AQP4基因多态性与视神经脊髓炎的相关研究

Correlation between Polymorphism of AQP4 Gene and Pathogenesis of Neuromyelitis Optica

目的:从基因水平研究分析AQP4基因外显子区域单核苷酸多态性位点(SNP)以及其在视神经脊髓炎(NMO)发病机制中的作用。方法:将145例研究对象分为4组:NMO及高危综合征组25例,多发性硬化组(MS)40例,脑梗死组(CI)40例,正常对照组(NC)40例。对所有入选对象的全血DNA进行AQP4基因的单核苷酸位点分析,并通过特异性的定点诱变技术对携带AQP4基因表达序列的p EGFP质粒进行定点诱变,根据相应SNP位点突变生成相应突变质粒,脂质体转染法将相应质粒转染细胞建立抗AQP4抗体检测细胞株。根据相关统计学数据分析AQP4突变位点细胞株与滴度的相互关系并分析其在NMO发病机制中的可能作用。结果:NMO组发现SNP位点2个,位于2号外显子,突变位点为R108T及I110N。3、4号外显子区域未发现SNP位点,多发性硬化组、脑梗死组及正常对照组未发现相应的单核苷酸位点。组间单核苷酸多态性位点的分布差异有统计学意义(P<0.05),组间抗体滴度水平差异具有统计学意义(P<0.05)。结论:不同的SNP位点突变可能会导致表达蛋白一级结构发生微小的但却可以改变其抗原性的变化,抗原抗体反应的强度不同可能是导致不同突变组间滴度差异的原因,推测基因水平的变化可能参与了NMO的发病机制。

Objective To study the single nucleotide polymorphism（ SNP） locus of AQP4 gene in exons domain,and explore its possible role in the pathogenesis of neuromyelitis optica（ NMO）. Methods Total 145 objects were divided into group NMO（ n = 25,including 23 cases of NMO,2 cases of high risk syndrome）,group multiple sclerosis（ MS）（ n = 40）,group cerebral infarction（ CI）（ n = 40）,normal control group（ NC）（ n = 40）. The SNP locus analysis of whole blood DNA were preformed in all the objects,and according to the corresponding SNP loci,the corresponding mutant p EGFP plasmids with AQP4 gene expression sequences were generated by specific site-directed mutagenesis. The mutant plasmids were transfected into cells by liposomes and the corresponding cell lines were established. The correlation between mutation loci and pathogenesis of NMO was analyzed by comparing the titers of different mutative cell lines. Results Two new SNP loci,R108 T and I110 N located at exon 2 were found in group NMO,but no SNP locus was found at exon 3 and 4. Moreover,no SNP locus was found in the other three groups. The distribution of loci between group NMO and the other groups was significant difference（ P 0. 05）,and the titres between single and double site mutation groups was different（ P 0. 05）. Conclusion Different SNP loci mutation could induce the weeny change of primary construction of AQP4 protein,which is enough to change its antigenicity,and then the different reactive intensity of antigen-antibody may induce the difference of titers during different mutative cell lines. Therefore we speculated that the level of gene changes may contribute to the pathogenesis of NMO.