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.

项目概要 肺动脉高压（PAH）是一种神秘而致病的疾病，人们的见解正在不断涌现 关于疾病的遗传易感性。全基因组关联研究（GWAS）已经确定了单一 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 仅是连锁不平衡 (LD) 中单倍型 SNP 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 也仅揭示统计关联，并且已被 确定 PAH 相关 fSNP 贡献的潜在机制具有挑战性，这些机制是 大多位于非编码区，与PAH发病有关。使用我们最近开发的后 GWAS 通过功能基因组学平台，我在 SOX17 基因的基因组位点中发现了一个非编码 fSNP rs4738801， 已知的内皮效应器越来越多地在 PAH 发病机制中得到研究。我发现转录 因子 FUBP1 以等位基因不平衡的方式与 rs4738801 风险等位基因 C 结合，与风险等位基因的亲和力较低 C 比非风险等位基因 G 更重要，提供了该 fSNP 如何调节 PAH 致病基因的潜在机制 SOX17 会增加 PAH 风险。 FUBP1 控制肺动脉中 PAH 相关的病理表型 内皮细胞（PAEC）。由主要获得性 PAH 引发缺氧的 FUBP1 及其靶标下调 基因 SOX17 在肺和 PAH 患者和小鼠模型中分离的肺 EC 中减少。 3.77- 在缺氧引起的 PAH 患者中发现了 fSNP rs4738801 风险等位基因 C 的倍数富集，支持 缺氧敏感通路（FUBP1-SOX17）与疾病易感基因相结合的致病机制 该疾病临床表现的基因型（风险等位基因 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 导致 PAH 的 SOX17 依赖性遗传易感性。我进一步假设下调 缺氧引起的 FUBP1-SOX17 的表达有助于 PAH 的获得性发病机制。为了检验这个假设，我建议 2 个具体目标： 1) 明确 fSNP rs4738801 在促进内皮功能障碍中的等位基因特异性作用 基因编辑 iPSC-EC 和 PAH 患者肺组织中的 PAH； 2) 确定 FUBP1 在控制中的作用 小鼠模型中的 SOX17 和 PAH。实现这些目标将有助于我最终的职业目标成为 一位从事多环芳烃功能基因组学研究的独立医师科学家。立即科学发展 目标包括： 1) 发展 PAH 遗传学和功能基因组学方面的专业知识； 2) 发展专业知识 iPSC-EC 生物学和 CRISPR-Cas9 基因编辑技术； 3) 培养体内基因表达技能 操作并精通动物模型中 PAH 的评估。拟议的培训计划将 为我提供了扩展知识库的机会，包括 PAH 的先进研究技术 发病。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过渡为独立调查员。