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SNP in human ARHGEF3 promoter is associated with DNase hypersensitivity, transcript level and platelet function, and Arhgef3 KO mice have increased mean platelet volume

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Zou, S 
Teixeira, AM 
Kostadima, M 
Astle, WJ 
Radhakrishnan, A 


Genome-wide association studies have identified a genetic variant at 3p14.3 (SNP rs1354034) that strongly associates with platelet number and mean platelet volume in humans. While originally proposed to be intronic, analysis of mRNA expression in primary human hematopoietic subpopulations reveals that this SNP is located directly upstream of the predominantly expressed ARHGEF3 isoform in megakaryocytes (MK). We found that ARHGEF3, which encodes a Rho guanine exchange factor, is dramatically upregulated during both human and murine MK maturation. We show that the SNP (rs1354034) is located in a DNase I hypersensitive region in human MKs and is an expression quantitative locus (eQTL) associated with ARHGEF3 expression level in human platelets, suggesting that it may be the causal SNP that accounts for the variations observed in human platelet traits and ARHGEF3 expression. In vitro human platelet activation assays revealed that rs1354034 is highly correlated with human platelet activation by ADP. In order to test whether ARHGEF3 plays a role in MK development and/or platelet function, we developed an Arhgef3 KO/LacZ reporter mouse model. Reflecting changes in gene expression, LacZ expression increases during MK maturation in these mice. Although Arhgef3 KO mice have significantly larger platelets, loss of Arhgef3 does not affect baseline MK or platelets nor does it affect platelet function or platelet recovery in response to antibody-mediated platelet depletion compared to littermate controls. In summary, our data suggest that modulation of ARHGEF3 gene expression in humans with a promoter-localized SNP plays a role in human MKs and human platelet function-a finding resulting from the biological follow-up of human genetic studies. Arhgef3 KO mice partially recapitulate the human phenotype.



Animals, Blood Platelets, Cell Differentiation, Cell Size, Cohort Studies, Female, Fetal Blood, Gene Expression Regulation, Humans, Male, Mean Platelet Volume, Megakaryocytes, Mice, Inbred C57BL, Mice, Knockout, Polymorphism, Single Nucleotide, Promoter Regions, Genetic, Rho Guanine Nucleotide Exchange Factors

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Public Library of Science (PLoS)
Medical Research Council (MR/L003120/1)
European Commission (282510)
British Heart Foundation (None)
British Heart Foundation (None)
CCF (None)
Financial support was provided by NIH grants DK094934, DK086267, and DK0724429 to DSK, HL102482 to PB, the Cardeza Foundation for Hematologic Research (PB), and American Heart Association Fellowship 14PRE20480196 to LMS. MF is supported by the BHF Cambridge Centre of Excellence RE/13/6/30180. PvdH is supported by the Landsteiner Foundation for Blood Transfusion Research grant LSBR1133. LAT was supported by Leukaemia & Lymphoma Research Fellowship 09018. Research in the Ouwehand laboratory is supported by EU-FP7 project BLUEPRINT (282510) and by program grants from the National Institute for Health Research (NIHR); and the British Heart Foundation under numbers RP-PG- 0310-1002 and RG/09/12/28096. The laboratory receives funding from NHS Blood and Transplant for facilities.