Functional Genomics of Platelet Aggregation Using iPS and Derived Megakaryocytes

描述（由申请人提供）： 常见疾病的致病机制仅在使用单核苷酸多态性（SNP）的全基因组关联研究（GWAS）中发现的遗传变异中得到少量阐明。反映止血和血栓形成的血小板途径是许多心血管疾病和相关急性事件的潜在底物。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，这些表型是在来自已知基因型和表型的个体的基于细胞的模型中测定的。在我们的天然血小板聚集和响应于低剂量阿司匹林的聚集的GWAS研究（GeneSTAR，阿司匹林响应性的遗传研究）中，64个基因座与全基因组显著性的天然血小板聚集相关（p<5 × 10 - 4），而57个基因座与血小板对阿司匹林的响应性相关，许多在两个种族中重复。尽管我们正在进行功能基因组学研究以阐明已知基因（PEAR 1，RGL 3和MET）的发现，但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。关于哪些基因基于SNP修饰上调或下调的机制解释是不确定的。在3个阶段中，我们将（1）从外周血单核细胞中产生多能干细胞（iPS），然后将这些干细胞分化为巨核细胞（2）使用新的混合方法有效地产生iPS和巨核细胞，以及（3）在GeneSTAR中从400名受试者中产生iPS和巨核细胞（200名白人，200名非裔美国人），基于源自天然和阿司匹林后血小板功能中GWAS信号的特定假设进行选择;使用全面的Affytron外显子阵列表征遗传mRNA转录物;使用质谱法测量感兴趣的转录物的蛋白质表达;检查每个GWAS信号的mRNA和蛋白质表达模式，以确定天然血小板表型中涉及的功能途径;并使用我们先前的基因分型和表型人群检查阿司匹林反应变化的功能基因组学。约翰霍普金斯大学的这个项目将由一个跨学科的专家研究小组进行。（I期和II期，PI，L Cheng，血液学部，医学部; III期PI，L Becker，GeneSTAR研究项目），相关性（参见说明）：关于巨核细胞和血小板功能过程的精确信息可能导致创新和定制的风险评估方法和新的治疗靶点，以预防首次和复发的心血管和相关急性血栓事件。此外，I期和II期发育研究将有助于新的知识，这将积极影响iPS衍生造血细胞输注这些细胞缺陷的患者。

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.