脂联素基因变异及表观遗传修饰在COPD发生中的作用研究

Chronic Obstructive Pulmonary Disease (COPD) exhibits a strong genetic component in its pathogenesis. Genetic studies have identified some common variants associated with increased risk for COPD. However, all collectively explain only very small fraction of the genetic variance of COPD. The ‘missing heritability’ could be explained by various factors, the rare variants and DNA methylation will certainly contribute. Rare variants are abundant in human genome, and some are known to exert larger effect sizes than the common variants. DNA methylation, particularly within regulatory genomic regions such as the promoter region, may alter the transcriptional state of the gene, thereby modifying its expression and the expression of gene-related products. Low-grade systemic inflammation is thought to contribute to the complex pathogenesis of COPD. Increased levels of various systemic inflammatory biomarkers, like CRP, IL-6 and fibrinogen, have been repeatedly reported in patients with stable COPD and rise even more during acute exacerbations.Accumulating evidence indicates that the fat tissue is involved in the development of low-grade systemic inflammation and that the adipose tissue is a key endocrine organ because of releasing a wide variety of bioactive substances. Dysregulated production of adipose-derived factors or adipokines with proinflammatory or anti-inflammatory activities can contribute to the pathogenesis of low-grade systemic inflammation. Adiponectin is almost exclusively synthesized by adipocytes, and adiponectin levels are reduced in the plasma of obese individuals. Adiponectin plays an important role in controlling inflammation. In this project, we primarily aimed to (1) Identify rare variants in adiponectin and test the associations with COPD. We will sequence adiponectin gene in 100 COPD patients and 100 controls. The identified variants will be screened in additional 900 COPD patients and 900 controls. We also test the rare variants’ function using strategies based on their locations in gene. (2) Investigate the DNA methylation difference of adiponectin between COPD patients and control individuals. We will obtain genomic DNA from peripheral blood mononuclear cells (PBMCs), which could reflect the status of the immune system. Compare the methylation profiles in the promoter region of each gene by EpiTech methyl quantitative PCR (qPCR) and further pryosequencing after bisulfite treatment. (3) Test the effect of non-synonymours variant(nsSNV) of adiponectin on macrophage polarization by site-directed mutagenetis. We hypothesized that nsSNV affected the adiponectin function modulates macrophage polarization. Macrophages, playing key role in COPD inflammation, exhibit diversity and plasticity; they can exhibit “classical activation”(M1), associated with pro-inflammatory responses, or “alternative activation”(M2), associated with anti-inflammatory responses.

脂肪组织合成的脂联素与COPD及其系统性炎症密切相关，我们首次报道了脂联素基因(AdipoQ)常见变异(频率>0.05)与COPD的关系(PloS One,2012)。最近发现基因组罕见变异(频率<0.01)及甲基化对复杂疾病具有重要影响，但目前缺乏AdipoQ的罕见变异及甲基化对COPD的影响。脂联素在COPD中兼具促炎和抗炎效应，这是否与AdipoQ基因变异和甲基化修饰有关？目前尚无研究。巨噬细胞在COPD炎症反应中发挥重要作用，脂联素可促使巨噬细胞向促炎M1型或抗炎M2型极化，这是否受AdipoQ基因非同义变异(nsSNV)的调节，对此目前不清楚。在本项目中，我们将①对AdipoQ深度测序，发现影响COPD及其系统性炎症的罕见变异，并阐明功能；②检测分析AdipoQ甲基化，阐明其对COPD的影响；③定点诱变AdipoQ基因nsSNV，阐明AdipoQ基因变异对巨噬细胞极化的影响。

脂联素（adiponectin）受体基因CDH13位于16号染色体16q上。我们在279例COPD患者及367健康对照人群中，筛查了CDH13基因10个Tag SNPs（rs4783244、rs12922394、rs11646011、rs11640875、rs1870843、rs4783266、 rs11640522、 rs11646849、 rs11860282及rs2549151）的分布频率，并分析了它们与COPD发生的相关性。发现rs4783244 和rs12922394的等位基因或基因型在COPD和对照组的分布频率存在显著差异。rs4783266-rs11640522-rs11646849-rs11860282构成的单倍型GTAC and ATGT与COPD发病风险增高显著相关。