Center for Pulmonary Vascular Biology and Medicine, Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, Division of Cardiology, Department of Medicine University of Pittsburgh School of Medicine and University of Pittsburgh Medical Center Pittsburgh PA.
School of Medicine Tsinghua University Beijing China.
J Am Heart Assoc. 2023 Apr 4;12(7):e027894. doi: 10.1161/JAHA.122.027894. Epub 2023 Mar 28.
Background Pulmonary arterial hypertension (PAH) is a complex, fatal disease where disease severity has been associated with the single nucleotide polymorphism (SNP) rs2856830, located near the human leukocyte antigen DPA1 (HLA-DPA1) gene. We aimed to define the genetic architecture of functional variants associated with PAH disease severity by identifying allele-specific binding transcription factors and downstream targets that control endothelial pathophenotypes and PAH. Methods and Results Electrophoretic mobility shift assays of oligonucleotides containing SNP rs2856830 and 8 SNPs in linkage disequilibrium revealed functional SNPs via allele-imbalanced binding to human pulmonary arterial endothelial cell nuclear proteins. DNA pulldown proteomics identified SNP-binding proteins. SNP genotyping and clinical correlation analysis were performed in 84 patients with PAH at University of Pittsburgh Medical Center and in 679 patients with PAH in the database. SNP rs9277336 was identified as a functional SNP in linkage disequilibrium (>0.8) defined by rs2856830, and the minor allele was associated with decreased hospitalizations and improved cardiac output in patients with PAH, an index of disease severity. SNP pulldown proteomics showed allele-specific binding of nuclear ACTN4 (alpha actinin 4) protein to rs9277336 minor allele. Both ACTN4 and HLA-DPA1 were downregulated in pulmonary endothelium in human patients and rodent models of PAH. Via transcriptomic and phenotypic analyses, knockdown of HLA-DPA1 phenocopied knockdown of ACTN4, both similarly controlling cell structure pathways, immune pathways, and endothelial dysfunction. Conclusions We defined the pathogenic activity of functional SNP rs9277336, entailing the allele-specific binding of ACTN4 and controlling expression of the neighboring HLA-DPA1 gene. Through inflammatory or genetic means, downregulation of this ACTN4-HLA-DPA1 regulatory axis promotes endothelial pathophenotypes, providing a mechanistic explanation for the association between this SNP and PAH outcomes.
背景 肺动脉高压（PAH）是一种复杂的致命疾病，疾病的严重程度与位于人类白细胞抗原 DPA1（HLA-DPA1）基因附近的单核苷酸多态性（SNP）rs2856830 相关。我们旨在通过鉴定与 PAH 疾病严重程度相关的功能性变异体的等位基因特异性结合转录因子和下游靶标，来定义与 PAH 相关的内皮病理表型和 PAH 的遗传结构。
方法和结果 通过电泳迁移率变动分析含有 SNP rs2856830 和 8 个连锁不平衡 SNP 的寡核苷酸，发现了通过人肺动脉内皮细胞核蛋白等位基因不平衡结合而具有功能的 SNP。DNA 下拉蛋白质组学鉴定了 SNP 结合蛋白。在匹兹堡大学医学中心的 84 名 PAH 患者和 数据库中的 679 名 PAH 患者中进行了 SNP 基因分型和临床相关性分析。SNP rs9277336 被确定为 rs2856830 定义的连锁不平衡（>0.8）的功能性 SNP，其次要等位基因与 PAH 患者的住院次数减少和心输出量增加相关，这是疾病严重程度的一个指标。SNP 下拉蛋白质组学显示核 ACTN4（α 辅肌动蛋白 4）蛋白对 rs9277336 次要等位基因的等位基因特异性结合。在人类患者和 PAH 啮齿动物模型的肺内皮中，ACTN4 和 HLA-DPA1 均下调。通过转录组学和表型分析，HLA-DPA1 的敲低模拟了 ACTN4 的敲低，两者均相似地控制着细胞结构途径、免疫途径和内皮功能障碍。
结论 我们定义了功能性 SNP rs9277336 的致病活性，包含 ACTN4 的等位基因特异性结合，并控制着邻近 HLA-DPA1 基因的表达。通过炎症或遗传手段，下调该 ACTN4-HLA-DPA1 调节轴促进内皮病理表型，为该 SNP 与 PAH 结局之间的关联提供了一种机制解释。
