【作者】 任韵清；

【导师】 张学军； 杨森； 刘建军；

【作者基本信息】 安徽医科大学 ， 皮肤病与性病学， 2009， 博士

【摘要】 背景白癜风是一种皮肤科常见复杂疾病,人群中患病率约为0.1%-2%。尽管白癜风的病因和发病机制尚不明了,近年来的大量研究表明遗传和免疫因素在白癜风发病中起到重要作用。目前已有文献报道了6个白癜风易感位点,相当多的候选基因研究亦成为白癜风遗传学研究的热点。在前期研究中,本课题组对143个白癜风家系共736个体(包括369名患者和367名无白癜风表型的正常对照)进行全基因组扫描连锁分析,证实了染色体22q12区域存在中国汉族人白癜风的易感位点。X盒结合蛋白(XBP1)基因位于该定位区域,是一种重要的转录因子。X盒为HLA II类基因启动子区域的一个调控因子,与正向转录因子结合后,增强下游基因的表达,而XBP1编码的蛋白就是结合于X盒的这种转录因子,HLA-DR及DP的表达在很大程度上受XBP1基因的影响。结合白癜风与HLA自身免疫的相关性,该基因可以看作是白癜风的一个非常重要的功能候选基因。目的通过一系列的遗传和功能研究系统性探讨位于染色体22q12区域的XBP1基因与中国汉族人群白癜风发病的相关性。方法首先在319个中国汉族白癜风患者(有家族史)及294个正常对照中,对XBP1基因的启动子区域、外显子、外显子和内含子交界区域测序以及部分内含子区域直接测序,进行SNP筛查;然后分别对另外两个独立样本(365病例/404对照和1402病例/1288对照,包括94个trios)进行大规模基因分型以验证初期得到的位于启动子区域的一个SNP位点的关联性;并采用序列特异性引物-多聚酶链反应法检测了HLA-DRB4*01,DRB1*04,DRB1*07,DRB1*12的基因频率分布,应用Logistic回归进行两个位点的交互作用分析;构建XBP1基因该SNP位点的重组质粒的荧光素酶双报告基因载体,转染人HaCat和Mel-RM细胞,进行瞬时表达分析,以检测此多态对基因表达调控作用;分别采用实时定量PCR和免疫组化法从mRNA和蛋白水平分别检测XBP1基因在白癜风患者皮损和非皮损中是否存在表达差异。结果①在319个白癜风患者和294个正常对照中对XBP1基因进行测序,共检出34个SNPs,其中位于启动子区域的SNP rs2269577等位基因频率在白癜风病例和对照间存在显著性差异(p_trend=0.007, OR=1.36, 95%CI=1.09–1.71);②在另外的365个白癜风患者和404个正常对照进行测序,结果仍提示该SNP位点有显著意义(p_trend =0.008, OR=1.31, 95%CI=1.07–1.59);③继续扩大样本量,在另外的独立样本(1402病例/1288对照,包括94个trios)的SNP关联分析,结果进一步证实rs2269577位点的等位基因频率在白癜风患者和正常对照间存在显著性差异(p=0.003,OR=1.18, 95%CI=1.06–1.32; TDT检验p=0.005, OR=1.93, 95%CI=1.21–3.07);合并分析全部2086例患者和1986例对照,所得结果显示rs2269577等位基因频率在病例和对照组间存在极显著相关(p_trend=2.94×10-6, OR=1.23, 95%CI=1.13–1.35);④在对1755个病例和1742个对照的HLA分型结果中发现,HLA-DRB1*07与白癜风存在显著相关(p= 4.53×10-21, OR=1.98, 95%CI=1.72-2.29),并与rs2269577存在交互作用(p=0.033);⑤荧光素酶报告基因表达结果显示rs2269577 C等位基因可以增强XBP1启动子的转录活性,含有C等位基因的质粒所诱导的转录活性明显高于含有G等位基因所诱导的转录活性(P<0.05);⑥对白癜风患者皮损和非皮损的XBP1 mRNA和蛋白水平的检测均提示白癜风皮损中XBP1的表达增高,且携带危险等位基因C的白癜风皮损表达比未携带等位基因C的皮损表达亦有增高趋势。结论XBP1基因启动子区域的多态位点rs2269577与中国汉族人群白癜风存在显著相关性。其危险等位基因C可能通过提高XBP基因的转录活性,促进基因的表达从而参与白癜风的发病机制。更多还原

【Abstract】 Background Vitiligo is a genetically complex skin disorder that afflicts 0.1%-2% of the population throughout the world. Although the etiology of vitiligo remains obscure, the inherited nature and its frequent association with autoimmune disease indicate that genetic and immunological components play a role in the pathogenesis of the disease. Up to date, linkage analysis has identified six significant susceptible loci harboring the genetic risk factors of vitiligo and many candidate genes have been proposed. Our previous genome wide linkage scan of 143 Chinese families identified a significant genetic susceptibility locus for generalized vitiligo on chromosome 22q12. X-box binding protein 1(XBP1) gene is within this locus. With a view to the autoimmunity feature of vitiligo supported by the significant linkage to the MHC region on 6p21-p22 and evidences for the association of HLA-DR with vitiligo, XBP1 is a strong biological candidate gene for vitiligo due to its plausible role in the development of the disease through its interaction with HLA-DR.Objective To systematic investigate the association between the XBP1 gene common variation and susceptibility to vitiligo in Chinese Population.Method We performed a series of genetic association and functional analyses. Firstly, we sequenced all the exons, exon-intron boundaries as well as some 5’and 3’flanking sequences of XBP1 in 319 cases and 294 controls of Chinese Hans. We then performed an association analysis by genotyping a putative regulatory polymorphism within the promoter region of XBP1 in other two independent samples involving 365 cases vs 404 controls and 1402 cases vs1288 controls, including 94 trios. We also genotyped HLA-DRB4*01,DRB1*04,DRB1*07,DRB1*12 alleles using PCR-SSP and tested the epistatic interaction between this variation and HLA-DR alleles using logistical regression analysis. Subsequently, we performed an in-vitro reporter assay analysis to evaluate whether the promoter sequences carrying different SNP alleles have differential promoter activity. Then we analyzed the expression of XBP1 mRNA and protein level in the paired lesional and non-lesional skins using quantitative real-time PCR and immunohistochemistry analysis, respectively.Result We sequenced all the exons, exon-intron boundaries as well as some 5’and 3’flanking sequences of XBP1 in 319 cases and 294 controls of Chinese Hans. Of the 8 common variants identified, the significant association was observed at rs2269577 (p_trend=0.007, OR=1.36, 95%CI=1.09–1.71), a putative regulatory polymorphism within the promoter region of XBP1. We then sequenced the variant in additional 365 cases and 404 controls and found the supporting evidence for the association (p_trend =0.008, OR=1.31, 95%CI=1.07–1.59). To further validate the association, we genotyped the variant in another independent sample of 1402 cases and 1288 controls, including 94 parent-child trios, and confirmed the association by both case-control analysis (p=0.003, OR=1.18, 95%CI=1.06–1.32) and the family-based TDT test (p= 0.005, OR=1.93, 95%CI=1.21–3.07). The analysis of the combined 2086 cases and 1986 controls provided highly significant evidence for the association (p_trend= 2.94×10-6, OR=1.23, 95%CI=1.13–1.35). We also found the HLA-DRB1*07 allele to be significantly associated with vitiligo (p= 4.53×10-21, OR=1.98, 95%CI= 1.72-2.29) and observed a significant interaction between rs2269577 and HLA-DRB1*07 (p=0.033). Our subsequent functional study showed that the risk-associated C allele of rs2269577 had a stronger promoter activity than the non-risk G allele (P<0.05), and there was an elevated expression of XBP1 in the lesional skins of patients carrying the risk-associated C allele.Conclusion Our study has found a common regulatory variant, rs2269577, in the promoter region of XBP1 that is associated with increased risk for vitiligo and this association is probably due to the transcriptional modulation of XBP1 expression which was shown to be important for the development of vitiligo lesion.更多还原