Variation in platelet function: the genetics of platelet gene expression

描述（由申请方提供）：人血小板反应性存在极端的个体间差异，这可能导致某些个体在动脉粥样硬化斑块破裂后冠状动脉和脑动脉闭塞，而其他个体在不闭塞血管的情况下修复伤口。然而，有一个缺乏了解的分子和遗传机制负责这种变化的血小板功能。我们先前的全基因组扫描和血小板RNA表达研究已经阐明了肾上腺素诱导的血小板聚集的机制，鉴定了mRNA表达差异，以及微RNA（miRNA）调节血小板分泌蛋白VAMP 8表达的意想不到的证据。其他分析表明，交替外显子剪接和基因组拷贝数的变化也有助于血小板基因表达的变化。虽然我们的血小板RNA分析研究包括29名受试者，但其鉴定参与血小板对肾上腺素以外激动剂反应的基因的能力有限。我们现在建议召回180名受试者进行新的血小板表型分析，制备去白细胞的血小板RNA和mRNA和miRNA的微阵列分析。严格的生物信息学和统计学方法将最大限度地减少假阳性，候选mRNA和miRNA将通过实验进行表征。这种方法将允许定义调节血小板对特定激动剂（ADP、CRP、胶原、TRAP、U46619、利托那韦、CD 9和肾上腺素）的反应的基因，以及多种血小板刺激物共有的基因，并允许评估性别和种族的影响（目标1）。在目标2-4中，我们将描述调节不同反应性血小板中mRNA水平的分子机制。我们的分析将确定血小板反应性和交替剪接外显子之间的关联，从而作为血小板蛋白（目标2）的功能域的屏幕。我们将确定血小板miRNA谱，并表征不同反应性血小板中差异表达的miRNA的功能（目的3）。通过将RNA表达数据与我们先前对这些相同受试者的GWAS基因分型相关联，我们将讨论拷贝数变异对血小板基因表达的作用（目的4）。我们还将生成公共网络工具，根据性别，种族和年龄提供血小板mRNA和miRNA的相对表达。这项研究将导致对血小板生理学的新见解，增强我们对血小板基因表达遗传学的理解，促进血栓形成风险的性别和种族特异性生物标志物的选择，并为其他血小板研究人员提供有用的工具。 公共卫生相关性：心血管疾病是美国死亡的主要原因，这项研究的目标是更好地了解心血管疾病中凝血的机制，以便开发更好的药物开发靶点和更好的疾病风险预测指标。

DESCRIPTION (provided by applicant): There is extreme inter-individual variation in human platelet reactivity, which likely contributes to occlusion of coronary and cerebral arteries upon atherosclerotic plaque rupture in some individuals, whereas other individuals repair the wound without occluding the vessel. However, there is a lack of understanding of the molecular and genetic mechanisms responsible for this variation in platelet function. Our previous genome wide scan and platelet RNA expression studies have elucidated mechanisms for epinephrine-induced platelet aggregation, identified mRNA expression differences, as well as unexpected evidence that microRNAs (miRNAs) regulate expression of the platelet secretory protein, VAMP8. Additional analyses suggest that alternate exon splicing and genomic copy number variation also contribute to variation in platelet gene expression. Although our platelet RNA profiling study included 29 subjects, it was limited in its ability to identify genes involved in the platelet response to agonists other than epinephrine. We now propose to recall 180 subjects for new platelet phenotyping, preparation of leukocyte-depleted platelet RNA and microarray profiling of mRNA and miRNA. Rigorous bioinformatic and statistical approaches will minimize false positives, and candidate mRNAs and miRNAs will be characterized experimentally. This approach will allow definition of genes regulating the platelet response to specific agonists (ADP, CRP, collagen, TRAP, U46619, ristocetin, CD9 and epinephrine), as well as genes common to multiple platelet stimuli, and permit assessment of the effects of gender and ethnicity (Aim 1). In Aims 2-4 we will characterize molecular mechanisms regulating mRNA levels in platelets of differing reactivity. Our analyses will identify associations between platelet reactivity and alternately spliced exons, thus serving as a screen for functional domains in the platelet protein (Aim 2). We will determine platelet miRNA profiles and characterize the function of differentially expressed miRNAs in platelets of differing reactivity (Aim 3). By correlating RNA expression data with our prior GWAS genotyping on these same subjects, we will address the role of copy number variation on platelet gene expression (Aim 4). We will also generate public web tools that provide the relative expression of platelet mRNAs and miRNAs according to gender, race and age. This research will result in new insights into platelet physiology, enhance our understanding of the genetics of platelet gene expression, facilitate the selection of gender and race-specific biomarkers for thrombosis risk, and provide useful tools for other platelet researchers. PUBLIC HEALTH RELEVANCE: Cardiovascular disease is the major cause of mortality in the U.S. The goal of this research is to understand better the mechanisms that contribute to blood clotting in cardiovascular disease in order to develop better targets for drug development and better predictors of disease risk.