Identification of a functional genetic variant driving racially dimorphic platelet gene expression of the thrombin receptor regulator, PCTP

Abstract

Platelet activation in response to stimulation of the Protease Activated Receptor 4 (PAR4) receptor differs by race. One factor that contributes to this difference is the expression level of Phosphatidylcholine Transfer Protein (PCTP), a regulator of platelet PAR4 function. We have conducted an expression Quantitative Trait Locus (eQTL) analysis that identifies single nucleotide polymorphisms (SNPs) linked to the expression level of platelet genes. This analysis revealed 26 SNPs associated with the expression level of PCTP at genome-wide significance (p < 5×10^-8). Using annotation from ENCODE and other public data we prioritised one of these SNPs, rs2912553, for functional testing. The allelic frequency of rs2912553 is racially-dimorphic, in concordance with the racially differential expression of PCTP. Reporter gene assays confirmed that the single nucleotide change caused by rs2912553 altered the transcriptional potency of the surrounding genomic locus. Electromobility shift assays, luciferase assays, and overexpression studies indicated a role for the megakaryocytic transcription factor GATA1. In summary, we have integrated multi-omic data to identify and functionalise an eQTL. This, along with the previously described relationship between PCTP and PAR4 function, allows us to characterise a genotype-phenotype relationship through the mechanism of gene expression.

Keywords: PAR4; PCTP; Platelets; eQTL; genetics.

Figure 1. Annotation of PCTP eQTLs

(A) (Top panel) Local Manhattan plot of platelet eQTL SNPs (diamonds) in PCTP with P < 5 × 10⁻⁸ (blue dashed line). X-axis: genomic coordinates, left Y-axis: −log₁₀(P-value). Color indicates R² linkage disequilibrium value relative to rs2912553. Red line indicates recombination rate (right Y-axis). (Bottom panel) Annotation tracks (from top to bottom): Platelet eQTL SNPs in PCTP, colors indicate R² LD value relative to rs2912553. PCTP gene structure. ChromHMM chromatin state predictions from K562 cells, ChIP-Seq peaks from CD34 cells (H3K4me1, RUNX1, GATA1). (B) Differential expression of PCTP by rs2912553 genotype. Box represent interquartile range, line represents mean, whiskers represent 1.5 × interquartile range. N=154, P-value calculated as described in Materials and Methods. (C) Allelic frequencies of rs2912553 among black (B) and white (W) subjects in PRAX1.

Figure 2. rs2912553 genotype confers differential transcriptional potency on the surrounding locus

(A) Schematic representation of cloned fragments surrounding rs2912553. (B) Luciferase activity of reporter fragments by size containing the T or C allele of rs2912553. (C) Luciferase activity of all four potential haplotypes of rs2912552 and rs2912553. Results were normalized to cotransfected β-gal expression and presented as +/− SD. N=3 each for each condition. P = 3 × 10⁻²², 2-way ANOVA for the effect of genotype on luciferase activity.

Figure 3. Differential protein binding based on rs2912553 genotype

(A) The highest molecular weight complex protein complex from K562 nuclear extracts (arrow) bound with more affinity to probes containing a T allele than a C allele. Representative gel. (B) Quantification of the highest molecular weight complex from 3 independent gels as described in (A). N=3 for each condition P = 0.02, 2-way ANOVA for the effect of genotype on shifted band intensity. (C) Addition of anti-GATA1 antibodies disrupted formation of the complex (arrow) whereas anti-GATA2 antibodies had no effect. Representative gel. (D) Quantification of three independent gels as described in (C). N=3 for each condition. P= 0.0004, 2-way ANOVA for effect of antibody on shifted band intensity. Error bars represent +/− SD

Figure 4. Transcriptional regulation of the rs2912553 locus by GATA1

(A) Western blot of K562 cells transfected with siRNA directed against GATA1. Blots were probed for GATA1 and GAPDH. (B) Attenuation of GATA1 expression lead to a reduction in luciferase activity driven by the rs2912553 locus. (C) Overexpression of GATA1 results in enhanced transcriptional activity from the rs2912553 locus. Luciferase results were normalized to co-transfected β-gal. (D) Western blot of 293 cells transfected with a FLAG-GATA1 expression vector or control. Blots were probed with antibodies directed against GATA1, PC-TP, and GAPDH. (E) Quantification three independent gels as described in (D). Error bars represent +/− SD N=3 for each condition. P-values calculated by 2-way T-test.