Identification of a specific intronic PEAR1 gene variant associated with greater platelet aggregability and protein expression

Abstract

Genetic variation is thought to contribute to variability in platelet function; however, the specific variants and mechanisms that contribute to altered platelet function are poorly defined. With the use of a combination of fine mapping and sequencing of the platelet endothelial aggregation receptor 1 (PEAR1) gene we identified a common variant (rs12041331) in intron 1 that accounts for ≤ 15% of total phenotypic variation in platelet function. Association findings were robust in 1241 persons of European ancestry (P = 2.22 × 10⁻⁸) and were replicated down to the variant and nucleotide level in 835 persons of African ancestry (P = 2.31 × 10⁻²⁷) and in an independent sample of 2755 persons of European descent (P = 1.64 × 10⁻⁵). Sequencing confirmed that variation at rs12041331 accounted most strongly (P = 2.07 × 10⁻⁶) for the relation between the PEAR1 gene and platelet function phenotype. A dose-response relation between the number of G alleles at rs12041331 and expression of PEAR1 protein in human platelets was confirmed by Western blotting and ELISA. Similarly, the G allele was associated with greater protein expression in a luciferase reporter assay. These experiments identify the precise genetic variant in PEAR1 associated with altered platelet function and provide a plausible biologic mechanism to explain the association between variation in the PEAR1 gene and platelet function phenotype.

Figure 1

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.

Figure 2

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.

Figure 3

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.