Identification of a specific intronic PEAR1 gene variant associated with greater platelet aggregability and protein expression by Nauder Faraday, Lisa R. Yanek, Xiao Ping Yang, Rasika Mathias, J. Enrique Herrera-Galeano, Bhoom Suktitipat, Rehan Qayyum, Andrew D. Johnson, Ming-Huei Chen, Geoffrey H. Tofler, Ingo Ruczinski, Alan D. Friedman, Arnaldur Gylfason, Unnur Thorsteinsdottir, Paul F. Bray, Christopher J. O'Donnell, Diane M. Becker, and Lewis C. Becker Blood Volume 118(12):3367-3375 September 22, 2011 ©2011 by American Society of Hematology

Gene variants in NTRK1 and PEAR1 associated with platelet function after aspirin treatment and linkage disequilibrium at the locus. Gene variants in NTRK1 and PEAR1 associated with platelet function after aspirin treatment and linkage disequilibrium at the locus. (A) EA ancestry and (B) African ancestry. In both figures, the top panel shows the strength of association between individual SNPs along the locus (genetic coordinates chromosome 1:155050166 to 155154850, NCBI build 36.3) and platelet function phenotypes. Col2 and 5 indicate aggregation to collagen 2 and 5 μg/mL, respectively; ADP2 and 10, aggregation to ADP 2 and 10μM, respectively; Epi2 and 10, aggregation to epinephrine 2 and 10μM, respectively. Red dotted line signifies Bonferroni corrected significance threshold (3.19 × 10−5); rs numbers across the top signify SNPs reaching the significance threshold (red font denotes significance for both ancestries, black font denotes significance for one ancestry). Chromosomal coordinates for the NTRK1 and PEAR1 genes within the locus are shown by blue bars. In both figures, the bottom panel shows linkage equilibrium among SNPs in the region. The pairwise strength of association between SNPs (D') is signified by depth of color of shaded areas (white = 0, dark red = 1.0). Areas within triangles signify LD blocks. Nauder Faraday et al. Blood 2011;118:3367-3375 ©2011 by American Society of Hematology

Platelet expression of PEAR1 protein is related to genetic variation in PEAR1 at rs12041331. Platelet expression of PEAR1 protein is related to genetic variation in PEAR1 at rs12041331. Western blotting analysis of platelet lysates (30 μg/lane) from randomly selected persons who varied by genotype (AA, AG, GG) at rs12041331. (A) Representative Western blots. (B) Summary results for platelet expression by genotype normalized to control (AG pooled mixture). (C) Platelet expression of PEAR1 protein assessed by quantitative ELISA. Results are displayed as mean ± SD; *P < .05 for difference among groups by ANOVA. Nauder Faraday et al. Blood 2011;118:3367-3375 ©2011 by American Society of Hematology

PEAR1 rs12041331 gene variant modifies expression of luciferase in a gene reporter assay. PEAR1 rs12041331 gene variant modifies expression of luciferase in a gene reporter assay. (A) PGL3 promoter vector construct showing the insertion site for a 186-bp sequence of intron1 of PEAR1 spanning the A allele or G allele of rs12041331. Luc+ indicates luciferase gene. (B) Expression of luciferase in MEG-01 cells cotransfected with plasmids containing PGL3 and β-galactosidase. (C) Expression of luciferase in HUVECs cotransfected with plasmids containing PGL3 and β-galactosidase. Luciferase expression is shown by relative light units of luciferase (RLUs) normalized to absorption (OD) of β-galactosidase activity. PGL3 basic indicates luciferase gene plasmid without promoter; PGL3-P, luciferase gene plasmid with SV40 promoter; PGL3-P-A, luciferase gene plasmid with SV40 promoter and A allele–containing insert cloned from PEAR1; PGL3-P-G, luciferase gene plasmid with SV40 promoter and G allele–containing insert cloned from PEAR1. Results are displayed as mean ± SD; *P < .001 for difference among groups by ANOVA; n = 3. Nauder Faraday et al. Blood 2011;118:3367-3375 ©2011 by American Society of Hematology