Genetic Architecture Underlying the Formation of Platelets: Lessons Learned from Population Studies and Rare Platelet Syndromes

Transfusion medicine / Platelets Genetic architecture underlying the formation of platelets: lessons learned from population studies and rare platelet syndromes

W.H. Ouwehand 1-3 ABSTRACT A. Rendon 1,2,4 The cellular composition of blood varies widely between healthy individuals. The normal ranges of on behalf of the BRIDGE, the blood cell indices are the result of the interplay between genetic and environmental factors, par - HAEMGEN and THROMBOGENOMICS ticularly diet and host-pathogen interactions. The blood cell indices are under strong heritable control consortia and in spite of the fact that this was known for some time, the number of known genetic loci causing this natural variation was, until recently, limited. With the completion of the sequencing of the human 1Department of Haematology, genome, and the cataloguing of common variants in the main populations of different ethnicity, it has University of Cambridge; become possible to develop typing arrays to scan the genome for common variants. With these 2National Health Service Blood and advances, the era of genome wide association studies commenced in 2007 and this has led to the dis - Transplant, Cambridge; covery of more than 140 common genetic variants that regulate the life cycle of red cells and platelets. 3Wellcome Trust Sanger Institute, These variants are signposts for known and many novel regulators of hematopoiesis. With the €1000 Wellcome Trust Genome Campus, whole genome becoming reality, high throughput next generation sequencing is rapidly entering into Hinxton, Cambridge; 4Biostatistics clinical practice for the ascertainment of the molecular diagnosis of inherited non-malignant and Unit, Medical Research Council, malignant hematologic disorders. Treatment protocols will become increasingly tailored on the basis Cambridge, UK of these molecular results. Correspondence: Learning goals Willem H. Ouwehand E-mail: [email protected] At the conclusion of this activity, participants should know that: - through global collaboration, over the last five years it has been possible to identify more than 140 genetic loci that regulate the formation of red cells and platelets, and two-thirds of these genes Hematology Education: are novel regulators of hematopoiesis; the education program for the - the €1000 whole genome is entering into the clinical arena and will, over the next five years, annual congress of the European transform the molecular diagnosis of malignant and non-malignant hematologic disorders; Hematology Association - sharing of genomic and clinical data using standard vocabulary, and at a scale far beyond that of the typical randomized clinical trials, will be required to bring the benefits of genomics to patient 2014;8:403-410 care; - hematology clinical care teams will increasingly work with experts in computational biology and statistical genomics to bring the advantages of modern genomics to the bedside.

The genome and sequence variation in a mainstream health system. These large- scale initiatives will lead to an exponential The sequencing of the coding fraction of increase in the aggregated number of genomes tens of thousands of human exomes and thou - analyzed and as a consequence the minor sands of whole genomes provides an exhaus - allele frequencies (MAFs) of each class of tive and increasingly accurate catalog of the variants, particularly the rare ones, will different types of DNA variants. Single become increasingly accurate. Most DNA nucleotide variants (SNVs) are the predomi - samples analyzed in large projects are from nant source of variation. So far, around 63 mil - populations of Northern European ancestry, lion SNVs have been identified, the majority but eventually accurate sequence variant cata - being rare. The two other main types of vari - logs for the vast majority of populations of dif - ants are copy number and structural variants ferent ethnicities will become available. (CNV and SV, respectively). These are far less Single nucleotide variants are categorized in frequent with about one CNV for each 10 bins of arbitrary MAFs ranging from common SNVs. The ongoing reduction in cost of ones (MAF>0.05), rare ones (MAF<0.05 and sequencing has prompted initiatives like the >0.001) to private ones (MAF <0.001). It is 1000Genomes project, 1 the UK10K project assumed that the majority of sequence variants (http://www.uk10k.org/) 2 and the NHS100K have a negligible effect on phenotype. whole genome sequencing (WGS) project. Genome wide association studies (GWAS), The aim of the latter project is to sequence the which commenced in 2007, have identified genome of the first 100,000 NHS patients nearly 12,000 common SNVs associated with (http://www.genomicsengland.co.uk/) by risk of diseases or disease-relevant quantita - 2018. 3 The main mission of the NHS100K tive risks. 4 A well-curated and searchable cata - project, which is being coordinated by log of common SNVs found to be associated Genomics England Ltd. is to introduce WGS with disease risks and traits can be found at Hematology Education: the education program for the annual congress of the European Hematology Association | 2014; 8(1) | 403 | 19 th Congress of the European Hematology Association

http://www.genome.gov/gwastudies 5 The effects of com - identified platelet genes encoding a protein already exten - mon variants discovered by GWAS on qualitative or quan - sively studied in myeloid biology. PIK3CG codes for the titative traits are typically small and range between 10- gamma chain of the pi3/pi4 kinase complex, which is 30%. Hence their discovery has required sizable meta- responsible for the synthesis of phosphatidylinositol- analysis bringing together GWAS and related clinical data 3,4,5-trisphosphate (PtdIns[3,4,5]P3, PIP3) by conversion from hundreds of thousands of disease cases and healthy of PtdIns-3,4-biphosphate. The formation of PIP3 by the controls. At the other end of the variation frequency spec - pi3/pi4 kinase complex is a key step in the signal transduc - trum, extremely rare alleles and mutations are found. In tion cascades leading to the release of calcium from the contrast to the common variants, a small fraction of this intracellular stores and controlling cellular movement, class of variants have large effects on phenotype and are adhesion, and contraction. All three cellular processes are causative of the thousands of Mendelian disorders of man. important for platelet formation and it is, therefore, entire - The human gene mutation database (http://www.hgmd. ly plausible to assume that genotype-driven differences in cf.ac.uk) attempts to capture high penetrance disease- PIK3CG transcript levels modify these processes, and causing variants and disease-modifying variants. So far, thereby the count and volume of platelets. Indeed, whole 152,355 variants have been recorded in the database, with genome expression studies of RNA samples obtained from 133,789 of these annotated as disease-causing, i.e. high healthy individuals confirmed the assumption that penetrance variants and 10,301 as likely to be disease- PIK3CG levels differ between groups sampled from the 10 causing. The remaining ones belong to the categories of three rs342293 genotype groups. However, the four disease-associated polymorphisms (n=2730) and disease- remaining platelet GWAS genes encode novel players associated functional polymorphisms. with hitherto unclear functional roles in hematopoiesis. This review considers the recently identified associa - These 2 initial studies with small sample numbers were tions between common variants and the formation of red followed by several meta-analysis of GWAS bringing cells and platelets by hematopoietic stem cells, how these together data of nearly 100,000 individuals, and in a step - variants exert their effect on blood cell formation, how wise manner, the number of platelet (Figure 1A) and red next generation sequencing (NGS) technologies are aiding cell (Figure 1B) GWAS loci increased to 68 and 75, 7,8,11-13 the discovery of novel mutations/rare variants which respectively. underlie severe inherited disorders of platelets and can be There are a number of similar observations between the applied to improve the diagnosis of this class of rare inher - results of the GWAS meta-analysis for red cells and ited disorders. platelets. First, at a large number of loci the sentinel SNV (the SNV with the lowest P-value of association) is local - ized within a gene or within 10 Kilobase (kb) of a gene- Discovery of common variant associated with red body. Therefore, as was the case for ARHGEF3 , TAOK1 cell and platelet formation and WDR66 , gene candidacy could be inferred from the GWAS results with a reasonable level of likelihood The number and volume of red cells and platelets varies (Figure 1A and B). Second, the overlap between the two widely in the healthy population. Studies in rodents, pri - gene lists is limited to five loci only ( TMCC2 and TRIM58 mates and twins showed a high level of heritability of on Chr1; HBS1L/MYB on Chr6; RCL1 on Chr9, and blood cell indices measured in a routine full blood count SH2B3 on Chr12), suggesting that the effect of most of the (FBC) analysis. However, until 2009 the number of genet - remaining identified SNVs is lineage-restricted. Third, the ic loci controlling the formation of platelets and red cells transcripts of the core gene set identified for both red cells was sparse. A study by Thein and colleagues was the first and platelets showed a highly significant enrichment of to identify a common SNV localized 5’ of the lineage-restricted or over-expressed gene transcripts (e.g. HBS1L/MYB genes as being associated with levels of fetal DNM3, GP1BA, ITGA2B, TPM1 for platelets and EIF5, hemoglobin in individuals of Northern European KIT, RCL1, SPT1A for red cells). Fourth, only approxi - 6 ancestry. More recent GWAS efforts have confirmed this mately one-third of the 140+ genes discovered by GWAS association and the same locus also exerts an effect on red encode known regulators of blood cell formation. Hence a 7,8 blood cell volume and platelet count. With the advent of large number of novel proteins with pivotal roles in increasingly affordable genome wide typing arrays, with hematopoiesis have been discovered (Figure 2) by associ - which up to a million common SNVs can be typed in a sin - ating the results of genome-wide typing of large popula - gle DNA test, the search for more common variants asso - tion samples with blood cell indices, which are routinely ciated with the volume and count of both red blood cells measured by a FBC. Not entirely unexpectedly, and in and platelets became feasible. In 2009, 2 studies showed keeping with the notion that GWAS-identified genes that a GWAS strategy could be successfully used to iden - encode a category of proteins with critical functional roles, tify novel loci controlling the formation of blood cells. we observed that 23 of the 68 platelet GWAS genes also Both studies were performed with the DNA samples from belonged to the category of Mendelian genes known to a relatively small number of healthy individuals and underlie inherited disorders. 11 Mutations in these genes revealed the first four SNVs for the volume and count of either cause disorders of the hematopoietic system or of platelets. Three of the four SNVs were localized in or IN other organ systems. close proximity to genes, identifying ARHGEF3 , TAOK1 The gene lists for red cells and platelets also showed and WDR66 as possible novel regulators of platelet forma - striking differences. All but 2 ( GCKR , THPO ) of the 68 tion. 9,10 The remaining fourth SNV rs342293 at 7q22.3, genes identified by the platelet GWAS are transcribed in which exerted also an effect on platelet function, was megakaryocytes, but nearly 25% of genes identified in the localized at an equidistance of the hypothetical gene red cell GWAS are not transcribed in erythroblasts or other FLJ36031 and PIK3CG . The latter was the only one of the hematopoietic cells. This may reflect the more complex

| 404 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2014; 8(1) Milan, Italy, June 12-15, 2014

Figure 1. (A) Genetic loci associated with the volume and count of platelets. Clockwise display of chromosomes 1 to 22. Names of candidate genes for platelet volume and count identified by the genome wide association study are presented. P-values of association for the sentinel single nucleotide variants are represented by the relative length of the centripetal black lines. (For further details see Gieger et al. 11 ) (B) Genetic loci associated with the volume and count of red cells. Clockwise display of chromosomes 1 to 22. Names of candidate genes for red cell volume, count and the other red cell indices identified by the genome wide association study are presented. P-values of association for the sentinel single nucleotide variants are represented by the relative length of the centripetal black lines. (For further details see van der Harst et al. 13 )

regulatory networks controlling the formation of red blood colleagues showed that the threonine form has a signifi - cells compared to the relatively simple thrombopoietin- cantly higher binding affinity for VWF than the methion - thrombopoietin receptor feedback loop that controls the ine one providing a credible molecular explanation for the mass of platelets. Red cell formation is influenced by a observed association between rs6065 and platelet count. large number of genes implicated in iron homeostasis and Interestingly, the platelet GWAS variant rs1558324 marks oxygen sensing, and it is reasonable to assume that a sub - the VWF gene as another candidate gene controlling the 14,15 set of genes involved in these processes are primarily tran - volume of platelets. The observation that common vari - scribed in non-hematopoietic tissues. Several of the ants in genes encoding a receptor essential for platelet known regulators of iron homeostasis ( HFE, TMPRSS6, function (GP1BA) and its ligand (VWF) are both modify - TFRC, TFR2 ) were identified in the red cell GWAS meta- ing the count and volume of platelets (Figure 2) is compat - analysis. 13 It can, therefore, be safely assumed that several ible with the notion that mutations in these genes are of the novel red cell GWAS genes of unknown function causative of Mendelian bleeding and platelet disorders. encode proteins important in either iron homeostasis or Loss-of-function mutations in GP1BA are causative of Bernard Soulier Syndrome, a rare autosomal recessive oxygen sensing. Further functional genomics studies are 16 warranted to test this assumption. macrothrombocytopenia with severe bleeding. Although most mutations in VWF cause a mild bleeding disorder with a normal platelet count, some dominant gain-of-func - Mechanisms underlying the observed associations tion mutations are causative of Type 2B VWF disease, which is frequently associated with thrombocytopenia. A quarter of the 140+ GWAS sentinel SNVs are either Mouse gene knockout studies for Gp1ba and Vwf have non-synonymous (ns) or in strong linkage disequilibrium confirmed the functional relevance of the GPIBA-VWF (LD) with an nsSNV (so called proxy SNVs). This obser - axis for platelet formation and hemostasis. The GWAS vation strongly suggests that at loci with nsSNVs, amino study for platelets revealed that more subtle changes at the acid polymorphisms underlie subtle differences in protein GP1BA and VWF loci effect their volume and count. function between the genotype groups. For example, For 39 platelet GWAS genes the sentinel SNVs or their GWAS variant rs6065, which regulates platelet count is proxys do not alter the amino acid sequence of the encod - localized in the GPIBA gene, which encodes glycoprotein ed proteins and alternative mechanisms have to account (GP)1BA, the platelet receptor for Von Willebrand Factor for the observed phenotypic differences. In the absence of (VWF). The GWAS variant rs6065 underlies a threo - amino acid differences, it is reasonable to postulate that nine145methionine polymorphism in the fifth leucine-rich differences in gene transcript levels between the genotype repeat domain of this receptor. A study by Ulrichts and groups drive the observed differences in blood cell count

Hematology Education: the education program for the annual congress of the European Hematology Association | 2014; 8(1) | 405 | 19 th Congress of the European Hematology Association

and volume. Genome wide studies have revealed the exis - and monocytes, obtained from large numbers of healthy tence of large numbers of non-coding SNVs with effects individuals, was used to identify eQTL SNVs. 10 Finally, on transcript levels of Cis-positioned genes. 17-21 These so- array expression studies were carried out as part of the called expression Quantitative Trait Loci (eQTL) SNVs Blueprint blood cell epigenome project, complemented by have been well documented at the genome-wide level for RNA sequencing (RNA-seq) studies of the eight main several blood cell types. Their effects are often specific for types of blood cell progenitors and precursors to enhance a single cell type indicating that they may be positioned in the catalog of transcript isoforms used by these cells. 26 elements of the genome that are only functional in a cer - From these genome-wide studies a picture has emerged tain cell type, but not in others. To investigate whether where several sentinel SNVs or their proxys are localized GWAS SNVs for platelets or red cells or their proxys are in cell type-specific regulatory elements. Wet-lab studies enriched at positions of cell type-specific enhancers a with the variants rs342293 at 7q22.3 and rs10914144 at comprehensive annotation of the genomes of megakary - 1q24.3 showed that they exert effects on the transcript lev - ocytes and erythroblasts was performed. This was els of the PIK3CG and DNM3 genes, respectively. 10,25 For achieved by exploiting the power of NGS. First, genomic the former, differences in PIK3CG transcript levels regions depleted of nucleosomes were experimentally between the three genotype groups is caused by the strong identified by formaldehyde-assisted identification of regu - effect of this variant on the binding of the transcription latory elements (FAIRE) in erythroblasts, megakaryocytes factor EVI1. The major (C) allele underlies robust binding and monocytes. 22,23 For megakaryocytes, this analysis was of EVI1, whilst the minor (G) allele does not bind EVI1. complemented by chromatin immunoprecipitation (ChIP) Lack of EVI1 binding at this nucleosome-depleted region for seven transcription factors (GATA1/2, FLI1, MEIS1, on 7q22.3 is associated with higher PIK3CG transcript NFE2, RUNX1, SCL) in human megakaryocytes 24 or in levels in platelets in GG individuals when compared with the megakaryocytic cell line CHRF-288-11. 25 Thirdly, CC individuals. 10 The GWAS variant rs10914144 localizes whole genome expression studies with RNA from platelets to the second intron of the DNM3 gene, but the functional

Figure 2. Cartoon of a megakaryocyte with the proteins encoded by the platelet GWAS genes. The proteins encoded by the GWAS genes for platelet volume and count are displayed in their respective cellular compartments (cell membrane, cytoplasm, nucleus, endoplasmic reticulum). For nine proteins the cellular localization is not known and these are placed together at the bottom-left of the cartoon. Two of the 68 GWAS genes ( GCKR, THPO ) are not transcribed in hematopoietic cells but are primarily transcribed in the liver. The VWF gene, which is transcribed in both endothelial cells and megakary - ocytes, has been for clarity and simplicity of the cartoon only be placed in endothelial cells. Protein domain structures were retrieved from the InterPro database at the European Bioinformatics Institute at https://www.ebi.ac.uk/interpro.

| 406 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2014; 8(1) Milan, Italy, June 12-15, 2014 annotation of the precursor cell genomes was not sugges - localized in a megakaryocyte-specific enhancer element in tive of a regulatory element at this position. Interestingly, the 5’ UTR of the RBM8A gene and the minor allele intro - it was noted that 10 kb upstream of the original GWAS duces a binding site for the transcription factor EVI1. The SNV there was a C/A variant at rs2038479 in high LD binding of EVI1 at this position is associated with less with the sentinel SNV and with an identical MAF. This effective transcription from the apparently intact RBM8A variant is localized in a region of open chromatin as shown gene causing an insufficiency of Y14, the protein encoded by a robust FAIRE signal, occupied by the transcription by RBM8A. Gene ablation in mice and knockdown in factor MEIS1 and RNA-seq of megakaryocytes showed zebrafish of Rbm8a is not compatible with life. The rela - the same region to be transcribed indicating the possibility tive insufficiency of Y14 in the megakaryocyte lineage, of a novel exon. Indeed, rs2038479 marked a novel alter - caused by the coming together of a deletion and the native transcription start site for DNM3, which is used repressive 5’UTR-SNV, leads to a selective maturation exclusively in megakaryocytes and not in other cell types block with a paucity of megakaryocytes, which is a classic that transcribe the gene. The novel megakaryocyte specif - hallmark of TAR. Because the effect of the minor allele is ic transcript lacks the canonical first exon leading to a only present in the megakaryocyte lineage, the differenti - shorter open reading frame encoding a DNM3 molecule, ation of the other myeloid lineages can proceed normally. which lacks its GTPase domain. 25 The differences in the Interestingly, our recent discovery that SMIM1 is the gene abundance of the levels of the novel transcript between that underlies the red blood cell group Vel provided a sim - rs2038479 genotype groups does influence the formation ilar example of the same novel genetic architecture of platelets and their volume, revealing an entirely new observed in TAR cases. A nonsense 17-bp deletion of one functional role for the Dynamin family of mechanochem - SMIM1 haplotype together with the major allele of red cell ical molecular spring molecules in platelet formation. GWAS variant rs1175550 on the alternate haplotype leads Further exploration and statistical analysis of the results to an extremely low level of SMIM1 transcript. This from the functional annotation studies of megakaryocytes results in a near absence of the Vel blood group protein and erythroblasts showed a similar mechanism of allelic from red cells. 39 This absence of protein does not lead to differences in the binding of nuclear proteins at other apparent pathobiology but it shows that a strong-acting platelet GWAS SNVs or their proxys at the following nine eQTL on one haplotype, if combined with a deletion on loci: FAR2, BAZ2A/ATP5B, RAD51B, LRRC16A, PDIA5, the alternate haplotype, can lead to the near absence of ABCC4, CTSZ/TUBB1, KALRN and LOC339862 .23 protein in a lineage-restricted manner. We postulate that this type of interaction between extremely rare mutations and common variants at the same locus may explain an Discovery of genes underlying Mendelian platelet 36 appreciable fraction of unresolved inherited disorders. and bleeding disorders The early success of gene discovery confirms the power, but also shows the limitations of whole exome sequencing To date, 32 genes have been identified underlying 24 27 (WES) as a tool to discover mutations for Mendelian distinct Mendelian platelet disorders. With the advent of platelet disorders. The challenges of analysis of the results NGS and other modern genomics platforms it has become obtained by WES of the remaining 500 BRIDGE BPD feasible to scan the coding fraction or the entire genome cases are substantial. First, the BRIDGE collection of from cases with molecularly unexplained bleeding and BPD cases is clinically more heterogeneous than GPS and platelet disorders (BPDs). Five new genes ( ANKRD26, TAR. Second, power of gene discovery is eroded by this NBEAL2, RBM8A, ACTN1, GFI1B ) for BPDs have been 28-34 phenotype heterogeneity, but it is hoped that this can be identified since 2011. To resolve the genetic basis of recovered by clustering of patients using detailed clinical additional rare inherited disorders of platelet count, vol - information and rich data from laboratory tests. The for - ume and function we have established the BRIDGE-BPD mer information is coded by assigning human phenotype collection of cases. So far, 716 BPD cases have been ontology (HPO) terms to the symptoms observed in enrolled in the BRIDGE sequencing program with the aim patients and by grading the severity of bleeding. Third, of discovering additional causal genes. So far, 519 sam - three of the inherited conditions at the ANKRD26 , RBM8A ples have been analyzed by whole exome sequencing. and SMIM1 loci could not have been revealed by WES Analysis of cases with grey platelet syndrome (GPS) and analysis only, as variants in regulatory elements are part of thrombocytopenia with absent radii (TAR) identified the observed genetic mechanisms. NBEAL2 28-30 and RBM8A 35-36 as the causative genes for these two syndromes. The GPS discovery is a classic example of an autosomal recessive platelet bleeding disor - Simplifying the diagnosis of inherited bleeding and der. Loss-of-function mutations on both NBEAL2 haplo - platelet disorders types leads to the formation of platelets which are devoid a of -granules and myelofibrosis develops in nearly all Finally, we are addressing the need of translating the GPS cases at a later age. Studies in Nbeal2 knockout mice application of NGS into the clinic. Currently, the diagnosis have recapitulated the phenotype observed in humans, of rare inherited BPDs is based on a plethora of biochem - although further molecular studies are required to define ical and platelet and coagulation factor function tests, the function of the NBEAL2 protein in granule formation many of which have a poor receiver operating characteris - and retention. 37,38 The study of TAR cases revealed an tic and considerable expertise is required to perform these entirely novel genetic mechanism where a submicroscopic laborious tests. Ideally, a single and affordable DNA- deletion of 1q21.1 on one parental haplotype is com - based test should be available to determine whether a clin - pounded by the minor allele of a SNV present in 1 in 30 ical case presenting with a BPD is caused by mutations in healthy controls, on the other haplotype. 35 This SNV is one of the known BPD genes. To achieve this, a working

Hematology Education: the education program for the annual congress of the European Hematology Association | 2014; 8(1) | 407 | 19 th Congress of the European Hematology Association group, ThromboGenomics (www.thrombogenomics. Prothrombotic Risk due to Genetic Variations in Platelet Genes: enhanced outside-in signaling through the Pro 33 vari - org.uk), has been established under the auspices of the ant of integrin á 3. Biol Med 2006;23:(5)505-13. International Society for Thrombosis and Haemostasis. 13. van der Harst P, Zhang W, Mateo Leach I, Rendon A, Verweij The working group is taking forward three initiatives. N, Sehmi J, et al. Seventy-five genetic loci influencing the human red blood cell. Nature. 2012;492(7429):369-75. First, a list of 51 candidate genes, including the 32 genes 14. Li CQ, Garner SF, Davies J, Smethurst PA, Wardell MR, underlying inherited platelet disorders and 19 genes Ouwehand WH. Threonine-145/Methionine-145 variants of underlying inherited coagulation disorders has been com - baculovirus produced recombinant ligand binding domain of piled. Second, an international group of experts work with GPIbà express HPA-2 epitopes and show equal binding of von Willebrand factor. Blood. 2000;95(1):205-11. the Locus Reference Genomic team at the European 15. Ulrichts H, Vanhoorelbeke K, Cauwenberghs S, Vauterin S, Informatics Institute to curate the variants in these genes Kroll H, Santoso S, et al. von Willebrand factor but not alpha- to clinical standard. Third, a pull-down platform for the thrombin binding to platelet glycoprotein Ibalpha is influ - enced by the HPA-2 polymorphism. Arterioscler Thromb sequencing of the exons of the 51 genes has been devel - Vasc Biol. 2003;23(7):1302-7. oped and validated. This assay will become available 16. Nurden AT, Nurden P. Inherited defects of platelet function. through the UK hemophilia centers and similar compre - Rev Clin Exp Hematol. 2001;5(4):314-34. 17. Stranger BE, Forrest MS, Dunning M, Ingle CE, Beazley C, hensive centers overseas during 2014. Thorne N, et al. Relative impact of nucleotide and copy num - ber variation on gene expression phenotypes. Science. 2007;315(5813):848-53. Conclusion 18. Rotival M, Zeller T, Wild PS, Maouche S, Szymczak S, Schillert A, et al. Integrating genome-wide genetic variations and monocyte expression data reveals trans-regulated gene In conclusion, six years of gene discovery and function - modules in humans. PLoS genetics. 2011;7(12):e1002367. al genomics have revolutionized our understanding of the 19. Heinig M, Petretto E, Wallace C, Bottolo L, Rotival M, Lu H, et al. A trans-acting locus regulates an anti-viral expression genetic architecture of megakaryopoiesis and platelet for - network and type 1 diabetes risk. Nature. 2010;467 mation. A large number of important novel regulators of (7314):460-4. both processes have been revealed. The discovery of novel 20. Dixon AL, Liang L, Moffatt MF, Chen W, Heath S, Wong genes implicated in rare BPDs supports an international KC, et al. A genome-wide association study of global gene expression. Nat Genet. 2007;39(10):1202-7. effort to bring to the clinic a comprehensive NGS platform 21. Fehrmann RS, Jansen RC, Veldink JH, Westra HJ, Arends D, for their affordable and rapid diagnosis. This will not only Bonder MJ, et al. Trans-eQTLs reveal that independent genet - remedy the considerable diagnostic delay for index cases, ic variants associated with a complex phenotype converge on intermediate genes, with a major role for the HLA. PLoS but also provide information for family planning purposes Genet. 2011;7(8):e1002197. if required. 22. Paul DS, Nisbet JP, Yang TP, Meacham S, Rendon A, Hautaviita K, et al. Maps of open chromatin guide the func - tional follow-up of genome-wide association signals: applica - References tion to hematological traits. PLoS genetics. 2011;7(6): e1002139. 23. Paul DS, Albers CA, Rendon A, Voss K, Stephens J, Haem 1. A map of human genome variation from population-scale Gen C, et al. Maps of open chromatin highlight cell type- sequencing. Nature. 2010;467(7319):1061-73. restricted patterns of regulatory sequence variation at hema - 2. Kaye J, Hurles M, Griffin H, Grewal J, Bobrow M, Timpson tological trait loci. Genome Res. 2013;23(7):1130-41. N, et al. Managing clinically significant findings in research: 24. Tijssen MR, Cvejic A, Joshi A, Hannah RL, Ferreira R, Forrai the UK10K example. Eur J Hum Genet. 2014 Jan 15. [Epub A, et al. Genome-wide analysis of simultaneous GATA1/2, ahead of print] RUNX1, FLI1, and SCL binding in megakaryocytes identi - 3. Moran N. 10,000 rare-disease genomes sequenced. Nature fies hematopoietic regulators. Dev Cell. 2011;20(5):597-609. Biotechnology. 2014. March 30. [Epub ahead of print] 25. Nurnberg ST, Rendon A, Smethurst PA, Paul DS, Voss K, 4. Genome-wide association study of 14,000 cases of seven Thon JN, et al. A GWAS sequence variant for platelet volume common diseases and 3,000 shared controls. Nature. marks an alternative DNM3 promoter in megakaryocytes 2007;447(7145):661-78. near a MEIS1 binding site. Blood. 2012;120(24):4859-68. 5. Welter D, MacArthur J, Morales J, Burdett T, Hall P, Junkins 26. Adams D, Altucci L, Antonarakis SE, Ballesteros J, Beck S, H, et al. The NHGRI GWAS Catalog, a curated resource of Bird A, et al. BLUEPRINT to decode the epigenetic signature SNP-trait associations. Nucleic Acids Res. 2014;42(Database written in blood. Nat Biotechnol. 2012;30:224-6. issue):D1001-6. 27. Nurden AT. Inherited abnormalities of platelets. Thrombosis 6. Thein SL, Menzel S, Peng X, Best S, Jiang J, Close J, et al. and haemostasis. 1999;82(2):468-80. Intergenic variants of HBS1L-MYB are responsible for a 28. Albers CA, Cvejic A, Favier R, Bouwmans EE, Alessi MC, major quantitative trait locus on chromosome 6q23 influenc - Bertone P, et al. Exome sequencing identifies NBEAL2 as the ing fetal hemoglobin levels in adults. Proc Natl Acad Sci causative gene for gray platelet syndrome. Nat Genet. USA. 2007;104(27):11346-51. 2011;43(8):735-7. 7. Soranzo N, Spector TD, Mangino M, Kuhnel B, Rendon A, 29. Gunay-Aygun M, Falik-Zaccai TC, Vilboux T, Zivony- Teumer A, et al. A genome-wide meta-analysis identifies 22 Elboum Y, Gumruk F, Cetin M, et al. NBEAL2 is mutated in loci associated with eight hematological parameters in the gray platelet syndrome and is required for biogenesis of HaemGen consortium. Nat. Genet. 2009 Nov;41(11):1182-90. platelet alpha-granules. Nat Genet. 2011;43(8):732-4. 8. Ganesh SK, Zakai NA, van Rooij FJ, Soranzo N, Smith AV, 30. Kahr WH, Hinckley J, Li L, Schwertz H, Christensen H, Nalls MA, et al. Multiple loci influence erythrocyte pheno - Rowley JW, et al. Mutations in NBEAL2, encoding a types in the CHARGE Consortium. Nat Genet. BEACH protein, cause gray platelet syndrome. Nat Genet. 2009;41(11):1191-8. 2011;43(8):738-40. 9. Meisinger C, Prokisch H, Gieger C, Soranzo N, Mehta D, 31. Stevenson WS, Morel-Kopp MC, Chen Q, Liang HP, Rosskopf D, et al. A genome-wide association study identifies Bromhead CJ, Wright S, et al. GFI1B mutation causes a three loci associated with mean platelet volume. Am J Hum bleeding disorder with abnormal platelet function. J Thromb Genet. 2009;84(1):66-71. Haemost. 2013;11(11):2039-47. 10. Soranzo N, Rendon A, Gieger C, Jones CI, Watkins NA, 32. Monteferrario D, Bolar NA, Marneth AE, Hebeda KM, Menzel S, et al. A novel variant on chromosome 7q22.3 asso - Bergevoet SM, Veenstra H, et al. A dominant-negative GFI1B ciated with mean platelet volume, counts, and function. mutation in the gray platelet syndrome. N Engl J Med. Blood. 2009;113(16):3831-7. 2014;370(3):245-53. 11. Gieger C, Radhakrishnan A, Cvejic A, Tang W, Porcu E, 33. Pippucci T, Savoia A, Perrotta S, Pujol-Moix N, Noris P, Pistis G, et al. New gene functions in megakaryopoiesis and Castegnaro G, et al. Mutations in the 5’ UTR of ANKRD26, platelet formation. Nature. 2011;480 (7376):201-8. the ankirin repeat domain 26 gene, cause an autosomal-dom - 12. Vijayan KV, Bray PF. Molecular Mechanisms of inant form of inherited thrombocytopenia, THC2. Am J Hum

| 408 | Hematology Education: the education program for the annual congress of the European Hematology Association | 2014; 8(1) Milan, Italy, June 12-15, 2014

Genet. 2011;88(1):115-20. 316-23. 34. Kunishima S, Okuno Y, Yoshida K, Shiraishi Y, Sanada M, 37. Kahr WH, Lo RW, Li L, Pluthero FG, Christensen H, Ni R, et Muramatsu H, et al. ACTN1 mutations cause congenital al. Abnormal megakaryocyte development and platelet func - macrothrombocytopenia. Am J Hum Genet. 2013;92(3):431- tion in Nbeal2(-/-) mice. Blood. 2013;122(19):3349-58. 8. 38. Deppermann C, Cherpokova D, Nurden P, Schulz JN, 35. Albers CA, Paul DS, Schulze H, Freson K, Stephens JC, Thielmann I, Kraft P, et al. Gray platelet syndrome and defec - Smethurst PA, et al. Compound inheritance of a low-frequen - tive thrombo-inflammation in Nbeal2-deficient mice. J Clin cy regulatory SNP and a rare null mutation in exon-junction Invest. 2013 Jul 1. [Epub ahead of print] complex subunit RBM8A causes TAR syndrome. Nat Genet. 39. Cvejic A, Haer-Wigman L, Stephens JC, Kostadima M, 2012;44(4):435-9, S1-2. Smethurst PA, Frontini M, et al. SMIM1 underlies the Vel 36. Albers CA, Newbury-Ecob R, Ouwehand WH, Ghevaert C. blood group and influences red blood cell traits. Nat Genet. New insights into the genetic basis of TAR (thrombocytope - 2013;45(5):542-5. nia-absent radii) syndrome. Curr Opin Gen Dev. 2013;23(3):

Hematology Education: the education program for the annual congress of the European Hematology Association | 2014; 8(1) | 409 |