【作者】 唐晓武；

【导师】 叶冬青； 潘发明；

【作者基本信息】 安徽医科大学 ， 流行病与卫生统计学， 2009， 博士

【摘要】 强直性脊柱炎(Ankylosing Spondylitis,AS)是一种血清学阴性,主要侵犯中轴骶骼关节的慢性炎症性自身免疫性疾病,其临床发病率较高,多见于青壮年男性,且有较高的家族聚集性。该病目前病因尚不明确,其发病被认为与多种因素有关,是一种十分复杂的疾病,又称为多因子疾病(multifactorial disease)。虽然针对AS的病因和发病机制,已经提出过很多学说,包括分子模拟学说、致关节炎多肽学说、HLA-B27分子非折叠蛋白理论假说等,但到目前为止,AS发生的确切原因尚不清楚。国内外大量研究表明,遗传因素是强直性脊柱炎发病的主导因素,通过全基因组扫描发现,多个区域与AS易感性有关[1,2]。其中以HLA-B27为代表的HLA区基因与AS的关联性最强,HLA-B27与AS的遗传易感性研究已经经历了30多年,在不同国家和地区都得到了证实,但确切的发病机理还不是十分清楚,而且整个HLA区域只能解释AS遗传易感性的40-50%,因此,非HLA区基因与AS易感性关联研究将是下一步研究的重点和热点。本研究旨在通过较大规模的强直性脊柱炎的遗传流行病学调查,借助人类基因组计划的研究成果,明确中国汉族人群易感区域易感基因(免疫球蛋白受体同系物基因)的单核苷酸多态性及单倍型与AS的遗传关联性,构建中国人群该易感家簇基因中与AS有关联意义的单核苷酸多态性(Single nucleotide polymorphism,SNP)和单倍型,并初步探讨基因和环境的交互作用。本研究采用经典病例对照设计方案,以问卷调查的方式,共收集169例HLA-B27阳性患者(107男性和62女性)和184个HLA-B27阳性健康对照(112个男性和72个女性)的一般流行病学资料和DNA血样,选择免疫球蛋白受体同系物家族基因中的FCRL1(1个功能SNP)、FCRL3(3个功能SNP)、FCRL4(1个功能SNP)、以及FCRL5(4个功能SNP)四个基因中的9个功能SNP(即改变氨基酸序列的非同义突变),通过PCR扩增后,用连接酶检测反应的方法筛查这些功能SNP位点的基因型及其在中国人群中的频率;并构建中国汉族人群中与AS有关联的SNPs和单倍型。用EPIdata 3.0软件建立数据库,数据经检错、整理后分别应用社会科学统计软件包(SPSS10.01)和经典的病例对照在线分析软件Shesis(http://analysis.bio-x.cn/myAnalysis.php)进行统计学分析。应用非条件多元Logistic回归方法分析AS可能存在的基因和环境交互作用。169个患者来自安徽医科大学第一附属医院的门诊和住院病人,平均年龄为26.38±6.32岁(其中最小为14岁,最大为78岁)。通过对FCRL中的五个基因中的9个SNP位点进行筛查,结果未发现FCRL1、FCRL3和FCRL4中的SNPs与AS有遗传关联性,而FCRL5基因中的2个SNPs(rs12036228和rs6427384)与AS存在遗传上的关联性,进一步分析提示rs12036228位点上C等位基因的出现增加AS发病的风险性,而rs6427384位点上T等位基因的出现增加AS发病的风险性,在对上述2个SNP位点进行单倍型分析后发现:携带有C-C单倍型的个体对AS发病具有保护性作用,而携带有C-T单倍型的个体则对AS的发病具有更高风险性。同时我们对中国汉族人群的HLA-B27及其亚型的结果进行筛查,研究结果提示在中国汉族人群中存在四种亚型(B * 2703,B * 2704, B * 2705和B * 2706),其中病例组中以B*2704和B*2705两个亚型较常见,分别占53.8%和34.9%,对照组中同样是B*2704和B*2705两个亚型较常见,病例组这两个亚型的比例高于对照组,但结果无统计学意义(χ2=7.014,P=0.056);另外对影响总体背痛程度因素进行单因素分析,没有发现对总体背痛程度有统计学意义的因素,故没有进一步进行多元分析。通对BASFI影响因素进行多因素分析结果提示性别、病程、学历、饮酒史可能是BASFI影响因素;同时我们将可能与AS发病有关的环境因素和可能易感SNP位点及交互项进行非条件logistic回归的拟合分析,结果显示:在调整其他自变量的情况下,最后进入模型的两个变量是有感染史和家庭居住环境,但没有发现FCRL5基因与上述环境因素之间存在交互作用。综上所述,我们认为在中国汉族人群中免疫球蛋白受体同系物家族基因(位于1q23-24区域)可能与强直性脊柱炎的遗传易感性有关,特别是FCRL5基因;同时还受到环境因素的作用,但本次研究未发现基因和环境因素对AS存在交互作用。至于不同HLA-B27亚型的AS在该区域的遗传易感性是否有差别将有待于进一步研究和证实。更多还原

【Abstract】 Ankylosing spondylitis (Ankylosing Spondylitis, AS) is a seronegative, chronic inflammatory autoimmune disease, which mainly affects the axial skeleton of the young men with a higher clinical incidence and family aggregation. The exact cause of the disease is currently unknown, it is generally accepted that AS is a very complex disease, also known as a multi-factorial diseases. Although many previous studies aimed at the etiology and pathogenesis of AS has been put forward many theories, including molecular simulation theory, theory of peptide-induced arthritis, HLA-B27 molecules of non-folded protein hypothesis, etc.But so far, the exact cause of AS remains unclear. However, a large number of domestic and overseas studies have shown that genetic factors take an important part in the pathogenesis of AS, a genome-wide scan have revealed multiple regions associated with AS susceptibility [1,2]. Among them, HLA region, represented by HLA-B27 gene, have robust association with AS, studies on HLA-B27 and the genetic susceptibility to AS has gone through more than 30 years, which have been confirmed in different countries and regions, but the exact pathogenesis of AS is still not very clear, and the entire HLA region can explain only a fraction (40 -50%) of AS genetic susceptibility, therefore, the non-HLA genes may also be associated with the susceptibility to AS.The purpose of this study was to explore the association of single nucleotide polymorphisms (SNPs) and haplotypes of Fc receptor-like genes with susceptibility to AS in a Chinese Han population, and to explore the interaction of genes and the environmental factors.Questionnaire investigation was performed to obtain information in this case-control study. we collected 169 cases of HLA-B27 positive AS patients (107 males and 62 females) from the department of rheumatology, the first affiliated hospital of Anhui Medical University, including outpatient and inpatient. 184 HLA-B27 positive healthy controls (112 male and 72 female) are also recruited from the same region. Their general epidemiological data and DNA samples were collected. in this study, we have chosen an important candidate family gene (immunoglobulin receptor homologue family gene) for AS . 9 non-synonymous mutation functional SNPs, from FCRL1 (1 SNP), FCRL3 (3 SNPs), FCRL4 (1 SNP) and FCRL5 (4 SNPs) respectively, are genotyped by PCR ligase detection reaction method to obtain their genotype frequency and haplotype in population. The database was established by EPIdata 3.0 software. The Social Science Statistical Package (SPSS10.01) and the classic case-control on-line analysis Software Shesis (http://analysis.bio-x.cn/myAnalysis.php) are used for statistical analysis.In total,169 patients are recruited, with an average age of 26.38±6.32 years, ranging form 14 to 78 years. through multiple SNP loci screening, except for FCRL5, FCRL1, FCRL3 and FCRL4 gene was not associated with AS genetic susceptibility.2 SNPs(rs12036228 and rs6427384) from FCRL5 gene is associated with AS. C allele in rs12036228 occurred more frequently in case group than in control group,and T allele in rs6427384 more frequently in case group. haplotype C-T, On the basis of the two SNPs above, occurres more frequently in case group than in control group,but haplotype C-C more frequently in control. Meanwhile, we analyzed the HLA-B27 gene subtypes and find that there are four subtypes (B * 2703,B * 2704, B * 2705 and B * 2706) in the Chinese Han population, in which B * 2704 and B * 2705 frequence in case group are more common than the others, accounting for 53.8% and 34.9% and the two subtypes is also more common in control group. The difference of HLA-B27 gene subtype frequence is not statistically significant between case group and control group ( 2=7.014, P = 0.056). In the study we find that environmental risk factors is not associated with the general back pain level in patients with AS. the results of multivariate analysis shows that Such factors as gender, disease duration, education, drinking history may be associated with the BASDFI of patients with AS. Meanwhile, the environmental factors and two SNPs associated with AS above is analyzed by non-conditional logistic regression analysis on the basis of other independent variables adjusted. the results showed that the two variables(infection history and family living environment) go into the model, but no interactions of FCRL5 gene with environmental factors was found associated with AS.In summary, our results suggested that FCRL family genes (located on 1q21-23 region), in particular FCRL5 gene, may be related to the genetic susceptibility to AS in the Chinese Han population. In addition, environmental factors also paly an important role in the onset of AS. However, no interactions of FCRL genes with environmental factors was found. Nevertheless, the sample size is relatively small, further studies in different populations are still needed.更多还原