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GeneSTAR NextGen Functional Genomics of Platelet Aggregation

ABSTRACT:

The causal mechanisms of common diseases and their therapies have been only marginally illuminated by genetic variants identified in genome wide association studies (GWAS) utilizing single nucleotide polymorphism (SNPs). Platelet activation pathways reflecting hemostasis and thrombosis are the underlying substrate for many cardiovascular diseases and related acute events. To overcome GWAS limitations, genomic studies are needed that 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 phenotypes and aggregation in response to low dose aspirin in 2200 subjects (GeneSTAR, Genetic Study of Aspirin Responsiveness), important genome wide "signals" (p<5x10-8) associated with native platelet aggregation and important "signals" associated with platelet responsiveness to aspirin were identified and replicated. Although we are currently performing functional genomics studies to elucidate our most promising findings in known genes (PEAR1, MET, PIKC3G), most "signals" occurred in intergenic regions or in introns. Mechanistic interpretation is limited by uncertainty as to which gene(s) are up- or down-regulated in the presence of most SNP modifications. In this 3 phase proposal, we will (1) create pluripotent stem cells (iPS) from peripheral blood mononuclear cells, and then differentiate these stem cells into megakaryocytes (2) develop an efficient strategy to produce iPS and megakaryocytes using a novel pooling method, and (3) produce iPS and megakaryocytes from 250 subjects in GeneSTAR (European Americans and 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 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 responsiveness to aspirin using our prior genotyped and phenotyped population. Precise information about the exact 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 thrombotic events.

PROVIDER: phs001074 | dbGaP |

SECONDARY ACCESSION(S): PRJNA312864PRJNA312865

REPOSITORIES: dbGaP

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			Action	DRS
	GapExchange_phs001074.v1.p1.xml	Xml
	dbGaPEx2.1.5.xsd	Other
	phs001074.v1-Documents.zip	Other
	Study_Report.phs001074.GeneSTAR_PlateletAggregation.v1.p1.MULTI.pdf	Pdf
	manifest_phs001074.GeneSTAR_PlateletAggregation.v1.p1.c2.DS-CVD-IRB-NPU-RD.pdf	Pdf

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Project description:Coronary artery disease (CAD) is the leading cause of human morbidity and mortality worldwide, underscoring the need to improve diagnostic strategies. Platelets play a major role, not only in the process of acute thrombosis during plaque rupture, but also in the formation of atherosclerosis itself. MicroRNAs are endogenous small non-coding RNAs that control gene expression and are expressed in a tissue and disease-specific manner. Therefore they have been proposed to be useful biomarkers. The aim of this study was to investigate whether differences in miRNA expression levels in platelets can be found (i) between patients with premature CAD and healthy controls and (ii) within healthy controls after and before aspirin and statin administration. In this case-control study we measured expression levels of platelet miRNAs using microarrays from 40 male patients with premature CAD and 40 age- and sex-matched healthy controls. Premature CAD was defined as a cardiac event before the age of 51 years. The patients were selected from the outpatient clinic of the Academic Medical Center (AMC) of Amsterdam, which is specialised in premature CAD. The control cohort was composed of 40 healthy Caucasian male volunteers, who were recruited by advertisement and who were matched with the cases for age and smoking habits. Individuals of this control cohort did not have a history of CVD, nor did they have a positive family history of CVD and they were not allowed to use any medication. Patients and controls were excluded when they suffered from diabetes. Most CAD patients use aspirin and statins as secondary prevention. The influence of these drugs on miRNA profiles is unknown. To assess the possible influence of medication on miRNA expression and to control for medication as confounding factor, we asked 27 volunteers in our control cohort to also use these drugs. We administered simvastatin 40 mg, once daily, for 6 weeks and during the last two weeks we added 100mg of acetyl salicylic acid, once daily. Blood samples including isolated platelets were collected at baseline in the absence of aspirin and statins and after six weeks of medication use. We also assessed platelet function using the Multiplate® Analyzer (Roche) in the absence of aspirin and statin use. In short, 300 µl whole blood was diluted with 300 µl 0.9% saline and stirred for 3 minutes at 37 ºC. Adenosine diphosphate (ADP) was added in a final concentration of 2.5 ?mol/L to initiate platelet aggregation. Aggregation was measured for 6 minutes and was reported in arbitrary aggregation units plotted against time. Also, the area under the aggregation curve (AUC) was measured. All samples were measured in the absence and presence of 200 ?mol/L indomethacin (20 min incubation with blood) to mimic the effect of aspirin use. We calculated the percentage reduction in AUC after incubation with indomethacin as an in vitro measure of the effect of aspirin use on whole blood platelet aggregation.

Project description:<p>The goal of this study was to identify genetic variants in a genome-wide association study (GWAS) that are associated with previously obtained platelet function phenotypes measured in each individual under baseline conditions and following 2 weeks of low dose aspirin (ASA). During this study we performed a high density 1 million-SNP genome scan on subjects from GeneSTAR (representing 800 2-generational families with a family history of premature coronary artery disease, 60% white and 40% African American, N=3232). We identified genomic loci associated with quantitative platelet phenotypes prioritized for their biological interest, determined associations between genomic loci and baseline platelet phenotypes (primary phenotypes are platelet aggregation in platelet rich plasma induced by collagen, adenosine diphosphate (ADP), arachidonic acid (AA), and epinephrine (Epi), determined associations between genomic loci and post-ASA platelet phenotypes, ie, measures of ASA "resistance" (primary phenotypes are as above, plus urinary levels of the prostaglandin metabolite, 11-dehydro-thromboxane B2 ), and compared peaks of association between genomic loci and three common baseline platelet phenotypes (collagen-, epinephrine-, and ADP-induced aggregation in platelet rich plasma) with associations found for these same phenotypes in the Framingham Heart Study. We also determined whether any significant genotype-phenotype associations in the 1 million SNP genome scan could be localized to any specific genes or potential genes of interest using publicly available databases and further examined whether candidate genes previously associated with a specific platelet phenotype are located in a genomic region of interest as determined from the SNP genome scan. We conducted replications of our findings, and are presently involved in larger scale meta-analysis of findings for pre-aspirin and post-aspirin associations.</p> <p>The study population is constituted of full siblings (SIBS) (ages 35-78 years) identified from The Johns Hopkins Sibling Study, now called GeneSTAR, the spouses of the SIBS, and their adult offspring (>21 years of age). They include 3200 individuals from approximately 300 African American and 500 white extended families. "Spouse" refers to the other parent of any SIB offspring, independent of marital status. Sibship sizes among families range from 1 to 16 (excluding index cases). On average, each SIB has 2 potentially eligible offspring. A family for this study is defined by all of the full sibships and the total numbers of offspring that come from all SIBS, rather than just the nuclear family.</p>

Project description:Background: Identifying individuals at heightened cardiovascular risk is a priority for reducing the global burden of cardiovascular disease. Aspirin is widely used to prevent cardiovascular events, though with variable results. Therefore, we hypothesized that aspirin exposure would reveal novel biological pathways relevant to the development of cardiovascular events. Methods: We administered aspirin, followed by peripheral blood RNA microarray profiling, in a discovery cohort of healthy volunteers (n = 50, HV1), followed by two validation cohorts of healthy volunteers (n = 53, HV2) or outpatient cardiology (OPC, n = 25) patients, in conjunction with platelet function testing with the platelet functions score (PFS, HV1 and HV2) or the VerifyNow Asprin (VN, OPC) test. Sets of coexpressed genes, or “Factors” were identified via Bayesian sparse factor analysis and associated with platelet function in HV1 and validated in HV2 and OPC. Validated factors were associated with death/MI in observational (n = 191) and case:control (n = 447) patient cohorts with available RNA data collected at the time of cardiac catheterization. Results: Factor analysis yielded 20 Factors, of which one, Factor 14, contained 60 genes and was associated with PFS in HV1 (r = -0.31, p-value = 0.03). Factor 14 was associated with platelet function with the same strength and direction in HV2 (r = -0.34, p-value = 0.02) and OPC (one-sided p-value for aspirin resistant vs. aspirin sensitive = 0.046), thus validating the association. Factor 14 was associated with death/MI in the two patient cohorts, odds ratio (OR) = 1.2, 95% CI [1.02-1.4], p-value = 0.01 and hazard ratio = 1.5, [1.2-1.9], p = 0.001, respectively, independent of known cardiovascular risk factors (combined OR = 1.2, CI = [1.02, 1.4], p = 0.03). Factor 14 and the expression of the Factor 14 transcript most highly correlative of PFS, ITGA2B, improved reclassification compared to traditional risk factors (category-free net reclassification index = 31% and 37%, p ≤ 0.0002 for both). Conclusions: By challenging humans subjects with aspirin, a medication used for cardiovascular risk reduction, we elucidated genes and pathways that may underlie platelet function and mechanisms responsible for cardiovascular death/MI. This accession represents the OPC cohort microarray data 26 subjects selected for microarray analysis were divided into three groups: aspirin resistant (AR, n=8, >550 ARU), high normal (HN, n=9, ARU 500-549) and aspirin sensitive (AS, n=9, ARU<500)

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GeneSTAR NextGen Functional Genomics of Platelet Aggregation

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