【作者】 吕永梅；

【导师】 杨森； 张学军；

【作者基本信息】 安徽医科大学 ， 皮肤病与性病学， 2011， 博士

【摘要】 引言:系统性红斑狼疮（systemic lupus erythematosus,SLE）是一种经典的自身免疫性疾病,以自身抗体产生、补体激活、免疫复合物沉积累及多种组织器官损伤为主的多基因遗传性疾病。由于可累及全身多个系统、器官,其患者的临床表现多种多样,且不同的患者之间有较大的差异,因此给临床的诊疗带来一定的困难。该病好发于女性,特别是育龄期妇女,男女的患病率之比约为1:9。其发病机制迄今尚未完全阐明,遗传因素和环境因素共同导致了该病的发生。SLE的临床表现,风险性和发病率在不同的种族、地区和性别间存在差异,提示遗传异质性在SLE临床表现的复杂性方面可能起重要的作用。拷贝数变异（copy number variants,CNVs）和单核苷酸多态性（single nucleotide polymorphisms, SNPs）是人类基因变异的两个重要遗传基础。目前,以Tag- SNPs为基础的关联分析研究（genome-wide association study, GWAS）在欧洲和亚洲SLE人群中确定了30多个SLE的易感基因/位点。但是,通过SNP- GWAS人们发现所识别的绝大部分常见变异仅能解释相对一小部分疾病风险,对疾病发生的解释贡献大约2%～15%,即使再进行大规模、大样本量的研究,遗传因素如何导致疾病的发病风险原因仍然解释不清。在SNP-GWAS的热潮过后,如何进一步挖掘SNP芯片数据的剩余价值是许多研究机构关心的问题。利用芯片数据进行CNV分析也是GWAS后期数据挖掘的一部分。我们通过illumina 610芯片平台分析CNV的结果不确定,故利用此平台不能有效分析相关的CNV。为了进一步对CNV认识和分析,我们通过候选基因的方法对与SLE强相关的C4基因进行拷贝数变异的相关性研究。CNV领域的一系列进展可以让我们重新审视CNV与疾病临床表型研究的必要性和可行性。最近,在多个不同人群SLE患者的GWAS研究中发现许多与SLE有关的易感基因,此研究除了为发现SLE遗传因素提供了直接证据外,同时也从基因组水平认识SLE的发病机制提供了重要线索。随后关于SLE基因型-表型的研究相继出现,不仅暗示了系统性红斑狼疮是一种异质性的疾病,而且进一步说明遗传因素在SLE的发病机制中有重要作用。基因拷贝数变异与疾病的临床表型相关性分析研究也说明基因拷贝数与疾病的临床表型相关。在此,我们行基因拷贝数变异与临床表型的分析进一步探索C4基因拷贝数变异与中国汉族SLE患者的临床表型之间的关系,对探索C4拷贝数变异在SLE发病过程中的特定作用及进一步阐明系统性红斑狼疮的发病机制具有重要意义。目的: 1)C4基因拷贝数（C4GCNs）在中国汉族正常人群中的分布状态。2) C4GCNs与中国汉族SLE患者的相关性分析。3)C4基因拷贝数变异多态性与SLE患者临床表型的相关性,进一步阐明系统性红斑狼疮的发病机制,并为下一步建立相应的动物模型和从功能上证实这些易感基因与特定表型相关提供相应的证据。方法:首先从安徽地区医院收集SLE患者和对照样本（1047例SLE患者和1056例正常对照）,按照相同的样本收集标准,利用TaqMan荧光定量PCR（TaqMan-based qRT-PCR）方法确定正常人群和SLE中C4 GCNs的分布状态,并进行相关性分析。其次,对与SLE强相关的C4 GCNs行病例-病例（如有关节炎的患者和无关节炎的患者）分析确定基因拷贝数相关的临床表型,然后再行病例-对照（如有关节炎的患者/无关节炎的患者和正常对照）分析,进一步确定基因拷贝数对不同临床表型所贡献的风险大小。用SPSS11.0软件进行统计分析P < 0.05认为有统计学意义。结果:1)C4 GCNs在正常人群中的分布:总C4基因拷贝数为2-8;其中C4A基因拷贝数为0-6,C4B基因拷贝数为0-5。2个拷贝的C4A和C4B占多数。C4A的拷贝数分布为:< 2的拷贝数样本为7.25%;2拷贝数样本为69.71%;> 2拷贝数样本为23.04 %。C4B的拷贝数分布:< 2的拷贝数样本为20.16%;2拷贝数样本为66.43%;> 2拷贝数样本为13.41%。4拷贝数的总C4为63.56%,其中2C4A-2C4B约占51.94 %。2)C4基因拷贝数在SLE患者中的分布:2个拷贝的C4A和C4B仍占多数。C4A的拷贝数分布偏向高拷贝侧:<2的拷贝数样本为13.10%;2拷贝数样本为67.10%;>2拷贝数样本为19.81%;C4B的拷贝数分布偏向低拷贝侧:<2的拷贝数样本为13.10%;2拷贝数样本为63.42% ;>2拷贝数样本为19.81%。通过与对照样本比较分析,在SLE患者中,低C4A基因拷贝数（<2）为SLE患者的危险因素(P=1.97×10^-5;OR: 1.93;95%CL:1.42-2.62);高C4A基因拷贝数（>2）没有显著性意义（P=0.08）;而C4B GCN与SLE无明显相关。C4基因拷贝数在SLE患者中:低拷贝（< 4）为SLE患者的危险因素（P=8.69×10-3;OR: 1.38; 95%Cl:1.09-1.76）;高拷贝数（>4）反而为SLE患者的保护性因素（P=0.04;OR: 0.79; 95%Cl:0.63-0.99）。C4基因拷贝数的增减主要与C4A GCN有关,与C4B GCN无明显关系。3)C4AGCN与血管炎有关（P=6.05×10-3）;C4A基因对多个临床表型均有贡献;未发现总C4基因拷贝数变异与某一临床表型相关;但对多个临床表型均存在不同程度的遗传压力。结论:本项研究不仅分析了C4 GCNs在中国汉族正常人群中的分布,而且进一步验证了C4 GCNs与SLE的相关性,同时也说明C4 GCNs在SLE的发病机制中起到重要作用;另外通过与欧洲人群SLE的C4 GCNs研究结果进行比较,发现影响SLE易感性的遗传变异在不同人种间存在差异,遗传异质性可能在SLE的易感性方面起着重要作用;进一步与临床表型的分析,C4A基因拷贝数可能与关节炎的发生密切相关,C4A和C4基因拷贝数变异对多个临床表型均存在不同程度的遗传压力。该项研究将加深我们对CNV遗传基础的认识,也为将来SLE机制研究提供启示。更多还原

【Abstract】 Introduction: Systemic lupus erythematosus （SLE） is a prototypic systemic autoimmune disease, characterized by a diverse array of autoantibody production, complement activation, immune complex deposition, tissues and organs damage influenced by both genetic and environmental factors. It can affect multiple systems and organs of the body and it is difficulty to give clinical diagnosis and treatment because of diverse clinical manifestations and differences between the patients. SLE affects predominantly in women （prevalence ratio of women to men is 9 to 1） and particularly during childbearing years. The pathogenesis of SLE has not yet been fully elucidated. It was concluded that genetic and environmental factors lead to the occurrence of the disease. There are marked disparities in incidence and prevalence of SLE patients worldwide, which varies in different ethnic and geographical populations. The prevalence of SLE ranges from 31 to 70 cases per 100,000 persons among Chinese and 7 to 71 in European populations. The ethnic and genetic heterogeneity may contribute to the complexity of its clinical manifestation.Copy number variation （CNV） and single nucleotide polymorphism （SNP） are two important human variations of human diversity in the genetic basis. At present, tag SNPs-based analysis of genome-wide association studies（GWAS）in European and Asian populations have identified more than thirty robust susceptibility genes and/or loci. However, susceptibility genes that were identified by SNP-GWAS can only explain a small part of the relative risk of disease, and interpretate of 2% to 15% disease pathogen. Even if more large-scale, large sample size been analyzed, they will still not explain all the main reason of genetic risk factors. After the boom of SNP-GWAS, many research institutions concerned that how to further identified the residual value data of SNP chip. CNV analysis is part of the late GWAS using microarray data. We found uncertain results of CNV through illumine-610 chip platform, which result in stagnation of leaning CNV using the platform. In order to further understand and analysis of CNV, we have analyzed C4 CNV which strong correlation with SLE though candidate gene approach.Many progresses in the area of CNV allow us to re-examine the necessity and feasibility studies beween the clinical phenotype and disease CNP. Recently, it was found that the number of susceptibility genes in many different populations of SLE patients through SLE GWAS study. The study not only provide direct evidence for genetic factors in SLE, but also learn the the pathogenesis of SLE from level of the genome, which provides an important clues to learn SLE. Furthermore, the studies of the genotype-phenotype on the SLE have emerged, the results not only implied that SLE is a heterogeneous disease, but also demonstrated that genetic factors have an important role in the pathogenesis of SLE. The correlation of gene copy number variations and disease phenotype indicates that gene copy number related to disease clinical phenotype. Here, we performed the analysis to explore the relationships between clinical phenotype and gene copy number variations in Chinese Han population, it is important to further explore the C4 copy number variation in the pathogenesis of SLE and further clarify the role of the pathogenesis of systemic lupus erythematosus.Object: 1) To find the distribution status of C4 gene copy number （C4GCNs） in the Chinese Han population. 2) To analyze the correlation with SLE in the Chinese Han population. 3)To investigate their relationships of C4 gene copy number variation with disease clinical phenotypes, to further clarify susceptibility genes underline the pathogenesis of SLE , then to establish the appropriate animal models and confirm the association with specific phenotypes which providing evidence from functional studies.Methods: First, we recruited all samples of SLE patients and controls from two hospitals （1099 cases and 1254 cases of SLE patients with normal controls） in this study, which according to the same standards for collection, TaqMan quantitative PCR （TaqMan-based qRT-PCR） were to performed to identified C4 GCNs the distribution status in the normal population and correlation analysis with SLE population. Second, we performed case-only analyses （e.g. presence of arthritis versus no arthritis） to identify which subphenotypes were associated with gene copy number. Then, we also performed subphenotype-control analyses （e.g. presence of arthritis or negative of arthritis versus health controls） to examine the risk conferred by the gene copy number on different subtypes of SLE. Furthermore, we performed case-only analyses （e.g. presence of arthritis versus no arthritis） to identify which subphenotypes were associated with gene copy number. P values were estimated using SPSS11.0 software, which below 0.05 were considered to be statistically significant.Results: 1) The results showed that the GCN varied from 2 to 8 for total C4, from 0 to 6 for C4A, and from 0 to 5 for C4B in the normal population. Two copies of C4A are the most common counts of GCN. The distribution of C4A copy number: the frequency of <2 copy number is 7.25%; 2 copy number is 69.71 %; > 2 copy number is 23.04%, the distribution of C4B copy number: the frequency of <2 copy number is 20.16%; 2 copy number is 66.43 %;> 2 copy number is 13.41%. Four copies of total C4 is 63.56%, of which 2C4A-2C4B is about 51.94%.2) It was found that the distribution of C4 gene copy number in SLE patients. The frequencies of two copies of C4A（69.71%） and C4B（66.43%）genes are in the majority. The C4A GCN distribution is skewed towards the side of high copy number （<2, 13.10%; 2, 67.10 %;> 2, 19.81%）, C4B GCN distribution is skewed towards the low side of copy number （<2, 13.10%; 2, 63.42 %;> 2, 19.81）. Compared with the control sample analysis, low C4A GCN （<2） is a risk factor in SLE patients (P = 1.97×10^-5; OR: 1.93; CL: 1.42-2 .62), higher C4A GCN （> 2） is no significant differences （P = 0.08）. However, C4B is not clearly related to SLE. Low copy number （<4） for the risk factor in patients with SLE (P = 8.69×10^-3; OR: 1.38; 95%CL: 1.09-1 .76); high copy number （> 4） is a protective factor for SLE （P = 0.04; OR: 0.79; 95% CL: 0.63-0.99）. Furthermore, there was significantly increase or decrease in the frequencies of the C4 GCN due to the variations of C4A but not C4B in SLE cases. 3) Significant associations were found for C4A gene copy number variation with arthritis (P = 6.05×10^-3), we examine the risks conferred by the C4A gene copy number on different subtypes of SLE; Significant associations were not found for C4 gene copy number variation with any clinical phenotype, however, we examine the risks conferred by C4 gene copy number on different subtypes of SLE.Conclusion: This study not only unfolds C4 GCNs in the Chinese normal population, but also validates that the C4 gene copy number was associated with SLE, and also identified that C4GCNs play an important role in the pathogenesis of SLE. Compared with the C4GCNs in European populations of SLE, we found that the susceptibility of copy number variation to SLE is difference in different ethnic, and the genetic heterogeneity of SLE susceptibility may play an important role. Through further analysis the clinical features, our study suggested that C4 gene copy number might be associated with arthritis, and C4 and C4A gene copy number play important roles in the development of SLE in Chinese Han population, which can conferred the risks on clinical features.The study will not only deepen our understanding of the genetic basis of the CNV, but also provide for the future mechanism to SLE.更多还原