Post-GWAS Functional Genomics Analysis to Define Pathogenic Mechanisms for Pulmonary Arterial Hypertension

GWAS 后功能基因组学分析确定肺动脉高压的致病机制

• 批准号: 10697364
• 负责人: Wei Sun
• 金额: $ 16.07万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10697364
• 关键词: Affinity; Alleles; Allelic Imbalance; Animal Model; Architecture; Basic Science; Binding; Biology; Blood Vessels; CRISPR/Cas technology; Cell Line; Clinical; Coupled; DNA Binding; DNA Sequence Alteration; Data; Dependence; Dependovirus; Development; Diagnostic; Disease; Disease susceptibility; Down-Regulation; Endothelium; Enterochromaffin Cells; Environment; Experimental Animal Model; Functional disorder; Future; Gene Expression; Genes; Genetic; Genetic Predisposition to Disease; Genomic approach; Genomics; Genotype; Goals; Haplotypes; Human; Hypoxia; Intervention; Knockout Mice; Knowledge; Link; Linkage Disequilibrium; Lung; Lung diseases; Mediating; Mentors; Methods; Molecular; Molecular Target; Mus; Mutation; Names; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Persons; Physicians; Predisposition; Proteins; Pulmonary Hypertension; Reporting; Research; Research Personnel; Research Project Grants; Research Technics; Resources; Risk; Role; SOX17 gene; Sampling; Scientist; Severities; Single Nucleotide Polymorphism; Structure of parenchyma of lung; Techniques; Technology; Testing; Training; Translational Research; Universities; Untranslated RNA; Validation; Work; career; career development; endothelial dysfunction; familial hypertension; functional genomics; genetic association; genome wide association study; genomic locus; genomic platform; human DNA; in vivo; induced pluripotent stem cell; insight; knock-down; knowledge base; lung hypoxia; mouse model; nanoparticle; nanopolymer; novel; overexpression; pulmonary arterial hypertension; pulmonary artery endothelial cell; risk variant; siRNA delivery; skills; stem cell biology; transcription factor; vector; virulence gene

PROJECT SUMMARY Pulmonary arterial hypertension (PAH) is an enigmatic and morbid disease where insights are emerging regarding genetic susceptibility to disease. Genome-wide Association Studies (GWAS) have identified single nucleotide polymorphisms (SNPs) that are associated with PAH risk and severity. Yet, the GWAS-reported SNPs are only tags of haplotype SNPs in linkage disequilibrium (LD). Thus, the tag SNP may simply be linked to the true disease-causing functional SNP (fSNP). GWAS also only reveal statistical associations, and it has been challenging to define the mechanisms underlying the contribution of the PAH-associated fSNPs, which are mostly located in the non-coding regions, to the pathogenesis of PAH. Using our recently developed post-GWAS functional genomics platform, I identified a non-coding fSNP rs4738801 in the genomic locus of SOX17 gene, a known endothelial effector increasingly being studied in PAH pathogenesis. I identified that the transcription factor FUBP1 binds to the rs4738801 risk allele C in an allele-imbalanced manner, with lower affinity to risk allele C than non-risk allele G, providing an underlying mechanism how this fSNP regulates the PAH pathogenic gene SOX17 and contributes to the PAH risk. FUBP1 controls PAH-associated pathophenotypes in pulmonary arterial endothelial cells (PAECs). Downregulated by the major acquired PAH trigger hypoxia, FUBP1 and its target gene SOX17 are decreased in lungs and isolated pulmonary ECs from PAH patients and mouse models. A 3.77- fold enrichment of fSNP rs4738801 risk allele C was found in patients with PAH induced by hypoxia, supporting a pathogenic mechanism of a hypoxia-sensitive pathway (FUBP1-SOX17) combined with a disease susceptible genotype (risk allele C) for clinical manifestation of this disease. Based on these data, I hypothesize that the allele-imbalanced binding of transcription factor FUBP1 to fSNP rs4738801 defines the genomic architecture contributing to the SOX17-dependent genetic susceptibility of PAH. I further postulate that the downregulation of FUBP1-SOX17 by hypoxia contributes to the acquired pathogenesis of PAH. To test this hypothesis, I propose 2 specific aims: 1) To define the allele-specific role of fSNP rs4738801 in promoting endothelial dysfunction in PAH in gene-edited iPSC-ECs and PAH patient lung tissues; and 2) To determine the role of FUBP1 in controlling SOX17 and PAH in mouse models. Accomplishing these aims will facilitate my enduing career goal of becoming an independent physician-scientist in PAH functional genomics research. Immediate scientific development objectives include: 1) To develop expertise in PAH genetics and functional genomics; 2) To develop expertise in iPSC-EC biology and CRISPR-Cas9 gene-editing techniques; and 3) To develop skills of in vivo gene expression manipulation and become proficient in the assessment of PAH in animal models. The proposed training plan will provide me with the opportunity to expand my knowledge base to include advanced research techniques in PAH pathogenesis. The resources and expertise of my mentors, contributors, and the rich research environment at the University of Pittsburgh will assure my successful transition to an independent investigator.

项目总结 肺动脉高压(PAH)是一种神秘而病态的疾病，人们对它的认识正在浮现。 关于疾病的遗传易感性。全基因组关联研究(GWAS)已经确定了单个 与PAH风险和严重性相关的核苷酸多态(SNPs)。然而，GWAS报告的SNPs 只是处于连锁不平衡(LD)的单倍型SNPs的标签。因此，标签SNP可以简单地链接到 真正的致病功能性SNP(FSNP)。Gwas也只揭示了统计上的关联，而且它已经 定义与多环芳烃相关的fSNP的潜在贡献机制具有挑战性，这些fSNP是 多位于非编码区，对PAH的发病起重要作用。使用我们最近开发的后GWA后 在功能基因组学平台上，我在SOX17基因的基因组座位上发现了一个非编码的fSNP rs4738801，a 已知的内皮效应在PAH发病机制中的研究日益增多。我确认这份抄本 FUBP1因子以等位基因不平衡的方式与rs4738801风险等位基因C结合，与风险等位基因亲和力较低 C比非危险等位基因G，提供了一种潜在的机制，该fSNP如何调节PAH致病基因 SOX17，并导致PAH风险。FUBP1控制PAH相关的肺动脉病理表型 内皮细胞(PAECs)。主要获得性PAH触发低氧下调FUBP1及其靶点 SOX17基因在PAH患者和小鼠肺组织和分离的肺内皮细胞中表达降低。A 3.77- 低氧、支持性PAH患者fSNP rs4738801风险等位基因C倍体丰富 低氧敏感途径(FUBP1-SOX17)与疾病易感性结合的致病机制 本病临床表现的基因分型(危险等位基因C)。根据这些数据，我假设 转录因子FUBP1与fSNP rs4738801的等位基因不平衡结合定义了基因组结构 与SOX17依赖的PAH遗传易感性有关。我进一步假设，下调监管 低氧诱导的FUBP1-SOX17表达参与了PAH的获得性发病机制。为了检验这一假设，我建议 2特定目的：1)确定fSNP rs4738801在促进血管内皮细胞功能障碍中的等位基因特异性作用。 基因编辑的IPSC-ECs和PAH患者肺组织中的PAH；以及2)确定FUBP1在控制 SOX17和PAH对小鼠模型的影响。实现这些目标将有助于我成为 多环芳烃功能基因组研究方面的独立内科医生兼科学家。近期科学发展 目标包括：1)发展多环芳烃遗传学和功能基因组学方面的专门知识；2)发展以下方面的专门知识 IPSC-EC生物学和CRISPR-Cas9基因编辑技术；3)发展体内基因表达技能 并精通动物模型中多环芳烃的评估。拟议的培训计划将 让我有机会扩展我的知识库，将PAH中的高级研究技术包括在内 发病机制。我的导师、贡献者的资源和专业知识，以及 匹兹堡大学将确保我成功地过渡到一名独立调查员。