Gene Expression and Regulatory Networks in Human Leukocytes

描述(由申请人提供)：用微阵列分析基因表达对人类健康具有巨大的潜力，可以阐明疾病的途径或提供生物标志物来监测疾病或其解决方案。该项目将使用高通量的基因分型、免疫表型和基因表达分析方法，研究控制人类免疫细胞基因表达的基础，以及自然遗传变异或衰老如何影响基因表达。在实践中，该项目将结合几种交叉信息的方法： 1)在已建立的免疫基因组(ImmGen)项目的样本/数据管道和强大的方案的基础上，以及手头已建立的不同种族的健康志愿者队列，将使用微阵列技术从600名健康志愿者的纯化的天然CD4淋巴细胞和单核细胞中生成全基因组表达谱。将为所有捐赠者建立密集的遗传图谱。这些结果将阐明人类基因组的变异如何影响免疫基因的表达，这对于理解导致免疫性或炎症性疾病易感性的基因变异至关重要。对这些丰富数据的计算分析将允许重建基因之间的调控联系，有助于建立通用模块和特定免疫细胞类型的模块。这些数据将由一个由10个人组成的受限子集产生的正交数据组来补充，在这个数据组中，我们将描述存在于人类血液中的28个仔细描绘的细胞群体的更大集合。这项工作还将受益于与ImmGen在小鼠身上进行的类似实验进行的强大的物种间比较。 2)此外，来自同一组28个已定义细胞群体的RNA将被微阵列探测，这些微阵列探索转录组的其他方面：i)microRNAs和其他非编码RNA；ii)外显子或剪接连接阵列，将建立人类血液白细胞差异剪接图谱。 3)高通量流式细胞术将在采集样本时确定来自相同捐献者的血细胞的组成和反应性，将免疫表型与基因表达和遗传变异联系起来。 4)遗传变异性决定了基因表达的基线水平，但也决定了对激活挑战的反应。对于来自相同捐赠者的样本，用于转录计数的纳米串技术将被用于分析代表T细胞或树突状细胞的反应特征的已定义基因集的转录反应，以精细地剖析对不同触发因素(树突状细胞的不同细菌配体，T细胞的不同细胞因子环境)的反应。 为了与本项目的资源方面保持一致，所有数据和解释都将公开进行 通过使用和发展ImmGen项目、布罗德研究所和国际储存库的现有网络体系结构，可在管理后迅速提供数据，允许公众查询和浏览数据。 相关性：利用基因组表达微阵列的DNA芯片进行探索，对人类健康、更好地了解疾病和用作诊断工具具有巨大的潜力。利用高通量基因组技术和计算生物学的结合，我们将对非裔美国人、亚洲人和欧洲人祖先的人类血细胞中的基因表达进行广泛的探索，询问这些谱是如何受到遗传变异或年龄的影响的。这些结果将为解释遗传和免疫学研究提供宝贵的参考基准。

DESCRIPTION (provided by applicant): Profiling of gene expression with microarrays holds great potential for human health, for illuminating disease pathways or providing biomarkers to monitor disease or its resolution. Using high-throughput approaches for genotyping, immunophenotyping and gene expression analysis, the project will examine the basis for the control of gene expression in human immune cells, and how it is influenced by natural genetic variation or aging. In practice, the project will combine several cross-informative approaches: 1) Building on the established sample/data pipelines and robust protocols of the Immunological Genome (ImmGen) project, and on established cohorts of ethnically diverse healthy volunteers at hand, microarray techniques will be used to generate whole-genome expression profiles from purified na¿ve CD4+ lymphocytes and monocytes from 600 healthy volunteers. A dense genetic map will be established for all donors. The results will elucidate how variation in the human genome affects the expression of immune genes, of key importance in understanding gene variants that bring susceptibility to immune or inflammatory disease. Computational analysis of these rich data will allow the reconstruction of regulatory connections between genes, helping to establish general modules and those specific of a given immune cell type. These data will be complemented by an orthogonal datagroup, generated from a restricted subset of 10 individuals, in which we will profile a larger set of 28 carefully delineated cell populations that exist in human blood. This work will also benefit from powerful interspecies comparison with similar experiments being performed in mice by ImmGen. 2) In addition, RNA from the same set of 28 defined cell populations will be probed with microarrays that explore other aspects of the transcriptome: i) microRNAs and other non-coding RNAs; ii) exon or splice junction arrays that will establish a map of differential splicing in human blood leukocyte. 3) The composition and reactivity of blood cells from the same donors will be established at the time of sample collection using high-throughput flow cytometry, correlating immune phenotypes with gene expression and genetic variation. 4) Genetic variability conditions the baseline levels of gene expression, but also the responsiveness to activating challenges. With samples from the same donors, Nanostring technology for transcript counting will be used to analyze the transcriptional response of defined gene sets, representing response signatures of T or dendritic cells, for a fine-grained dissection of responses to different triggers (different bacterial ligands for dendritic cells, different cytokine environment for T cells). In keeping with the resource aspect of this project, all data and interpretations will be made publicly available rapidly upon curation, allowing public querying and browsing of the data, by using and evolving the existing web architectures of the ImmGen project, of the Broad Institute, and of international repositories. RELEVANCE: Exploration with DNA chips of the genome's expression microarrays holds great potential for human health, to better understand disease and to serve as diagnostic tools. Using a combination of high-throughput genomic techniques and computational biology, we will perform a broad exploration of gene expression in human blood cells across groups of African-American, Asian and European ancestry, asking how these profiles are affected by genetic variation or by age. These results will provide an invaluable reference benchmark for the interpretation of genetic and immunological studies.