Functional Genomics of Platelet Aggregation Using iPS and Derived Megakaryocytes

使用 iPS 和衍生巨核细胞进行血小板聚集的功能基因组学

• 批准号: 8690135
• 负责人: Lewis C Becker
• 金额: $ 233.83万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-05 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8690135
• 关键词: Acute; Affect; African American; Aspirin; Biological Assay; Blood Platelets; Cardiovascular Diseases; Cardiovascular system; Cells; Development; Disease; Dose; Event; Exons; Genes; Genetic; Genomics; Genotype; Hematology; Hematopoietic; Hemostatic function; Individual; Instruction; Intercistronic Region; Introns; Knowledge; Lead; Mass Spectrum Analysis; Measures; Medicine; Megakaryocytes; Messenger RNA; Methods; Missense Mutation; Modeling; Modification; Molecular; Pathway interactions; Patients; Pattern; Peripheral Blood Mononuclear Cell; Phase; Phenotype; Platelet aggregation; Pluripotent Stem Cells; Population; Process; Race; Recurrence; Research; Research Personnel; Risk Assessment; Signal Transduction; Single Nucleotide Polymorphism; Stem cells; Thrombosis; Transcript; Transfusion; Variant; base; functional genomics; genetic variant; genome wide association study; genome-wide; induced pluripotent stem cell; innovation; interest; mRNA Expression; new therapeutic target; novel; prevent; programs; protein expression; response

DESCRIPTION (provided by applicant): The causal mechanisms of common diseases are only marginally illuminated by genetic variants found in genome wide association studies (GWAS) using single nucleotide polymorphism (SNPs). Platelet pathways reflecting hemostasis and thrombosis are the underlying substrate for many cardiovascular diseases and related acute events. To overcome GWAS limitations, genomic studies must integrate molecular surrogates for platelet-related phenotypes assayed in cell-based models derived from individuals of known genotypes and phenotypes. In our GWAS study of native platelet aggregation and aggregation in response to low dose aspirin (GeneSTAR, Genetic Study of Aspirin Responsiveness), 64 loci were associated with native platelet aggregation at genome wide significance (p<5x10"^) while 57 were associated with platelet responsiveness to aspirin, many replicated in both races. Although we are performing functional genomics studies to elucidate findings in known genes (PEAR1, RGL3, and MET), most signals were in intergenic regions (38%), or in introns (55%), with only 1.6% producing missense mutations in exons. Mechanistic interpretation is uncertain re which gene(s) are up- or down-regulated based on SNP modifications. In 3 phases, we will (1) create pluripotent stem cells (iPS) from peripheral blood mononuclear cells, and then differentiate these stem cells into megakaryocytes (2) efficiently produce iPS and megakaryocytes using a novel pooling method, and (3) produce iPS and megakaryocytes from 400 subjects in GeneSTAR (200 whites, 200 African Americans), selected based on specific hypotheses derived from GWAS signals in native and post aspirin platelet function; characterize genetic mRNA transcripts using a comprehensive Affymetrix exon array; measure protein expression for transcripts of interest using mass spectrometry; examine mRNA and protein expression patterns for each GWAS signal to determine the functional pathway(s) involved in native platelet phenotypes; and examine the functional genomics of variations in aspirin response using our prior genotyped and phenotyped population. This project at Johns Hopkins will be conducted by an interdisciplinary group of expert investigators. (Phase 1 and II, PI, L Cheng, Hematology Division, Dept of Medicine; Phase III PI, L Becker, GeneSTAR Research Program), RELEVANCE (See instructions): Precise information about the functional processes in megakaryocytes and platelets may lead to innovative and tailored approaches to risk assessment and novel therapeutic targets to prevent first and recurrent cardiovascular and related acute thrombotic events. Further, Phase I and II developmental research will contribute to new knowledge that would positively affect the transfusion of iPS-derived hematopoietic cells in patients with such cell deficiencies.

描述（由申请人提供）： 使用单核苷酸多态性 (SNP) 进行的全基因组关联研究 (GWAS) 中发现的遗传变异只能勉强阐明常见疾病的因果机制。反映止血和血栓形成的血小板通路是许多心血管疾病和相关急性事件的基础。为了克服 GWAS 的局限性，基因组研究必须整合在源自已知基因型和表型个体的细胞模型中测定的血小板相关表型的分子替代物。在我们针对低剂量阿司匹林反应的天然血小板聚集和聚集的 GWAS 研究中（GeneSTAR，阿司匹林反应性遗传学研究），64 个位点在全基因组范围内与天然血小板聚集相关（p<5x10"^），而 57 个位点与血小板对阿司匹林的反应性相关，其中许多位点在两个种族中都得到了复制。尽管我们正在进行功能基因组学研究以阐明已知基因（PEAR1、 RGL3 和 MET），大多数信号位于基因间区域（38%）或内含子（55%），只有 1.6% 在外显子中产生错义突变。根据 SNP 修饰，哪些基因上调或下调的机制解释是不确定的。分三个阶段，我们将 (1) 从外周血单核细胞中创建多能干细胞 (iPS)，然后分化 将这些干细胞转化为巨核细胞 (2) 使用新颖的混合方法有效地产生 iPS 和巨核细胞，以及 (3) 从 GeneSTAR 中的 400 名受试者（200 名白人，200 名非洲裔美国人）中产生 iPS 和巨核细胞，这些受试者是根据来自天然和阿司匹林后血小板功能的 GWAS 信号的特定假设进行选择的；使用综合 Affymetrix 表征遗传 mRNA 转录本 外显子阵列；使用质谱法测量感兴趣转录本的蛋白质表达；检查每个 GWAS 信号的 mRNA 和蛋白质表达模式，以确定参与天然血小板表型的功能途径；并使用我们之前的基因分型和表型人群检查阿司匹林反应变异的功能基因组学。约翰·霍普金斯大学的这个项目将由一个由专家研究人员组成的跨学科小组进行。 （第一阶段和第二阶段， PI，L Cheng，医学系血液科； III 期 PI，L Becker，GeneSTAR 研究计划），相关性（参见说明书）：有关巨核细胞和血小板功能过程的精确信息可能会带来创新和定制的风险评估方法和新的治疗目标，以预防首次和复发性心血管及相关急性血栓事件。此外，第一期和第二期开发研究 将有助于产生新知识，从而对患有此类细胞缺陷的患者输注 iPS 衍生造血细胞产生积极影响。