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: 10524975
• 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/10524975
• 关键词: Address; 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; Polymers; 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; base; 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; novel; overexpression; pulmonary arterial hypertension; pulmonary artery endothelial cell; risk variant; siRNA delivery; skills; stem cell biology; transcription factor; vector

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.

项目摘要 肺动脉高压（PAH）是一种神秘的病态疾病， 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风险和严重程度相关的核苷酸多态性（SNP）。然而，GWAS报告的SNP 是连锁不平衡（LD）中单倍型SNPs的唯一标签。因此，标签SNP可以简单地连接到标签SNP。 真正致病功能性SNP（fSNP）。GWAS也只揭示了统计学上的关联， 定义PAH相关fSNP的作用机制具有挑战性， 大多位于非编码区，与PAH的发病有关。使用我们最近开发的后GWAS 在功能基因组学平台上，我在SOX 17基因的基因组位点上鉴定了一个非编码fSNP rs 4738801， 已知的内皮效应物在PAH发病机制中的研究越来越多。我发现这份抄本 因子FUBP 1以等位基因不平衡的方式与rs 4738801风险等位基因C结合，对风险等位基因具有较低的亲和力 C比非风险等位基因G，提供了该fSNP如何调节PAH致病基因的潜在机制 SOX 17，并导致PAH风险。FUBP 1控制肺动脉中PAH相关的病理表型 内皮细胞（PAECs）。由主要获得性PAH触发缺氧下调的FUBP 1及其靶点 基因SOX 17在肺和来自PAH患者和小鼠模型的分离的肺EC中减少。A 3.77- 在缺氧诱导的PAH患者中发现fSNP rs 4738801风险等位基因C的倍数富集，支持 缺氧敏感性通路（FUBP 1-SOX 17）的致病机制与易受 基因型（风险等位基因C）的临床表现，这种疾病。根据这些数据，我假设 转录因子FUBP 1与fSNP rs 4738801的等位基因不平衡结合定义了基因组结构 有助于PAH的SOX 17依赖性遗传易感性。我进一步假设下调 缺氧诱导FUBP 1-SOX 17基因表达的改变参与了PAH的获得性发病机制。为了验证这个假设，我建议 2个具体目的：1）确定fSNP rs 4738801在促进内皮功能障碍中的等位基因特异性作用， 基因编辑的iPSC-EC和PAH患者肺组织中的PAH;以及2）确定FUBP 1在控制PAH中的作用。 SOX 17和PAH在小鼠模型中的作用实现这些目标将有助于我实现职业目标， PAH功能基因组学研究方面的独立医生兼科学家。近期科学发展 目标包括：1）培养PAH遗传学和功能基因组学方面的专业知识; 2）培养以下方面的专业知识： iPSC-EC生物学和CRISPR-Cas9基因编辑技术;以及3）开发体内基因表达的技能 操作，并成为在动物模型中PAH的评估熟练。拟议的培训计划将 为我提供了扩展知识基础的机会，包括PAH的先进研究技术 发病机制我的导师，贡献者的资源和专业知识，以及丰富的研究环境， 匹兹堡大学将确保我成功转型为独立调查员