There is increasing evidence that genome-wide association (GWA) studies represent a powerful approach to the identification of genes involved in common human diseases. We describe a joint GWA study (using the Affymetrix GeneChip 500K Mapping Array Set) undertaken in the British population, which has examined approximately 2,000 individuals for each of 7 major diseases and a shared set of approximately 3,000 controls. Case-control comparisons identified 24 independent association signals at

Elevated blood pressure is a common, heritable cause of cardiovascular disease worldwide. To date, identification of common genetic variants influencing blood pressure has proven challenging. We tested 2.5m genotyped and imputed SNPs for association with systolic and diastolic blood pressure in 34,433 subjects of European ancestry from the Global BPgen consortium and followed up findings with direct genotyping (N≤71,225 European ancestry, N=12,889 Indian Asian ancestry) and in silico comparison (CHARGE consortium, N=29,136). We identified association between systolic or diastolic blood pressure and common variants in 8 regions near the CYP17A1 (P=7×10−24), CYP1A2 (P=1×10−23), FGF5 (P=1×10−21), SH2B3 (P=3×10−18), MTHFR (P=2×10−13), c10orf107 (P=1×10−9), ZNF652 (P=5×10−9) and PLCD3 (P=1×10−8) genes. All variants associated with continuous blood pressure were associated with dichotomous hypertension. These associations between common variants and blood pressure and hypertension offer mechanistic insights into the regulation of blood pressure and may point to novel targets for interventions to prevent cardiovascular disease.

We have genotyped 14,436 nonsynonymous SNPs (nsSNPs) and 897 major histocompatibility complex (MHC) tag SNPs from 1,000 independent cases of ankylosing spondylitis (AS), autoimmune thyroid disease (AITD), multiple sclerosis (MS) and breast cancer (BC). Comparing these data against a common control dataset derived from 1,500 randomly selected healthy British individuals, we report initial association and independent replication in a North American sample of two new loci related to ankylosing spondylitis, ARTS1 and IL23R, and confirmation of the previously reported association of AITD with TSHR and FCRL3. These findings, enabled in part by increased statistical power resulting from the expansion of the control reference group to include individuals from the other disease groups, highlight notable new possibilities for autoimmune regulation and suggest that IL23R may be a common susceptibility factor for the major 'seronegative' diseases.

Summary. Background: Anagrelide is a selective inhibitor of megakaryocytopoiesis used to treat thrombocytosis in patients with chronic myeloproliferative disorders. The effectiveness of anagrelide in lowering platelet counts is firmly established, but its primary mechanism of action remains elusive. Objectives and Methods: Here, we have evaluated whether anagrelide interferes with the major signal transduction cascades stimulated by thrombopoietin in the hematopoietic cell line UT-7/mpl and in cultured CD34 + -derived human hematopoietic cells. In addition, we have used quantitative mRNA expression analysis to assess whether the drug affects the levels of known transcription factors that control megakaryocytopoiesis. Results: In UT-7/ mpl cells, anagrelide (1 lM) did not interfere with MPLmediated signaling as monitored by its lack of effect on JAK2 phosphorylation. Similarly, the drug did not affect the phosphorylation of STAT3, ERK1/2 or AKT in either UT-7/mpl cells or primary hematopoietic cells. In contrast, during thrombopoietin-induced megakaryocytic differentiation of normal hematopoietic cultures, anagrelide (0.3 lM) reduced the rise in the mRNA levels of the transcription factors GATA-1 and FOG-1 as well as those of the downstream genes encoding FLI-1, NF-E2, glycoprotein IIb and MPL. However, the drug showed no effect on GATA-2 or RUNX-1 mRNA expression. Furthermore, anagrelide did not diminish the rise in GATA-1 and FOG-1 expression during erythropoietin-stimulated erythroid differentiation. Cilostamide, an exclusive and equipotent phosphodiesterase III (PDEIII) inhibitor, did not alter the expression of these genes. Conclusions: Anagrelide suppresses megakaryocytopoiesis by reducing the expression levels of GATA-1 and FOG-1 via a PDEIII-independent mechanism that is differentiation context-specific and does not involve inhibition of MPL-mediated early signal transduction events.

To cite this article: Ahluwalia M, Butcher L, Donovan H, Killick-Cole C, Jones PM, Erusalimsky JD. The gene expression signature of anagrelide provides an insight into its mechanism of action and uncovers new regulators of megakaryopoiesis. J Thromb Haemost 2015; 13: 1103-12.Summary. Background: Anagrelide is a cytoreductive agent used to lower platelet counts in essential thrombocythemia. Although the drug has been known to selectively inhibit megakaryopoiesis for many years, the molecular mechanism accounting for this activity is still unclear. Objectives and Methods: To address this issue we have compared the global gene expression profiles of human hematopoietic cells treated ex-vivo with and without anagrelide while growing under megakaryocyte differentiation conditions, using high-density oligonucleotide microarrays. Gene expression data were validated by the quantitative polymerase chain reaction and mined to identify functional subsets and regulatory pathways. Results: We identified 328 annotated genes differentially regulated by anagrelide, including many genes associated with platelet functions and with the control of gene transcription. Prominent among the latter was TRIB3, whose expression increased in the presence of anagrelide. Pathway analysis revealed that anagrelide up-regulated genes that are under the control of the transcription factor ATF4, a known TRIB3 inducer. Notably, immunoblot analysis demonstrated that anagrelide induced the phosphorylation of eIF2a, which is an upstream regulator of ATF4, and increased ATF4 protein levels. Furthermore, salubrinal, an inhibitor of eIF2a dephosphorylation, increased the expression of ATF4-regulated genes and blocked megakaryocyte growth. Conclusions: These findings link signaling through eIF2a/ATF4 to the antimegakaryopoietic activity of anagrelide and identify new potential modulators of megakaryopoiesis.