【作者】 刘湘；

【导师】 胡丽华；

【作者基本信息】 华中科技大学 ， 临床检验诊断学， 2010， 博士

【摘要】 强直性脊柱炎(Ankylosing Spondylitis, AS)是一种与免疫相关的复杂疾病,受遗传和环境等多种因素的共同影响。多个研究显示遗传因素在AS发病中起着十分重要的作用。位于6号染色体短臂上的人类主要组织相容性符合体(major histocompatibility complex, MHC)是与AS相关的主要遗传位点。MHCⅠ编码的人类白细胞抗原B27(human leukocyte antigen-B27, HLA-B27)是迄今所知的、与疾病相关中最强的HLA抗原。全基因组扫描发现除HLA-B27基因外,还有其他的非MHC基因参与AS的发病机制,由于AS属于自身免疫性疾病范畴,一些和免疫相关基因与疾病的关联性研究,已成为研究AS遗传背景的一大亮点,例如白介素(interleukin, IL)基因、程序性凋亡(programmed cell death, PDCD)基因等。然而受多种因素影响,造成这些研究结果并不完全一致,尤其是各种基因单核苷酸多态性(single nucleotide polymorphisms, SNP)与AS的关系尚未完全澄清,有待进一步研究。本课题首先采用聚合酶链反应-序列特异性引物(polymerase chain reaction-sequence-specific primer, PCR-SSP)法研究了中国湖北地区HLA-B27基因亚型与AS的相关性,然后再应用聚合酶链反应-限制性内切酶片段长度多态性(polymerase chain reaction-restriction fragment length polymorphisms, PCR-RFLP)技术对PDCD-1基因的3个SNP进行了AS疾病的关联研究,最后,应用meta分析探讨了白介素-23受体(interleukin-23 receptor, IL-23R)基因中10个SNP与AS的相关性。本研究共分以下三部分：目的：分析在中国湖北地区HLA-B27基因亚型与AS的相关性,探讨HLA-B27基因亚型在AS发病中的作用,同时评价HLA-B27基因亚型检测的临床意义。方法：收集HLA-B27阳性的研究对象共346例。其中AS患者190例,男129名,女61名,平均年龄(39±9)岁,健康对照者156名,来男81名,女75名,平均年龄(30±11)岁。采用病例对照研究方案,通过PCR-SSP进行HLA-B27基因亚型检测。结果：共检出6种亚型,分别是B*2702、B*2703、B*2704、B*2705、B*2706及B*2713。在AS患者与健康对照组中,都以B*2704亚型(72.1%和56.4%)为主,其次为B*2705亚型(21.6%和31.4%),两组中均检测到频率较低的B*2702亚型(0.5%和1.3%)及B*2703亚型(5.3%和8.3%),而B*2713亚型只在AS组测到1例(0.5%),B*2706亚型只在健康对照组测到4例(2.6%)。AS患者组与健康对照组相比,尽管B*2703及B*2705亚型在对照组频率较高,但两组间的差异均无统计学意义(χ2=1.301,P>0.05；χ2=4.032,P>0.05),OR值和95%可信区分别为为0.611和0.601,0.262-0.701和0.371-0.937。B*2704亚型在两组中所占比例均为最高且组间存在显著性差异(χ2=9.279,P<0.01),OR值为1.997,95%可信区为1.279-3.119,显示B*2704亚型与AS发病危险度相关。结论：中国湖北地区人群中B*2704和B*2705为主要亚型,其中B*2704与AS呈强相关,B*2706可能与AS呈负相关,其他低频率基因亚型与AS的相关性有待进一步探讨,HLA-B27基因亚型可作为AS早期诊断的重要参考指标。另外本研究所使用的PCR-SSP法能检测出29个HLA-B27亚型(B*2701-B*2730,B*2722除外),能满足当前对HLA-B27基因亚型检测的需要。目的：分析PDCD-1基因SNP在AS患者与健康对照人群中的分布,比较两组人群分布差异,探讨PDCD-1基因多态性与AS易感性的关联,以期对深入研究AS的病因、发病机制提供一些理论依据。方法：采用病例对照研究方案,收集408名研究对象的血标本,其中包含216名AS患者标本。通过PCR-RFLP技术对PDCD-1基因的3个SNP位点(rs11568821,rs2227981，rs2227982)进行分型,χ2检验比较患者组及对照组基因型和等位基因频率的分布差异,运用SHESIS在线软件进行LD和基因相关性分析。结果：位点一(rs11568821)在所有AS患者和健康对照组中仅发现GG纯合子基因型,呈非多态性；位点二(rs2227981)在AS患者和健康对照组中均检出CC纯合子、CT杂合子和TT纯合子三种基因型,但在两组中该位点基因型和等位基因分布频率相近,χ2检验差异无显著性(P=0.145)；位点三(rs2227982)在AS患者和健康对照组中均检出TT纯合子、CT杂合子和CC纯合子三种基因型,且基因型和等位基因分布频率在两组中具有显著性差异(P=0.025),AS患者组具有更高的CT杂合子基因型(P=0.026,OR=1.542,95%CI=1.051-2.261)和T等位基因的频率(P=0.004,OR=1.553,95%CI=1.144-2.109)。运用SHESIS在线软件分析rs2227981和rs2227982,发现两者存在较强程度连锁(D＇=0.729)；在该两个位点构建了4种单体型,CT单体型的频率在AS患者组(21.6%)高于健康对照组(13.9%)(P=0.002,OR=1.712,95%CI=1.222-2.397),CC单体型在健康对照组(57.1%)比AS患者组(44.6%)更普遍(P=0.000,OR=0.603,95%CI=0.467-0.780)。结论：在中国汉族人群中, rs2227982中CT杂合子和T等位基因、特定单体型CT (PDCD-1.5/1.9)与AS发病呈正相关,CC单体型与AS发病呈负相关,PDCD-1基因多态性与AS之间存在遗传相关性。PDCD-1基因多态性与AS的相关性还需在更大规模不同人群中研究,以便得到更多证据目的：对国内外有关IL-23R基因多态性与AS的研究结果作meta分析,综合评价IL-23R基因SNP与AS的关系,为人群AS易感基因的评估提供依据。方法：电子检索Medline数据库和中国期刊全文数据库,根据一定纳入标准进行质量评价以及数据提取,卡方检验各个研究之间的异质性,采用Review Manager 4.2软件进行统计分析。结果：共有6篇文献符合纳入标准,被研究的人群涉及9类,基因型频率分布全部符合Hardy-Weinberg遗传平衡,Egger检验均未发现发表偏倚。共对10个IL-23R SNP位点(rsll209026,rsl004819,rsl0489629,rsll465804,rsl34315,rsl0889677,rsl 1209032,rsl495965,rs7517847,rs2201841)等位基因的各项研究进行meta分析,齐性检验中除rs10889677外,均未发现异质性(P>0.05),因此rs10889677采用随机效应模型,其它位点采用固定效应模型对等位基因频率进行分析,综合结果表明所有位点等位基因在两组之间分布均存在差异(P<0.05),5个位点(rsll209026、rsl0489629、rsll465804、rsl343151、rs7517847)等位基因在对照组分布频率高于AS组,另5个位点反之。结论：IL-23R SNP与AS呈正相关或负相关,被研究的10个等位基因中,与AS呈负相关性(对患者起保护作用)的位点有：rs11209026(G>A)、rsl0489629(G>A)、rs11465804(T>G)、rsl343151(C>T)、rs7517847(T>G)。其余位点与AS均呈正相关。meta分析可获得更高的统计学效能,为进一步的研究提供方向和思路。更多还原

【Abstract】 Ankylosing Spondylitis (AS) is an immune-related complex disease caused by a combination of genetic and environmental factors. A lot of studies show that genetic factors play an important role in the development of AS.The MHC (major histocompatibility complex) on chromosome 6p is strongly linked and associated with AS. The association of HLA-B27 (human leukocyte antigen-B27) coded by MHCⅠwith AS is the strongest among genetic association with other disease. Genome-wide screening studies have led to the identification of several non-MHC genes possibly linked to AS. Due to autoimmune mechanisms participating in the pathogeneisis of AS, studies on the disease association with certain genes involved in the autoimmune response may highlight the genetic background of AS, such as the genes IL (interleukin), PDCD (programmed cell death) and et al. However, the results are not unified and repeated because of many reasons, especially relation between SNPs (single nucleotide polymorphisms) and AS. Therefore, a more thorough search for polymorphisms in the locus will be needed.In this study, we firstly investigated the the association of the B27 subtypes with AS in the HuBei population of China by PCR-SSP (polymerase chain reaction-sequence-specific primer). Then, by employing PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphisms), we analyzed the association of PDCD-1 (programmed cell death) SNP and haplotypes with AS. At last, a meta-analysis was conducted to determine the contribution of IL-23R (interleukin-23 receptor) gene SNP previously implicated in AS susceptibility. The present study can be divided into the following three parts. OBJECTIVE To analyze the association of the B27 subtypes with AS in the HuBei population of China, investigate the role of B27 subtypes in the etiology of AS, and evaluate the usefulness of HLA-B27 subtype in clinical diagnosis of AS.METHODS A total of 190 patients with AS and a control group of 156 subjects were recruited for the study. Of 190 AS patients,129 were men and 61 women, with a mean age of 39±9. HLA-B27 subtypes were confirmed by PCR-SSP.RESULTS Six B27 subtypes were determined:B*2702,03,04,05,06 and B*13. HLA-B*2704 (patients 72.1% vs. controls 56.4%) and HLA-B*2705 (patients 21.6% vs. controls 31.4%) were the two high frequency genotypes in controls and patients. There were significant differences in the distribution of B*2704 subtypes between patients with AS and controls (χ2=9.279, P<0.01, OR=1.997,95% CI=1.279-3.119). Compared with the controls, the AS patients had high frequency of B*2704 and low frequency of B*2705. B*2703 was detected in 10 (5.3%) patients and in 13 (8.3%) controls. B*2702, B*2706 and B*2713 were relatively rare. Other HLA-B27 subtypes were relatively rare. One B*2713 was solely detected in AS group but not in controls, four B*2706 were found only in controls.CONCLUSION B*2704 was the dominant subtypes followed by B*2705. B*2704 is strongly associatied with AS. B*2706 may have a negative association with AS. Subtyping for HLA-B27 by PCR-SSP has been proved to be suitable for clinical application. It is useful to diagnose AS in clinic. OBJECTIVE To investigate the association of PDCD-1 polymorphisms and haplotypes with AS in Chinese Han population.METHODS In a case-control association study, three SNP, PDCD-1.3 G/A, PDCD-1.5 C/T and PDCD-1.9 T/C, were genotyped in 216 AS patients and 264 healthy controls using PCR-RFLP assay.RESULTS All genotype distributions in the patients and in the controls were in Hardy-Weinberg equilibrium. The associations of genotypes and alleles with AS were analyzed. The genotype distributions of PDCD-1.9 were significantly different between the patients with AS and the controls (P=0.025). The frequencies of TC genotype and T allele of PDCD-1.9 were higher in the patients than those in the controls (P=0.026 and 0.004). No association for PDCD-1.5 in AS was found, and PDCD-1.3 was nonpolymorphic in Chinese Han population. Moreover, significant LD was found between PDCD-1.5 and PDCD-1.9 (D’=0.729). Four haplotypes between SNPs PDCD-1.5 and PDCD-1.9 were constructed. The frequency of the CT haplotype was higher in the AS patients (21.6%) than the controls (13.9%) (P=0.002, OR=1.712,95%CI=1.222-2.397). The CC haplotype was more common in controls (57.1%) than in patients (44.6%) (P=0.000, OR=0.603,95% CI=0.467-0.780).CONCLUSION The results support a genetic association between the PDCD-1 polymorphism and susceptibility to AS in Chinese Han population. A more thorough search in other populations will be needed. OBJECTIVE To determine the contribution of IL-23R gene SNP previously implicated in AS susceptibility in different populations worldwide.METHODS An electric search was performed in PubMed and Chinese periodical full-text database. Association between IL-R23 SNP and AS was examined. Data on allele frequency was extracted. Heterogeneity, power and publication bias were explored. Odds ratio Was summarized. All were perfermed by Review Manager 4.2 software.RESULTS Six studies of IL-R23 SNP were enrolled. The alleles frequency of 10 SNP (rs11209026, rs1004819, rs10489629, rs11465804, rs134315,rs10889677, rs11209032,rs1495965, rs7517847, rs2201841) were analyzed in AS cases and controls recruited in 9 different centres. No heterogeneity and publication bias were observed (P>0.05) except rs10889677. Thus, random effect mode (DerSimonian-Laird test) was used to calculate OR of rs10889677, fixed effect mode (Peto test) was used in others SNPs. All allele distributions were significantly different between the patients with AS and the controls (P<0.05). The alleles frequency of 5 SNP (rs11209026, rs10489629, rs11465804, rs1343151, rs7517847) was higher in the controls than in AS patients.CONCLUSION This study confirms that IL-23R SNP is associated with susceptibility to AS. Meta-analysis is a useful tool by providing sufficiently large sample sizes to produce robust findings.更多还原