Wang Qing-Song, Xiao Hong-Qiong, Chen Hua-Xian, Liu Yan-Ping, Ding Xu-Dong
Department of Rehabilitation Medicine, Xiangyang No. 1 People's Hospital, Hubei University of Medicine, Xiangyang, Hubei 441000, P.R. China.
Exp Ther Med. 2017 Dec;14(6):6017-6021. doi: 10.3892/etm.2017.5267. Epub 2017 Oct 6.
The single nucleotide polymorphism (SNP) site within the aquaporin (AQP)-4 gene exons and its possible role in the pathogenesis of neuromyelitis optica (NMO) were studied. From March 2010 to June 2012, 72 patients with NMO from Xiangyang No. 1 People's Hospital, Hubei University of Medicine were enrolled in the NMO group. At the same time, 80 patients with multiple sclerosis (MS) were enrolled in our study as the MS group. Blood samples were collected and DNA was extracted for analysis of SNP sites of gene. Specific site-directed mutagenesis method was used for site-directed mutagenesis on plasmid enhanced green fluorescence protein carrying gene. Mutant plasmids were constructed and used for transfecting cell lines. The differences of anti-AQP4 antibody level in the cell line were analyzed. The possible correlation between gene SNP sites and the pathogenesis of NMO were analyzed. In the NMO group, 6 SNP sites in gene were located in exons 2 and 5. These included R108T, I110N, E280R, D281R, P295R and E317M. There was no SNP site in exons 1, 3 and 4. In the MS group, no SNP site was found in gene. R108T, I110N, R108T/I110N, E280R/D281R, P295R and E317M cell lines were constructed in the NMO group, and anti-AQP4 antibody in the serum was compared between R108T/I110N, E280R/D281R and E317M cell lines and the original HEK293T cell line. The difference was statistically significant (P<0.05). The positive rate of anti-AQP4 antibody titer in serum was compared between R108T, I110N, R108T/I110N, E280R/D281R, P295R and E317M cell lines in the NMO group and the original cell line in the MS group. In conclusion, SNP sites in gene in patients with NMO may lead to some conformational changes in AQP4 protein. This affects the antigenicity of AQP4 protein. The different intensity of antigen-antibody reaction may cause the differences of titer observed between the different mutant cell lines.
研究了水通道蛋白（AQP）-4基因外显子内的单核苷酸多态性（SNP）位点及其在视神经脊髓炎（NMO）发病机制中的可能作用。2010年3月至2012年6月，将湖北医药学院附属襄阳市第一人民医院的72例NMO患者纳入NMO组。同时，将80例多发性硬化症（MS）患者纳入本研究作为MS组。采集血样并提取DNA，用于分析该基因的SNP位点。采用特异性定点诱变方法对携带该基因的质粒增强绿色荧光蛋白进行定点诱变。构建突变体质粒并用于转染细胞系。分析细胞系中抗AQP4抗体水平的差异。分析该基因SNP位点与NMO发病机制之间的可能相关性。在NMO组中，该基因的6个SNP位点位于外显子2和5。这些位点包括R108T、I110N、E280R、D281R、P295R和E317M。在外显子1、3和4中未发现SNP位点。在MS组中，该基因未发现SNP位点。在NMO组构建了R108T、I110N、R108T/I110N、E280R/D281R、P295R和E317M细胞系，并比较了R108T/I110N、E280R/D281R和E317M细胞系与原始HEK293T细胞系血清中抗AQP4抗体的差异。差异具有统计学意义（P<0.05）。比较了NMO组中R108T、I110N、R108T/I110N、E280R/D281R、P295R和E317M细胞系与MS组原始细胞系血清中抗AQP4抗体滴度的阳性率。总之，NMO患者该基因中的SNP位点可能导致AQP4蛋白发生一些构象变化。这影响了AQP4蛋白的抗原性。抗原-抗体反应强度的不同可能导致不同突变细胞系之间观察到的滴度差异。
