在转录因子NF- 1结合位点;同样,当 16 6 1位点的等位基因是G而非A时,存在转录因子c -EBPβ结合位点,刀豆蛋白A(ConA)、植物血凝素(PHA)能促进NF -1和c- EBPβ的这种位点特异性转录活性。结论 MG患者CTLA 4表达异常,启动区C T 1 772 和A G 1 6 6 1 多态性可导致无效转录,T→C 1 772 的突变能影响基因的剪接,干扰蛋白的表达和功能,阻止了负性调节信号的传递而致发病。

Objective To study the expression of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) in patients with myasthenia gravis (MG) and molecular immunology pathogenesis of MG caused by CTLA-4 gene promoter polymorphism. Methods ELISA assay was used to determine the expression level of serum sCTLA-4. Two independent single nucleotide polymorphisms (SNP) of CTLA-4 at position -1772 and position -1661 in the promoter were analyzed by RFLP. Transcription factor NF-1 and c/EBPβ binding site were confirmed by CHIP assay. Results sCTLA-4 levels in patients serum are correlated with the haplotype and genotype. Patients with CT -1772 genotype or CT -1772 AA -1661 haplotypes have higher levels of sCTLA-4 than patients with TT -1772 or TT -1772 AA -1661. The frequency of the CT -1772 genotype is significantly higher in MG patients and especially, in MG with thymoma as comparing with controls. Meanwhile, the frequency of the G -1661 allele and GG -1661 genotype is lower in MG patients. These SNPs change the sequence of transcription factor binding sites: NF-1 and c/EBPβ. T→C -1772 mutation deletes NF-1 binding site while A→G -1661 mutation creates c/EBPβ binding site, which is confirmed by CHIP assay. DNA variants lose site-specific binding activity of transcription factor regulated by lectin. Conclusion Patients with MG have an aberrant expression of the CTLA-4 products and C/T -1772 and A/G -1661 polymorphisms which result in inefficient transcription of CTLA-4 gene. T→C -1772 mutation also affect gene splicing. The two SNPs may function as an etiological factor to disease susceptibility.