Discovery of genes controlling the formation and function of platelets

Platelets are the smallest cell in the blood and maintain vessel wall integrity and haemostasis by initiating repair at places of vessel wall damage. On the flipside platelets are drivers of thrombus formation in coronary arteries after plaque rupture. The number, volume and pro-thrombotic function of platelets vary widely in the population, but each value is tightly regulated within narrow ranges at the individual level. Each of the three traits are independent risk factors for coronary artery disease (CAD) and a high platelet count, large platelets and hyperactive ones are associated with poorer clinical outcomes in CAD, particularly in the post myocardial infarction setting. Environment like diet and other lifestyle factors explain about a quarter of the observed variation in the values of the three traits at the population level. This suggests that the remaining three quarters of variation is therefore controlled by our genes and indeed results from studies in rodents, primates and twins confirm this notion. Platelets are formed in the bone marrow by the megakaryocyte, an extremely rare and the largest precursor of blood cells. Megakaryocytes are derived from haematopoietic stem cells by a process of lineage commitment and proliferation. Endomitosis, which means normal duplication of DNA as occurs during mitosis, but not being followed by cell division underlies polyploidisation which is unique to megakaryocytes and explains their large size. A single megakaryocyte forms about 4,500 platelets and the formation occurs at the megakaryocyte-endothelial cell interface through the formation of so-called proplatelets localised at the tips of long protrusions originating from the megakaryocyte body. The molecular mechanisms controlling the formation of platelets and their pro-thrombotic function are only partially understood. We therefore set out to identify new players in megakaryopoiesis and platelet formation and used modern genomics approaches to achieve this. We scanned the genome of nearly 68,000 healthy individuals for differences by genome-wide typing of common single nucleotide variants (SNP). A Genome Wide Association Study (GWAS) encompassing about 1.2 million of these common SNP identified 68 variants associated with the volume and count of platelets at genome-wide significance level. These SNP mark regions of the genome which contains genes encoding proteins important for megakaryopoiesis and the formation and function of platelets. The effects of a quarter of the observed associations on platelet phenotype are explained by coding SNP that alter the primary amino acid sequence of the candidate proteins. It is assumed that these amino acid polymorphisms alter the function of the respective proteins resulting in the differences in count and volume observed between the three genotype groups. The remaining associated SNP were however localised in the non-coding fraction of the genome and we postulated that they modify gene transcription. To investigate this assumption we applied computational biology approaches and wet-lab genome-wide and locus-focussed functional studies. First we performed chromatin immunoprecipitation combined with massive parallel sequencing (ChIP-seq) for 6 transcription factors (FLI1, GATA1, GATA2, MEIS1, RUNX1, SCL) in primary megakaryocytes to determine the sides where these proteins bind to the genome. Second we applied formaldehyde assisted identification of regulatory elements (FAIRE) in primary erythroblasts, megakaryocytes and monocytes resulting in a high quality catalogue of the nucleosome-depleted regions in these three cell types. Third we derived the precursor cells for red cells and platelet, erythroblasts and megakaryocytes respectively by culturing human haematopoietic stem cells in the presence of erythropoietin and thrombopoietin, the 2 lineage-specific growth factors. We retrieved RNA from the cultured cells at sequential time points and determined the transcriptome landscape by analysing the RNA samples on whole genome expression arrays. From these 3 studies a picture has emerged of allelic differences in the binding of several key transcription factors like MEIS1 and EVI1 at association SNP. This differential binding is associated with allelic differences in transcript levels between the three genotype groups for genes adjacent to or at the association SNP, for example for ABCC4, DNM3 and PIK3CG. Detailed studies at the DNM3 locus showed that the association SNP, a MEIS1 binding site and a RNA transcript together marked an alternative promoter which is only operational in megakaryocytes, but not in any other cell type known to transcribe this gene. Furthermore two thirds of the 68 GWAS genes for platelet volume and count were hitherto unknown to be implicated in haematopoiesis and platelet biology. To delineate the function of these novel genes in haematopoiesis and megakaryopoiesis we applied silencing of gene orthologues in zebrafish. Not unexpectedly we observed that these novel regulators exert their effect at different points in the process of blood cell formation regulating lineage choice and proliferation. The GWAS studies in healthy individuals are complemented by the genetic analysis of DNA samples from patients with rare bleeding and platelet disorders as an alternative approach to discover novel regulators. For this to be successful we have brought together through the NIHR BioResource-Rare Diseases the largest collection of patients with Mendelian bleeding and platelet disorders. Sequencing of the entire coding fraction of the genome or so called exome of the first 100 patients has revealed so far four novel genes encoding proteins unknown to be implicated in megaDiscovery of genes controlling the formation and function of platelets