Variation in platelet function: the genetics of platelet gene expression

描述(由申请人提供)：人的血小板反应性在个体之间存在极大的差异，这可能导致一些人在动脉粥样硬化斑块破裂时闭塞冠状动脉和脑动脉，而另一些人则在不阻塞血管的情况下修复伤口。然而，对导致这种血小板功能变化的分子和遗传机制缺乏了解。我们之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，确定了mRNA表达的差异，以及意外的证据表明，microRNAs(MiRNAs)调节血小板分泌蛋白VAMP8的表达。其他分析表明，外显子的交替剪接和基因组拷贝数的变化也是导致血小板基因表达变化的原因之一。虽然我们的血小板RNA图谱研究包括29名受试者，但它识别参与血小板对肾上腺素以外的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鉴定，制备去白细胞的血小板RNA，并对mRNA和miRNA进行微阵列分析。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mRNAs和miRNAs将进行实验表征。这一方法将允许定义调节特定激动剂(ADP、CRP、胶原、TRAP、U46619、瑞斯托菌素、CD9和肾上腺素)的血小板反应的基因，以及多种血小板刺激的共同基因，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选择性剪接外显子之间的关联，从而筛选出血小板蛋白中的功能区域(目标2)。我们将测定血小板miRNA图谱，并鉴定不同反应性的血小板中差异表达的miRNA的功能(目标3)。通过将RNA表达数据与我们先前对这些相同受试者的GWA型进行关联，我们将解决拷贝数变化对血小板基因表达的作用(目标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mRNAs和miRNAs的相对表达。这项研究将对血小板生理学有新的认识，增进我们对血小板基因表达遗传学的理解，促进性别和种族特异性血栓形成风险生物标志物的选择，并为其他血小板研究人员提供有用的工具。

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.