Functional Genomics of Platelet Aggregation Using iPS and Derived Megakaryocytes

描述(由申请人提供)： 在使用单核苷酸多态(SNPs)的全基因组关联研究(GWAS)中发现的遗传变异只略微阐明了常见疾病的致病机制。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血小板相关表型的分子替代物，这些表型是从已知基因类型和表型的个体衍生的基于细胞的模型中分析的。在我们关于低剂量阿司匹林对天然血小板聚集和聚集的研究中(GeneSTAR，阿司匹林反应性的遗传学研究)，基因座与天然血小板聚集在全基因组意义上相关(p&lt；5x10“^)，而57个基因座与血小板对阿司匹林的反应性相关，其中许多在两个种族中都是重复的。虽然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)的发现，但大多数信号位于基因间隔区(38%)或内含子(55%)，只有1.6%的信号产生外显子的错义突变。基于SNP修饰，机制解释不确定哪个基因(S)表达上调或下调。我们将分三个阶段：(1)从外周血单核细胞中培养出多能干细胞，然后将其分化为巨核细胞；(2)利用一种新的混合方法高效地产生多能干细胞和巨核细胞；以及(3)从GeneSTAR(200名白人，200名非裔美国人)的400名受试者(200名白人和200名非裔美国人)中产生多能干细胞和巨核细胞，这些细胞和巨核细胞是基于特定的假设而选择的，这些假设来自于天然和阿司匹林后的血小板功能；使用一个全面的Affymetrix外显子阵列来表征遗传的mRNAs；使用质谱仪测量感兴趣的转录本的蛋白质表达；检查每个GWAs信号的mRNAs和蛋白质表达模式，以确定与天然血小板表型有关的功能途径(S)；并使用我们先前的基因分型和表型人群来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调查人员组成的跨学科小组进行。(第一阶段和第二阶段，Pi，L程，医学系血液科；第三阶段，Pi，L Becker，GeneSTAR研究计划)，相关性(参见说明)：关于巨核细胞和血小板功能过程的准确信息可能导致风险评估的创新和定制方法，以及预防首次和复发心血管及相关急性血栓形成事件的新治疗靶点。此外，第一阶段和第二阶段的开发研究将有助于新的知识，这将积极影响iPS来源的造血细胞在此类细胞缺陷患者中的输注。

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.