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),
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.
|Smith, Cory; Abalde-Atristain, Leire; He, Chaoxia et al. (2015) Efficient and allele-specific genome editing of disease loci in human iPSCs. Mol Ther 23:570-7|
|Wang, Ying; Cheng, Linzhao; Gerecht, Sharon (2014) Efficient and scalable expansion of human pluripotent stem cells under clinically compliant settings: a view in 2013. Ann Biomed Eng 42:1357-72|
|Smith, Cory; Gore, Athurva; Yan, Wei et al. (2014) Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. Cell Stem Cell 15:12-3|
|Bai, Hao; Xie, Yin-Liang; Gao, Yong-Xing et al. (2013) The balance of positive and negative effects of TGF-* signaling regulates the development of hematopoietic and endothelial progenitors in human pluripotent stem cells. Stem Cells Dev 22:2765-76|
|Wang, Ying; Chou, Bin-Kuan; Dowey, Sarah et al. (2013) Scalable expansion of human induced pluripotent stem cells in the defined xeno-free E8 medium under adherent and suspension culture conditions. Stem Cell Res 11:1103-16|