Genome-Wide Association of Native and Post Aspirin Platelet Function Phenotypes

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.

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.

• Study Weblinks:
  • GeneSTAR
• Study Design:
  • Prospective Longitudinal Cohort
• Study Type:
  • Family
  • Cohort

• BioProject
• PubMed
• Sequence Read Archive
• BioSample
• MeSH
• PMC

Inclusion: Persons were eligible if they had been a participant in GeneSTAR (examining the effect of 81 mg of aspirin daily for 2 weeks on platelet function with pre and post aspirin platelet testing). All subjects were: 1.) offspring of a person from a sibship identified in the SIB study, > 21 years of age, 2.) a full sibling from a sibship identified in The Johns Hopkins Sibling Study. In the latter case, the SIB had a spouse (or co-parent of any offspring) or at least one offspring available to participate. All of these subjects underwent a GWAS to be included in the database.

Exclusion: For the parent study these included the following: 1.) the presence of any coronary heart disease or vascular thrombotic event, 2.) any bleeding disorder or any hemorrhagic event in the past (stroke, gastrointestinal bleed), 3.) use of any anticoagulants or anti-platelet agents (i.e. warfarin, persantin, clopidogrel), 4.) chronic or acute nonsteroidal anti-inflammatory agents, including COX-2 inhibitors that could not be discontinued, 5.) recent active gastrointestinal disorder, 6.) current pharmacotherapy for a gastrointestinal disorder, 7.) pregnancy or risk of pregnancy during the treatment trial, 8.) recent menorrhagia, 9.) known aspirin intolerance or allergic side effects, 10.) serious medical disorders, including autoimmune diseases, renal or hepatic failure, cancer or HIV-AIDS, 11.) chronic or acute use of glucocorticosteroid therapy or any drug that may interfere with the measured outcomes, 12.) serious psychiatric disorders, and, 13.) unable to independently make a decision to participate.

The GeneSTAR study GWAS analyses were based on a prior NIH-sponsored study (Program in Gene Environment Interactions, PROGENI) examining gene environment interactions on platelet function. Families with a history of known premature coronary disease participated in a 2 week trial of platelet function prior to and following aspirin therapy, 81 mg/day. Platelet function was measured ex vivo, using various agonists and other measures, and DNA was banked. The first iteration of the genetic study of platelets was in PROGENI, and the study then proceeded on to STAMPEED.

The GeneSTAR GWAS study was carried out as planned under an award as part of STAMPEED (SNP Genotyping and Multiple Phenotyping in Existing Epidemiologic Databases). In the first year, a fully curated platelet function phenotype and family structure database and Data Dictionary were created. We used principal components analysis and components emerged that related closely to our biological hierarchy and explained nearly 80% of the variance in the overall phenotypes. We completed heritabilities, distributions, and associations among all platelet variables. Genotype-phenotype associations for each SNP were examined at Hopkins separately by race. The majority of our analyses were done at Washington University Division of Statistical Genetics (Province, MA) A locus on chromosome 1 (corresponding to the gene PEAR1) was analyzed by both groups and gave similar results.

In white subjects, we found 32 SNPs of genome-wide significance (< 5x10-8) for native platelet function phenotypes and 29 SNPs of genome-wide significance for post-ASA platelet phenotypes. For African Americans, the numbers of genome-wide significant associations were 32 for native platelet phenotypes and 28 for post-ASA phenotypes.

We tabulated SNPs in which there was a genome-wide significance level in one race, and replication at a nominal p value or better in the other race (p < 0.05), or SNPs in which the significance level in one race was < 10-11, with or without replication in the other .The most interesting associations occurred in PEAR1. PEAR1 (platelet endothelial aggregation receptor-1) is a platelet transmembrane receptor with unknown agonist, discovered in 2005, activated by platelet-platelet contact. In a recent candidate gene study, we found an association between a SNP in the promoter region of the PEAR1 gene with multiple native and post-ASA aggregation phenotypes in both whites and African Americans, This has been confirmed in the white Framingham Heart Study cohort (Johnson AD et al, Nature Genetics).

We completed a meta-analysis of native platelet aggregation phenotypes with the Framingham Heart Study Offspring Cohort (Johnson A, Nature Genetics 42:608-613, 2010). We identified associations of seven loci with platelet aggregation in subjects of European ancestry. Evidence of replication was found for all loci in the African American cohort in GeneSTAR. In addition to these 7 loci, 15 other loci were identified in the meta-analysis as having association P values of 5.0x10-8<p<1.0x10-4 in the European ancestry cohort with P<0.05 in the African American cohort.

We have continued fine mapping and sequencing regions of interest, and are completing more work in replications with the HAPI Heart Study at the University of Maryland. Attempts to identify PEAR1 mRNA in platelet lysates were unsuccessful. PEAR1 appears to represent an important molecule in platelet aggregation and could represent an important new therapeutic target for drug development. The study is continuing to move toward a basic science explanation of the findings to date. Because of the nature of the original informed consent in PROGENI, we are submitting only aggregate data to dbGaP, with individual level data available via a special application process to the Principal Investigators at Johns Hopkins.

• Primary Phenotype: Platelet Aggregation
• Blood Physiological Phenomena
• Cardiovascular Physiological Phenomena

• Principal Investigator
  • Lewis C. Becker, MD. Johns Hopkins University School of Medicine, Baltimore, MD, USA.
• Funding Sources
  • HL087698-03(HL06-012). National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA.