rs1354034 is the leading SNP and an eQTL for ARHGEF3 expression.

<p>(A) Visual summary of the statistical evidence for variant-phenotype association at the locus of rs1354034 from data generated by the TEMPLE targeted sequencing study. It shows the variant specific P-values from Fisher’s exact tests against null hypotheses that genotype frequencies are the same in the high and low platelet count samples. The color of each point represents the <i>r</i>² between the corresponding variant and rs1354034, estimated from the TEMPLE genotypes. The continuous line indicates the local recombination rate estimated from HapMap data. (The tool used to generate the plot is available at doi:10.1093/bioinformatics/btq41.) (B) Shown is rs 1354034 (blue X) and its LD structure in +/- 250 kb window. There is no SNP in linkage disequilibrium (threshold <i>r</i>² >0.8) found 250k bp up- or downstream of the rs1354034 locus (data from 1000 Genome Project), suggesting rs1354034 is the leading SNP of ARHGEF3. (C) A strong association of the rs1354034 genotype with ARHGEF3 mRNA levels (p = 1.37x10^-15, ordinary linear regression). This association stayed significant even after accounting for subject age, gender and race (p = 2.39 x 10⁻¹¹, multiple linear regression). Primary data for 5C in online <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178095#pone.0178095.s003" target="_blank">S1 Table</a>.</p

<i>Arhgef3</i> KO mice have enlarged platelets, and normal platelet activation.

<p>(A) Peripheral blood was taken from 4 week old littermate mice with the indicated genotypes. <i>Arhgef3</i> KO mice have significantly larger platelets (MPV) but normal platelet counts. (B) Mean forward side scatter (FSC) of platelets (gated as CD41/CD61+ from whole blood) confirmed that KO mice have larger platelets. (C) Flow cytometry for Integrin α2bβ3 conformational change (JONA MFI) and P-selectin exposure of platelets from littermate mice in response to ADP, thromboxane (U46), thrombin and PAR4-AP stimulation. There are no significant differences between WT and <i>Arhgef3</i> KO platelets with any of the agonists tested. (3 independent experiments, n = 4/genotype). (D) Tail bleeding times of WT and <i>Arhgef3</i> KO littermates showed no differences in bleeding time. (WT n = 10, KO n = 9).</p

rs1354034 is located in a DNase I hypersensitive region in primary human MKs.

<p>Shown is the chromatin locus containing the ARHGEF3 gene. rs1354034 (orange arrow) is located in an open-chromatin (DNase I peak) non-coding region upstream of the transcription start site of ENST00000296315 (the most commonly transcribed mRNA in MK, indicated by an asterisk). Green: DNase 1 peaks, Purple: H3K27Ac ChIP peaks, Blue: intron/exon boundaries, Bottom: major ENST’s identified for ARHGEF3.</p

ARHGEF3 expression in primary murine and human cells.

<p>(A-B) ARHGEF3 mRNA and protein levels were assessed in megakaryocytes derived in vitro from WT E13.5 fetal liver cells. A 3% BSA gradient was used to separate megakaryocytes (MKs). Both CD41 (positive control) and ARHGEF3 expression are increased in the pellet fractions compared to the 3% BSA fraction (p < 0.005). (C) RNA sequencing data from public a database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178095#pone.0178095.ref012" target="_blank">12</a>] shows ARHGEF3 upregulation during human CD34+ differentiation down the MK lineage. (D) Barplots representing transcript isoform expression levels across eight human hematopoietic cell types.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178095#pone.0178095.ref013" target="_blank">13</a>] ARHGEF3 expression (represented by the total height of the barplots) increases during megakaryocytic lineage differentiation and the most abundant transcript isoform in megakaryocytes is ENST00000296315. The figure includes Ensembl v70 annotated transcripts and those annotated as non-protein coding or nonsense mediated decay have been grouped together. [HSC: hematopoietic stem cell, MPP: multipotent progenitor, CLP: common lymphoid progenitor, CMP: common myeloid progenitor, GMP: granulocyte monocyte progenitor, MEP: megakaryocyte erythrocyte progenitor, EB: erythroblasts and MK: megakaryocytes.]</p