Translational genomics in gout: From GWAS signal to mechanism

摘要 痛风在美国很普遍(占成年人口的3.9%)，不仅直接影响人们的生活，而且 也与心脏代谢性疾病并存。痛风由不可预测的急性炎症或 尿酸单钠(MSU)晶体激活单核细胞NLRP3炎性小体引起的耀斑 高尿酸血症患者，随后产生IL-1，并招募大量 炎症细胞进入受影响的关节。表观遗传重编程和基因转录改变作为回应 可溶尿酸盐水平升高是这种增强的细胞对次级刺激的反应性的机制， 被称为先天训练免疫力。为了更好地了解控制疾病进展的因素 高尿酸血症与痛风我们已经完成了一项针对痛风的非常大的全基因组相关性研究。 识别数百个与痛风相关的基因座。基因座包括长非编码RNA(LncRNAs)，它具有 不同的功能，包括调节基因表达，沉积表观遗传修饰和 染色体结构的组织。有些是免疫基因启动的lncRNAs(IPLs)，它直接 多个启动子上的转录机器。从这些基因座我们发现了一条关键的致病途径 我们将对其分子遗传学进行研究。 控制其激活的进程。芯片通路参与表观基因组DNA的调控。 底物的甲基化、组蛋白修饰和代谢。它可能会使人们更容易患痛风 通过使先天免疫系统对MSU晶体产生高度反应。我们假设 从痛风中发现的遗传位点通过调节基因表达来控制这一途径的活性， 包括通过调节表观基因组。在检验这一假设的过程中，我们将从三个方面理解 该途径的分子调控，为今后针对该途径进行改良研究奠定了基础 痛风的管理。在目标1中，我们将使用实验系统，包括斑马鱼痛风模型，以 了解调控区域在哪里表达，以及痛风相关基因座如何影响NLRP3- 炎症小体激活。斑马鱼是半透明的，可以开发创新的模式，还有 与人类以及功能相当的巨噬细胞和中性粒细胞的基因保守率为82%。在AIM 2 我们将比较MSU晶体刺激的单核细胞的转录组和表观基因组 痛风风险等位基因的高负荷和低负荷，以便进一步深入了解痛风的分子调控 路径，并确定下游路径。单个基因将在细胞系中被敲除。在AIM 3中 我们将使用我们已建立的管道来了解lncRNAs(即ipl)如何连接转录机制。 以先天免疫基因的启动子在特定的基因位点上控制痛风发作的激活。 我们的研究将加深我们对痛风耀斑机制及其遗传基础的了解，并最终 指出可能允许痛风的新治疗方法的研究领域。

SUMMARY Gout is prevalent in the US (3.9% of the adult population) and not only directly impacts peoples’ lives but is also co-morbid with cardiometabolic disease. Gout consists of unpredictable episodes of acute inflammation or flares resulting from monocyte NLRP3 inflammasome activation by monosodium urate (MSU) crystals in people with hyperuricemia, the subsequent production of IL-1, and recruitment of large numbers of inflammatory cells into the affected joint. Epigenetic reprogramming and altered gene transcription in response to elevated levels of soluble urate are mechanisms of this enhanced cellular reactivity to the secondary stimuli, known as innate trained immunity. To better understand the factors controlling the progression from hyperuricemia to gout we have completed a very large genome-wide association study (GWAS) in gout, identifying several hundred gout-associated loci. Loci include long-noncoding RNAs (lncRNAs) that have diverse functions including regulation of gene expression, deposition of epigenetic modifications and organization of chromosome architecture. Some are immune gene-priming lncRNAs (IPLs) that direct transcriptional machinery over multiple promoters. From these loci we have identified one key causal pathway (clonal hematopoiesis of indeterminate potential (CHIP)) for which we will study the molecular genetic processes controlling its activation. The CHIP pathway is involved in control of the epigenome - DNA methylation, histone modification and metabolism of substrates. It may make people more susceptible to gout by causing the innate immune system to be hyper-responsive to MSU crystals. We hypothesize that the genetic loci identified from the gout GWAS control activity of this pathway by regulation of gene expression, including through regulation of the epigenome. In testing this hypothesis, in three Aims, we will understand the molecular control of the pathway and provide a basis for future studies in targeting this pathway for improved management of gout. In Aim 1 we will use experimental systems, including a zebrafish model of gout, to understand where regulatory regions are expressed and how the gout-associated loci influence NLRP3- inflamasome activation. Zebrafish are translucent, allowing development of innovative models, and there is 82% conservation of genes with humans and functionally equivalent macrophages and neutrophils. In Aim 2 we will compare the transcriptome and epigenome in MSU crystal-stimulated monocytes of individuals with high and low burdens of gout risk alleles in order to gain further insights into the molecular regulation of the pathways, and to identify downstream pathways. Individual genes will be knocked down in a cell line. In Aim 3 we will use our established pipeline to understand how lncRNAs (i.e. IPLs) connect transcriptional machinery to the promoters of innate immune genes at specific loci in the molecular control of activation of the gout flare. Our studies will deepen our knowledge of the mechanisms of gout flares and its genetic basis, and ultimately point to areas of research that may allow for novel treatments in gout.