Noncoding SNPs decrease expression of FABP5 during COPD exacerbations

Differential susceptibility to recurrent infection and exacerbation in COPD is not well understood. Here we investigated the potential impact of genetic variation in Fatty Acid Binding Protein 5 (FABP5), a metabolic regulator we previously demonstrated to be downregulated in COPD and further downregulated in patients reporting one or more exacerbation. Through negative binomial analysis of the COPDGene SNP dataset, we identified 5 novel linked single nucleotide polymorphisms (SNPs) across the FABP5 locus that were significantly associated with severe exacerbations in a Non-Hispanic White cohort (rs4338057, rs12549270, rs202275, rs202277, and rs202279). We integrated multiple sources of previously published data to prioritize SNPs most likely to exert regulatory function. Ultimately, rs202275 emerged as the lead candidate due to unique alignment with the loading site of a bidirectional RNA Pol II signature of active enhancer utilization in our recently published patient-derived airway epithelial PRO-seq dataset, suggesting a critical regulatory role for the region harboring this variant. Using Micro-C genome-wide chromosome conformation capture, we found that the region harboring rs202275 makes three-dimensional physical contacts with the FABP5 transcription start site (TSS), suggesting the SNP region could regulate FABP5 transcription through a looping mechanism. To examine SNP function, we analyzed previously deposited gene-array data (GEO Accession #GSE42057) from peripheral blood mononuclear cell (PBMC) samples and found that COPD patients carrying the rs202275 risk allele (T) express significantly lower levels of FABP5 compared to non-carrier patients. Seahorse real-time mitochondrial respiration assays in freshly isolated PBMCs further revealed disrupted oxidative phosphorylation in cells from patients carrying the rs202275 risk allele. As macrophage polarization from pro-inflammatory toward pro-resolving phenotype is dependent on oxidative metabolism, the functional impact of the rs202275 risk allele and associated reduction in FABP5 transcription may contribute to increased or prolonged systemic inflammation, thereby increasing susceptibility to exacerbation in COPD. Vehicle-treated BEAS-2B airway epithelial cells were used for Micro-C to evaluate genome-wide three-dimensional chromatin conformation under basal conditions.慢性阻塞性肺疾病（Chronic Obstructive Pulmonary Disease, COPD）患者对复发性感染与急性加重的差异易感性机制尚未得到充分阐明。本研究探讨了脂肪酸结合蛋白5（Fatty Acid Binding Protein 5, FABP5）遗传变异的潜在影响——该代谢调控因子此前已被证实于COPD患者体内表达下调，且在报告过至少一次急性加重的患者中表达进一步降低。 通过对COPDGene单核苷酸多态性（Single Nucleotide Polymorphism, SNP）数据集开展负二项分布分析，我们在非西班牙裔白人队列中鉴定出FABP5基因座上的5个全新连锁单核苷酸多态性位点，其与重度急性加重显著相关（rs4338057、rs12549270、rs202275、rs202277及rs202279）。 我们整合多组已发表数据，以筛选出最有可能发挥调控功能的SNP。最终，rs202275成为核心候选位点，因其与我们近期发表的患者来源气道上皮细胞PRO-seq（Precision Run-On Sequencing）数据中活性增强子利用的双向RNA聚合酶II（RNA Polymerase II, RNA Pol II）结合位点精准匹配，提示该变异所在区域具有关键调控作用。 借助全基因组染色体构象捕获Micro-C技术，我们发现rs202275所在区域与FABP5的转录起始位点（Transcription Start Site, TSS）存在三维物理相互作用，提示该SNP区域可通过染色质环化机制调控FABP5的转录。 为探究该SNP的功能，我们分析了此前公开的外周血单个核细胞（Peripheral Blood Mononuclear Cell, PBMC）基因芯片数据（基因表达综合数据库（Gene Expression Omnibus, GEO）登录号GSE42057），结果显示携带rs202275风险等位基因（T）的COPD患者，其FABP5表达水平显著低于非携带者。 对新鲜分离的PBMC进行Seahorse实时线粒体呼吸功能检测进一步证实，携带rs202275风险等位基因的患者细胞存在氧化磷酸化功能紊乱。 由于巨噬细胞从促炎表型向促消退表型的极化依赖于氧化代谢，rs202275风险等位基因的功能影响及伴随的FABP5转录下调，可能会加剧或延长全身性炎症，进而增加COPD患者的急性加重易感性。本研究使用载体处理的BEAS-2B气道上皮细胞开展Micro-C实验，以评估基础状态下的全基因组三维染色质构象。