AQP4基因多态性与视神经脊髓炎遗传易感性的相关研究

Studies on association of AQP4 gene polymorphism and Neuromyelitis Optica genetic susceptibility

目的初步探讨AQP4外显子区域基因多态性与我国视神经脊髓炎(NMO)患者遗传易感性的关系。方法用常规酚/氯仿法从患者及正常对照组全血标本中提取基因组DNA,应用PCR扩增AQP4外显子区基因,再对PCR产物进行纯化及正反双向测序,然后通过与GeneBank公布的人类AQP4基因序列比较,寻找阳性突变位点,并比较各阳性突变位点在NMO、MS、CI、NC中突变率的差异。然后通过特异性的定点诱变技术对pEGFPN1-AQP4质粒诱导相应位点的突变生成相应的突变AQP4质粒,采用脂质体转染法,将相应的质粒转染进入HEK-293T细胞稳定表达后进行NMO-IgG抗体滴度的检测。应用SPSS11.5统计软件进行相关数据分析。结果我们发现视神经脊髓炎组5号外显子区域有单核苷酸多态性(SNP)位点4个,突变的位点为C9414T、G9416C、A9458C、C9524A,而多发性硬化、脑梗死及正常对照组均未发现相应的SNP位点,同时NMO患者1号外显子区域未发现SNP位点。对不同的SNP位点突变细胞株进行相关检测后发现,组间SNP位点分布的差异有统计学意义(P<0.05),组间血清抗体滴度水平具有统计学差异(P<0.05)。结论 AQP4基因5号外显子区域共发现了4个阳性突变位点,AQP4基因编码区的变异或多态性可影响靶蛋白AQP4的表达,进而可能参与NMO的发病机制。

Objective To investigate the correlation between polymorphism in the exon region of AQP4 gene and ge- netic susceptibility of Neuromyelitis Optiea （NMO） patients in China. Methods 72 Neuromyelitis Optiea patients （NMO） ,68 multiple sclerosis patients（MS） , 100 cerebral infarction patients（CI） and 100 normal people as control group were investigated. Genomic DNA was abstracted from the whole blood of specimen. PCR programme were performed to am- plify the exon 1 and exon 5 regions of AQP4 gene,and PCR products were purified and sequenced bidirectionally. The se- quences were then blasted with AQP4 gene of human species from Genebank to search for positive mutations,and the differ- ences of mutations were analyzed among NMO,MS,CI and NC groups. PEGFP-N1-AQP4 plasmid was used to induce muta- tions to generate the mutations of AQP4 plasmid by using specific site-directed mutagenesis method. The plasmid was then transfeeted into HEK-293T cells through the lipofeetion method and NMO-IgG antibody titers were detected after the cells expressed stably. Data were analyzed with SPSSll. 5 software. Results 4 SNP loei（C9414T,G9416C,A9458C,C9524A） were found in exon 5 of AQP4 gene in NMO group,neither in MS,CI nor NC group. No SNP was found in exon 1 in NMO group. It was found that both the distribution of SNPs and the serum antibody titers statistically differentiated among the four groups （ P 〈 0.05 ） after detecting the cell lines of different SNP mutations. Conclusions At least 4 SNP loci were found in exon 5 of AQP4 gene. It can be infered that the mutation or polymorphism of the coding region of AQP4 gene can influence on the expression of target protein AQP4,and thus may be involved in the pathogenesis of NMO.