中国汉族人类风湿关节炎外周血基因表达谱研究
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摘要
类风湿关节炎（rheumatoid arthritis, RA）是以持续性滑膜炎、全身性炎症及自身抗体的出现为特征的一组疾病，这类自身抗体以类风湿因子（rheumatoid factor, RF）和抗环瓜氨酸多肽（CCP）抗体（anti-cyclic citrullinated peptide antibody, anti-CCPantibody）为代表。RA是最常见的风湿性疾病之一，在我国，RA患病率为0.2-0.4%。该病病程迁延，致残率高，严重影响患者的生活质量，同时高额的医疗费用给患者和国家造成沉重的经济负担。因此，提高RA发病机制的研究水平，从而推进临床诊断和治疗水平是降低RA致残率，改善患者生活质量和保护社会劳动力的重要方法，是国家亟待解决的重大问题。
虽然RA发病的发病机制迄今未明，但是公认为RA的发病是由遗传和环境因素（如：吸烟）共同发挥作用而致病。RA是在环境因素，如吸烟和感染的影响下发生免疫紊乱导致活化的CD4+T细胞和MHC-Ⅱ型抗原递呈细胞浸润滑膜，并在细胞因子的参与下发病。遗传学因素可以解释RA大约50%的易感性。迄今为止，通过基因组学的研究，特别是全基因组关联分析研究（GWAS）的引入后，已发现40余个RA易感基因，为疾病的发病机制研究和风险预测提供了依据。同时，对这些发现的基因进行进一步挖掘，从群体遗传学中易感位点过渡到实际运用于诊疗的临床分子标记，完成真正的从实验室成果向临床诊疗手段的转化成了当今RA研究的当务之急。作为基因水平和蛋白水平的桥梁，转录水平的研究显得非常重要。
目前国际上在转录组水平研究RA的文章数量不菲，但是有以下局限性：一，目前尚无中国汉族人转录组水平的研究。绝大多数研究是基于欧裔人群的数据，而既往的遗传学研究证实类风湿关节炎是具有种族特异性的疾病。二，大多数研究使用的方法为芯片杂交技术。基因芯片技术需要根据已知基因来设计探针，只限用于已知序列，因为无法检测新的mRNA。基因芯片技术的灵敏度也非常有限,难以检测低丰度的目标和重复序列，这就需要更多的样本来进行研究。同时，它也很难检测出异常转录产物。RNA测序（RNA sequencing, RNA-Seq）技术为解决第二个问题提供了良好的平台。相对于芯片杂交技术，RNA测序技术能够提供更精确的数字化信号，更高的检测通量以及更广泛的检测范围。
本研究将运用RNA测序技术，在mRNA水平探索RA免疫遗传学发病机制：对已发现的易感基因进行进一步挖掘，力图从群体遗传学中易感位点过渡到实际运用于诊疗的临床分子标记，完成真正的从实验室成果向临床诊疗手段的转化。
第一部分基于RNA-Seq技术类风湿关节炎外周血基因表达谱
目的：基于RNA-seq技术检测中国汉族人类风湿、关节炎患者与正常对照外周血差异表达的基因，探讨汉族人RA的发病机制。
方法：提取5例处于活动期的抗CCP抗体阳性女性类风湿关节炎患者与性别匹配5例正常对照（Normal Control, NC）的外周血单个核细胞mRNA，使用Truseq RNAsample preparation kit（Illumina）构建测序文库，应用HiSeq2000系统进行测序分析，使用Cufflinks软件进行RNA表达量的计算，Cuffdiff软件默认参数进行显著性差异表达分析（P值<0.05并且表达差异2倍以上）。表达差异显著的基因进行聚类分析、GO分析和KEGG Pathway分析。通过测序结果与既往类风湿关节炎转录组报道文献比对、与既往报道的全基因关联分析研究（GWAS）基因比对确定下一步进行验证的候选基因。
结果：32106个转录本在RA组或NC组中至少有一个样本表达，P值<0.05且表达差异2倍以上的转录本274个（138个转录本表达上调；136个转录本表达下调）。聚类分析显示显著差异表达的转录本能够将测序样本明确分为2类；274个差异表达的转录本进行GO分析，富集通路为：神经系统相关通路：轴突、神经元的发育，突触的装配、结构和激活；免疫系统相关通路：多肽、多聚糖抗原的MHC-II分子处理和递呈，T细胞的分化、激活和增殖，单核细胞的增殖，细胞对细菌成分（如内毒素）的反应；蛋白泛素化等等。根据比对既往文献及GO分析的结果选取43个基因作为验证试验的候选基因。
结论：测序实验差异基因的GO分析提示固有免疫和适应性免疫共同参与类风湿关节炎的发病发展；环境因素细菌感染特别是内毒素在类风湿关节炎发病发展中的作用；蛋白泛素化在发病机制中的研究值得深入探讨。
第二部分大样本验证类风湿关节炎相关基因
目的：在大样本人群中验证测序结果和既往报道类风湿关节炎相关基因，寻找可应用于临床诊治的分子标记。
方法：收集类风湿关节炎患者外周血101例，正常对照55例，并登记相关临床资料。通过Trizol法提取总RNA，逆转录为cDNA。使用实时荧光定量PCR技术首先在小样本（15例RA患者和15例健康对照）中进行验证中检测43个基因表达情况；最终筛选9个基因在大样本（86例RA和40例NC）中进行基因表达情况的检测。根据临床资料对患者进行分层及关联分析，统计基因表达的差异。
结果：小样本验证实验中证实以下基因在病例组和正常对照组中差异表达显著：GPRIN3(р<0.01)、CXCR2(р<0.01)、FCGBP(р<0.05)、CD14(р<0.05)、RCAN1(р<0.05)、IKZF3(р<0.05)、BIRC3(р<0.05)、AFF3(р<0.05)、TSC1(р<0.05)、TYK2(р<0.05)、PADI4(р<0.05)。选取其中感兴趣的9个基因在大样本实验中进行验证：TSC1(р<0.01)、CD14(р<0.01)、AFF3(р<0.01)、BIRC3(р<0.01)、GPRIN3(р<0.05)、FCGBP(р<0.05)、RCAN1(р<0.05)差异表达显著，IKZF3、CXCR2表达无差异。其中，抗环瓜氨酸抗体阴性患者TSC1表达下降较抗环瓜氨酸抗体阳性患者明显(р<0.05)。中晚期患者CD14表达下降较早期患者下降明显(р<0.05)。
结论：TSC1和BIRC3是首次报道的与汉族人RA相关的基因；除CD14、AFF3基因介导的免疫相关性通路，BIRC3、TSC1基因参与的蛋白异寡聚化及BIRC3介导的凋亡与抗凋亡在类风湿关节炎病理过程中可能具有重要作用。
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised bypersistent synovitis, systemic inflammation, and autoantibodies (rheumatoid factor andanti-cyclic citrullinated peptide antibody) with progressive disability. The cause of theinitiation and progression of RA remain unknown, though advances in understanding thepathogenesis of the disease have been made.
The genome-wide assay of gene expression and genetic variation to identify geneswhich are expressed differently in affected individuals is a powerful tool to discover thethe underlying changes of the diseases. Gene expression profiling has been used to studyRA, including to predict responsiveness to therapy, to identify biomarkers for diagnosis,therapy and prognosis. Both peripheral blood mononuclear cells (PBMCs) and synovialtissues of affected joints are used in such studies. Those microarray-based studies arelimited by hybridization noise, less sensitivity and depending on the exisitng knowledgeabout genome sequence. Recently RNA-Seq is developed to be a revolutionary approch fortranscriptomics which does not rely on probe design and use tag-based sequencingtechnologies. Previous studies have documented marked differences in the prevalence ratesof rheumatoid arthritis and specific SNPs by race and ethnicity, suggesting the geneexpression profile might vary among different races. Therefore, We undertook RNA-Sequsing Illumina HiSeq2000system on PBMCs from5Han Chinese patients with active RAand5age-matched and gender-matched controls. This study identified a number ofcandidate genes which were validated by quantitative real-time PCR (qRT-PCR) in adifferent sample population later. The immunological nature of some candidate geneshighlights likely immune processes altered in the development of RA. Further,identification of gene expression patterns corresponding to disease status can be used todevelop predictive.
Part I: Research on the immunogenetic mechanism of rheumatoid arthritis based onRNA-Seq technology
Objective: To identify differentially expressed genes in PBMCs from Han Chinesepatients with rheumatoid arthritis compared with healthy individuals, providing advancedclues for the pathogenesis of RA in Han Chinese patients.
Methods: RNA was extracted from PBMCs collected from5patients with activedisease and5gender-matched and age-matched controls. Expression profiles of these cellswere determined using RNA-Seq technology. Cuffdiff Two Origins of Blastemal Progenitors program in cufflinks was used to obtain differential expressed genes withsignificant expression. Cluster analysis, KEGG Pathway and Gene Ontology (GO) analysiswere taken respectively for the candidate genes with differential expression over2-foldchange. In comparison with genes which have been reported, candidate genes were chosenfor next validation experiment.
Results: RNA-Seq analysis identified32106transcripts in at least one sample and274transcripts were significantly differentially expressed between RA patients andcontrols with a p value <0.05and a significant fold-change>2. Of these,138transcriptswere overexpressed and136were underexpressed. Cluster analysis showed gooddelineation between patients with RA and healthy controls to confirm the validity of thisRNA-Seq experiment. GO analysis of the significant274transcripts revealed that theenrichment in immune system: antigen processing and presentation of peptide orpolysaccharide antigen via MHC class II, T cell activation, differentiation and proliferation,cellular response to molecule of bacterial origin, cellular response to lipopolysaccharide,ect; nervous system: synapse assembly, synapse structure and activity, synapseorganization, regulation of axonogenesis, central nervous system neuron development;reflex, ect; regulation of protein ubiquitination involved in ubiquitin-dependent protein.Combining GO analysis data and the genes reported,43candidate genes were identified.
Conclusion: Data from GO analysis implies both innate and adaptive immunity playan important role in the pathogenesis of RA; Bacteria infection, one of the environmentfactors, may lead to a breakdown of immune tolerance; protein ubiquitination is worth tofurther study in the pathophysiology of RA.
Part II: Validation on candidate genes by quantitative reverse transcription-PCR(qRT-PCR)
Objective: To validate the candidate genes by quantitative reverse transcription-PCR(qRT-PCR) and find biological remarker.
Methods: Peripheral blood samples were collected into EDTA tubes between6:00and8:00, Characteristics of the all study subjects were collected at the same time. Store per250ul blood sample in750ul Trizol() at-80°C. TRIzol() extraction followed by cDNAsynthesis. Candidate genes identified from the RNA-Seq study were validated byqRT-PCR in a smaller sample cohort (15cases versus15controls), then validated in alarger sample cohort (86cases versus40controls). Analyse gene expression leveldifference according to clinical characteristics in RA group. Evaluate the association between the differential gene expression profiling of RA patients with their autoimmuneresponse [anti-cyclic citrullinatedpeptide (CCP) antibodies, rheumatoid factor(RF)],disease activity [C reative protein(CRP), erythrocyte sedimentation rate (ESR), DiseaseActivity Score using28joint counts (DAS-28)], disease duration, and treatment(glucocorticoid) features.
Results:11genes were downregulated significantly in patients with RA compared tohealthy controls in the smaller sample cohort：GPRIN3(р<0.01)、CXCR2(р<0.01)、FCGBP(р<0.05)、CD14(р<0.05)、RCAN1(р<0.05)、IKZF3(р<0.05)、BIRC3(р<0.05)、AFF3(р<0.05)、TSC1(р<0.05)、TYK2(р<0.05)、PADI4(р<0.05).7genes werevalidated to downregulate in patients with RA compared to healthy controls in the largersample cohort: TSC1(р<0.01)、CD14(р<0.01)、AFF3(р<0.01)、BIRC3(р<0.01)、GPRIN3(р<0.05)、FCGBP(р<0.05)、RCAN1(р<0.05). But there was no difference offollowing genes expression level between patients and healthy controls: IKZF3、CXCR2.In the context of RA, TSC1expression level was downregulated more significantly inACPA positive cases than ACPA negative cases (р<0.05) and. CD14expression levelwas downregulated more significantly in non-early RA patients than early RA patientspatients(р<0.05) and significantly associated with the stage of disease.
Conclusion: TSC1and BIRC3are the first reported genes related to Han Chinesepatients with RA. Signaling pathways for validated genes may play roles in thepathogenesis of RA: CD14cooperates with Toll receptor to mediate the innate immuneresponse to bacterial lipopolysaccharide; AFF3activates transcription that may function inlymphoid development; BIRC3and TSC1mediate protein heterooligomerization; BIRC3regulates of apoptosis and anti-apoptosis.
虽然RA发病的发病机制迄今未明，但是公认为RA的发病是由遗传和环境因素（如：吸烟）共同发挥作用而致病。RA是在环境因素，如吸烟和感染的影响下发生免疫紊乱导致活化的CD4+T细胞和MHC-Ⅱ型抗原递呈细胞浸润滑膜，并在细胞因子的参与下发病。遗传学因素可以解释RA大约50%的易感性。迄今为止，通过基因组学的研究，特别是全基因组关联分析研究（GWAS）的引入后，已发现40余个RA易感基因，为疾病的发病机制研究和风险预测提供了依据。同时，对这些发现的基因进行进一步挖掘，从群体遗传学中易感位点过渡到实际运用于诊疗的临床分子标记，完成真正的从实验室成果向临床诊疗手段的转化成了当今RA研究的当务之急。作为基因水平和蛋白水平的桥梁，转录水平的研究显得非常重要。
目前国际上在转录组水平研究RA的文章数量不菲，但是有以下局限性：一，目前尚无中国汉族人转录组水平的研究。绝大多数研究是基于欧裔人群的数据，而既往的遗传学研究证实类风湿关节炎是具有种族特异性的疾病。二，大多数研究使用的方法为芯片杂交技术。基因芯片技术需要根据已知基因来设计探针，只限用于已知序列，因为无法检测新的mRNA。基因芯片技术的灵敏度也非常有限,难以检测低丰度的目标和重复序列，这就需要更多的样本来进行研究。同时，它也很难检测出异常转录产物。RNA测序（RNA sequencing, RNA-Seq）技术为解决第二个问题提供了良好的平台。相对于芯片杂交技术，RNA测序技术能够提供更精确的数字化信号，更高的检测通量以及更广泛的检测范围。
本研究将运用RNA测序技术，在mRNA水平探索RA免疫遗传学发病机制：对已发现的易感基因进行进一步挖掘，力图从群体遗传学中易感位点过渡到实际运用于诊疗的临床分子标记，完成真正的从实验室成果向临床诊疗手段的转化。
第一部分基于RNA-Seq技术类风湿关节炎外周血基因表达谱
目的：基于RNA-seq技术检测中国汉族人类风湿、关节炎患者与正常对照外周血差异表达的基因，探讨汉族人RA的发病机制。
方法：提取5例处于活动期的抗CCP抗体阳性女性类风湿关节炎患者与性别匹配5例正常对照（Normal Control, NC）的外周血单个核细胞mRNA，使用Truseq RNAsample preparation kit（Illumina）构建测序文库，应用HiSeq2000系统进行测序分析，使用Cufflinks软件进行RNA表达量的计算，Cuffdiff软件默认参数进行显著性差异表达分析（P值<0.05并且表达差异2倍以上）。表达差异显著的基因进行聚类分析、GO分析和KEGG Pathway分析。通过测序结果与既往类风湿关节炎转录组报道文献比对、与既往报道的全基因关联分析研究（GWAS）基因比对确定下一步进行验证的候选基因。
结果：32106个转录本在RA组或NC组中至少有一个样本表达，P值<0.05且表达差异2倍以上的转录本274个（138个转录本表达上调；136个转录本表达下调）。聚类分析显示显著差异表达的转录本能够将测序样本明确分为2类；274个差异表达的转录本进行GO分析，富集通路为：神经系统相关通路：轴突、神经元的发育，突触的装配、结构和激活；免疫系统相关通路：多肽、多聚糖抗原的MHC-II分子处理和递呈，T细胞的分化、激活和增殖，单核细胞的增殖，细胞对细菌成分（如内毒素）的反应；蛋白泛素化等等。根据比对既往文献及GO分析的结果选取43个基因作为验证试验的候选基因。
结论：测序实验差异基因的GO分析提示固有免疫和适应性免疫共同参与类风湿关节炎的发病发展；环境因素细菌感染特别是内毒素在类风湿关节炎发病发展中的作用；蛋白泛素化在发病机制中的研究值得深入探讨。
第二部分大样本验证类风湿关节炎相关基因
目的：在大样本人群中验证测序结果和既往报道类风湿关节炎相关基因，寻找可应用于临床诊治的分子标记。
方法：收集类风湿关节炎患者外周血101例，正常对照55例，并登记相关临床资料。通过Trizol法提取总RNA，逆转录为cDNA。使用实时荧光定量PCR技术首先在小样本（15例RA患者和15例健康对照）中进行验证中检测43个基因表达情况；最终筛选9个基因在大样本（86例RA和40例NC）中进行基因表达情况的检测。根据临床资料对患者进行分层及关联分析，统计基因表达的差异。
结果：小样本验证实验中证实以下基因在病例组和正常对照组中差异表达显著：GPRIN3(р<0.01)、CXCR2(р<0.01)、FCGBP(р<0.05)、CD14(р<0.05)、RCAN1(р<0.05)、IKZF3(р<0.05)、BIRC3(р<0.05)、AFF3(р<0.05)、TSC1(р<0.05)、TYK2(р<0.05)、PADI4(р<0.05)。选取其中感兴趣的9个基因在大样本实验中进行验证：TSC1(р<0.01)、CD14(р<0.01)、AFF3(р<0.01)、BIRC3(р<0.01)、GPRIN3(р<0.05)、FCGBP(р<0.05)、RCAN1(р<0.05)差异表达显著，IKZF3、CXCR2表达无差异。其中，抗环瓜氨酸抗体阴性患者TSC1表达下降较抗环瓜氨酸抗体阳性患者明显(р<0.05)。中晚期患者CD14表达下降较早期患者下降明显(р<0.05)。
结论：TSC1和BIRC3是首次报道的与汉族人RA相关的基因；除CD14、AFF3基因介导的免疫相关性通路，BIRC3、TSC1基因参与的蛋白异寡聚化及BIRC3介导的凋亡与抗凋亡在类风湿关节炎病理过程中可能具有重要作用。
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised bypersistent synovitis, systemic inflammation, and autoantibodies (rheumatoid factor andanti-cyclic citrullinated peptide antibody) with progressive disability. The cause of theinitiation and progression of RA remain unknown, though advances in understanding thepathogenesis of the disease have been made.
The genome-wide assay of gene expression and genetic variation to identify geneswhich are expressed differently in affected individuals is a powerful tool to discover thethe underlying changes of the diseases. Gene expression profiling has been used to studyRA, including to predict responsiveness to therapy, to identify biomarkers for diagnosis,therapy and prognosis. Both peripheral blood mononuclear cells (PBMCs) and synovialtissues of affected joints are used in such studies. Those microarray-based studies arelimited by hybridization noise, less sensitivity and depending on the exisitng knowledgeabout genome sequence. Recently RNA-Seq is developed to be a revolutionary approch fortranscriptomics which does not rely on probe design and use tag-based sequencingtechnologies. Previous studies have documented marked differences in the prevalence ratesof rheumatoid arthritis and specific SNPs by race and ethnicity, suggesting the geneexpression profile might vary among different races. Therefore, We undertook RNA-Sequsing Illumina HiSeq2000system on PBMCs from5Han Chinese patients with active RAand5age-matched and gender-matched controls. This study identified a number ofcandidate genes which were validated by quantitative real-time PCR (qRT-PCR) in adifferent sample population later. The immunological nature of some candidate geneshighlights likely immune processes altered in the development of RA. Further,identification of gene expression patterns corresponding to disease status can be used todevelop predictive.
Part I: Research on the immunogenetic mechanism of rheumatoid arthritis based onRNA-Seq technology
Objective: To identify differentially expressed genes in PBMCs from Han Chinesepatients with rheumatoid arthritis compared with healthy individuals, providing advancedclues for the pathogenesis of RA in Han Chinese patients.
Methods: RNA was extracted from PBMCs collected from5patients with activedisease and5gender-matched and age-matched controls. Expression profiles of these cellswere determined using RNA-Seq technology. Cuffdiff Two Origins of Blastemal Progenitors program in cufflinks was used to obtain differential expressed genes withsignificant expression. Cluster analysis, KEGG Pathway and Gene Ontology (GO) analysiswere taken respectively for the candidate genes with differential expression over2-foldchange. In comparison with genes which have been reported, candidate genes were chosenfor next validation experiment.
Results: RNA-Seq analysis identified32106transcripts in at least one sample and274transcripts were significantly differentially expressed between RA patients andcontrols with a p value <0.05and a significant fold-change>2. Of these,138transcriptswere overexpressed and136were underexpressed. Cluster analysis showed gooddelineation between patients with RA and healthy controls to confirm the validity of thisRNA-Seq experiment. GO analysis of the significant274transcripts revealed that theenrichment in immune system: antigen processing and presentation of peptide orpolysaccharide antigen via MHC class II, T cell activation, differentiation and proliferation,cellular response to molecule of bacterial origin, cellular response to lipopolysaccharide,ect; nervous system: synapse assembly, synapse structure and activity, synapseorganization, regulation of axonogenesis, central nervous system neuron development;reflex, ect; regulation of protein ubiquitination involved in ubiquitin-dependent protein.Combining GO analysis data and the genes reported,43candidate genes were identified.
Conclusion: Data from GO analysis implies both innate and adaptive immunity playan important role in the pathogenesis of RA; Bacteria infection, one of the environmentfactors, may lead to a breakdown of immune tolerance; protein ubiquitination is worth tofurther study in the pathophysiology of RA.
Part II: Validation on candidate genes by quantitative reverse transcription-PCR(qRT-PCR)
Objective: To validate the candidate genes by quantitative reverse transcription-PCR(qRT-PCR) and find biological remarker.
Methods: Peripheral blood samples were collected into EDTA tubes between6:00and8:00, Characteristics of the all study subjects were collected at the same time. Store per250ul blood sample in750ul Trizol() at-80°C. TRIzol() extraction followed by cDNAsynthesis. Candidate genes identified from the RNA-Seq study were validated byqRT-PCR in a smaller sample cohort (15cases versus15controls), then validated in alarger sample cohort (86cases versus40controls). Analyse gene expression leveldifference according to clinical characteristics in RA group. Evaluate the association between the differential gene expression profiling of RA patients with their autoimmuneresponse [anti-cyclic citrullinatedpeptide (CCP) antibodies, rheumatoid factor(RF)],disease activity [C reative protein(CRP), erythrocyte sedimentation rate (ESR), DiseaseActivity Score using28joint counts (DAS-28)], disease duration, and treatment(glucocorticoid) features.
Results:11genes were downregulated significantly in patients with RA compared tohealthy controls in the smaller sample cohort：GPRIN3(р<0.01)、CXCR2(р<0.01)、FCGBP(р<0.05)、CD14(р<0.05)、RCAN1(р<0.05)、IKZF3(р<0.05)、BIRC3(р<0.05)、AFF3(р<0.05)、TSC1(р<0.05)、TYK2(р<0.05)、PADI4(р<0.05).7genes werevalidated to downregulate in patients with RA compared to healthy controls in the largersample cohort: TSC1(р<0.01)、CD14(р<0.01)、AFF3(р<0.01)、BIRC3(р<0.01)、GPRIN3(р<0.05)、FCGBP(р<0.05)、RCAN1(р<0.05). But there was no difference offollowing genes expression level between patients and healthy controls: IKZF3、CXCR2.In the context of RA, TSC1expression level was downregulated more significantly inACPA positive cases than ACPA negative cases (р<0.05) and. CD14expression levelwas downregulated more significantly in non-early RA patients than early RA patientspatients(р<0.05) and significantly associated with the stage of disease.
Conclusion: TSC1and BIRC3are the first reported genes related to Han Chinesepatients with RA. Signaling pathways for validated genes may play roles in thepathogenesis of RA: CD14cooperates with Toll receptor to mediate the innate immuneresponse to bacterial lipopolysaccharide; AFF3activates transcription that may function inlymphoid development; BIRC3and TSC1mediate protein heterooligomerization; BIRC3regulates of apoptosis and anti-apoptosis.
引文
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