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1.  Variants near TERC are associated with mean telomere length. 
Nature genetics  2010;42(3):197-199.
We conducted genome-wide association analyses of mean leukocyte telomere length in 2,917 subjects and follow-up replication analyses in 9,492 and identified a locus on 3q26 encompassing the telomerase RNA component TERC, with compelling evidence for association (rs12696304, combined P value 3.72×10−14). Each copy of the minor allele of rs12696304 was associated with ≈75 base pairs shorter mean telomere length equivalent to ≈3.6 years of age-related attrition of mean telomere length.
doi:10.1038/ng.532
PMCID: PMC3773906  PMID: 20139977
2.  Elevated Levels of Procoagulant Plasma Microvesicles in Dialysis Patients 
PLoS ONE  2013;8(8):e72663.
Cardiovascular (CV) death remains the largest cause of mortality in dialysis patients, unexplained by traditional risk factors. Endothelial microvesicles (EMVs) are elevated in patients with traditional CV risk factors and acute coronary syndromes while platelet MVs (PMVs) are associated with atherosclerotic disease states. This study compared relative concentrations of circulating MVs from endothelial cells and platelets in two groups of dialysis patients and matched controls and investigated their relative thromboembolic risk. MVs were isolated from the blood of 20 haemodialysis (HD), 17 peritoneal dialysis (PD) patients and 20 matched controls. Relative concentrations of EMVs (CD144+ ve) and PMVs (CD42b+ ve) were measured by Western blotting and total MV concentrations were measured using nanoparticle-tracking analysis. The ability to support thrombin generation was measured by reconstituting the MVs in normal plasma, using the Continuous Automated Thrombogram assay triggered with 1µM tissue factor. The total concentration of MVs as well as the measured sub-types was higher in both patient groups compared to controls (p<0.05). MVs from HD and PD patients were able to generate more thrombin than the controls, with higher peak thrombin, and endogenous thrombin potential levels (p<0.02). However there were no differences in either the relative quantity or activity of MVs between the two patient groups (p>0.3). Dialysis patients have higher levels of circulating procoagulant MVs than healthy controls. This may represent a novel and potentially modifiable mediator or predictor of occlusive cardiovascular events in these patients.
doi:10.1371/journal.pone.0072663
PMCID: PMC3732282  PMID: 23936542
3.  New gene functions in megakaryopoiesis and platelet formation 
Gieger, Christian | Radhakrishnan, Aparna | Cvejic, Ana | Tang, Weihong | Porcu, Eleonora | Pistis, Giorgio | Serbanovic-Canic, Jovana | Elling, Ulrich | Goodall, Alison H. | Labrune, Yann | Lopez, Lorna M. | Mägi, Reedik | Meacham, Stuart | Okada, Yukinori | Pirastu, Nicola | Sorice, Rossella | Teumer, Alexander | Voss, Katrin | Zhang, Weihua | Ramirez-Solis, Ramiro | Bis, Joshua C. | Ellinghaus, David | Gögele, Martin | Hottenga, Jouke-Jan | Langenberg, Claudia | Kovacs, Peter | O’Reilly, Paul F. | Shin, So-Youn | Esko, Tõnu | Hartiala, Jaana | Kanoni, Stavroula | Murgia, Federico | Parsa, Afshin | Stephens, Jonathan | van der Harst, Pim | van der Schoot, C. Ellen | Allayee, Hooman | Attwood, Antony | Balkau, Beverley | Bastardot, François | Basu, Saonli | Baumeister, Sebastian E. | Biino, Ginevra | Bomba, Lorenzo | Bonnefond, Amélie | Cambien, François | Chambers, John C. | Cucca, Francesco | D’Adamo, Pio | Davies, Gail | de Boer, Rudolf A. | de Geus, Eco J. C. | Döring, Angela | Elliott, Paul | Erdmann, Jeanette | Evans, David M. | Falchi, Mario | Feng, Wei | Folsom, Aaron R. | Frazer, Ian H. | Gibson, Quince D. | Glazer, Nicole L. | Hammond, Chris | Hartikainen, Anna-Liisa | Heckbert, Susan R. | Hengstenberg, Christian | Hersch, Micha | Illig, Thomas | Loos, Ruth J. F. | Jolley, Jennifer | Khaw, Kay Tee | Kühnel, Brigitte | Kyrtsonis, Marie-Christine | Lagou, Vasiliki | Lloyd-Jones, Heather | Lumley, Thomas | Mangino, Massimo | Maschio, Andrea | Leach, Irene Mateo | McKnight, Barbara | Memari, Yasin | Mitchell, Braxton D. | Montgomery, Grant W. | Nakamura, Yusuke | Nauck, Matthias | Navis, Gerjan | Nöthlings, Ute | Nolte, Ilja M. | Porteous, David J. | Pouta, Anneli | Pramstaller, Peter P. | Pullat, Janne | Ring, Susan M. | Rotter, Jerome I. | Ruggiero, Daniela | Ruokonen, Aimo | Sala, Cinzia | Samani, Nilesh J. | Sambrook, Jennifer | Schlessinger, David | Schreiber, Stefan | Schunkert, Heribert | Scott, James | Smith, Nicholas L. | Snieder, Harold | Starr, John M. | Stumvoll, Michael | Takahashi, Atsushi | Tang, W. H. Wilson | Taylor, Kent | Tenesa, Albert | Thein, Swee Lay | Tönjes, Anke | Uda, Manuela | Ulivi, Sheila | van Veldhuisen, Dirk J. | Visscher, Peter M. | Völker, Uwe | Wichmann, H.-Erich | Wiggins, Kerri L. | Willemsen, Gonneke | Yang, Tsun-Po | Zhao, Jing Hua | Zitting, Paavo | Bradley, John R. | Dedoussis, George V. | Gasparini, Paolo | Hazen, Stanley L. | Metspalu, Andres | Pirastu, Mario | Shuldiner, Alan R. | van Pelt, L. Joost | Zwaginga, Jaap-Jan | Boomsma, Dorret I. | Deary, Ian J. | Franke, Andre | Froguel, Philippe | Ganesh, Santhi K. | Jarvelin, Marjo-Riitta | Martin, Nicholas G. | Meisinger, Christa | Psaty, Bruce M. | Spector, Timothy D. | Wareham, Nicholas J. | Akkerman, Jan-Willem N. | Ciullo, Marina | Deloukas, Panos | Greinacher, Andreas | Jupe, Steve | Kamatani, Naoyuki | Khadake, Jyoti | Kooner, Jaspal S. | Penninger, Josef | Prokopenko, Inga | Stemple, Derek | Toniolo, Daniela | Wernisch, Lorenz | Sanna, Serena | Hicks, Andrew A. | Rendon, Augusto | Ferreira, Manuel A. | Ouwehand, Willem H. | Soranzo, Nicole
Nature  2011;480(7376):201-208.
Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function.
doi:10.1038/nature10659
PMCID: PMC3335296  PMID: 22139419
4.  Kv1.3 is the exclusive voltage-gated K+ channel of platelets and megakaryocytes: roles in membrane potential, Ca2+ signalling and platelet count 
The Journal of Physiology  2010;588(Pt 9):1399-1406.
A delayed rectifier voltage-gated K+ channel (Kv) represents the largest ionic conductance of platelets and megakaryocytes, but is undefined at the molecular level. Quantitative RT-PCR of all known Kv α and ancillary subunits showed that only Kv1.3 (KCNA3) is substantially expressed in human platelets. Furthermore, megakaryocytes from Kv1.3−/− mice or from wild-type mice exposed to the Kv1.3 blocker margatoxin completely lacked Kv currents and displayed substantially depolarised resting membrane potentials. In human platelets, margatoxin reduced the P2X1- and thromboxaneA2 receptor-evoked [Ca2+]i increases and delayed the onset of store-operated Ca2+ influx. Megakaryocyte development was normal in Kv1.3−/− mice, but the platelet count was increased, consistent with a role of Kv1.3 in apoptosis or decreased platelet activation. We conclude that Kv1.3 forms the Kv channel of the platelet and megakaryocyte, which sets the resting membrane potential, regulates agonist-evoked Ca2+ increases and influences circulating platelet numbers.
doi:10.1113/jphysiol.2010.188136
PMCID: PMC2876798  PMID: 20308249
5.  A common variant in low density lipoprotein receptor-related protein 6 gene (LRP6) is associated with LDL-cholesterol 
Objective
A rare mutation in low density lipoprotein receptor-related protein 6 gene (LRP6) was identified as the primary molecular defect underlying monogenic form of coronary artery disease. We hypothesised that common variants in LRP6 could predispose subjects to elevated LDL-cholesterol (LDL-C).
Methods and Results
12 common (minor allele frequency ≥0.1) single nucleotide polymorphisms in LRP6 were genotyped in 703 individuals from 213 Polish pedigrees (Silesian Cardiovascular Study families). The family-based analysis revealed that the minor allele of rs10845493 clustered with elevated LDL-C in offspring more frequently than expected by chance (p=0.0053). The quantitative analysis restricted to subjects free of lipid-lowering treatment confirmed the association between rs10845493 and age-, sex- and BMI-adjusted circulating levels of LDL-C in families as well as 2 additional populations - 218 unrelated subjects from Silesian Cardiovascular Study replication panel and 1138 individuals from Young Men Cardiovascular Association cohort (p=0.0268, p=0.0476 and p=0.0472, respectively). In the inverse variance weighted meta-analysis of the 3 populations each extra minor allele copy of rs10845493 was associated with 0.14 mmol/L increase in age-, sex- and BMI-adjusted LDL-C (SE=0.05, p=0.0038).
Conclusions
Common polymorphism in the gene underlying monogenic form of coronary artery disease impacts on risk of LDL-C elevation.
doi:10.1161/ATVBAHA.109.185355
PMCID: PMC2814817  PMID: 19667113
gene; genetics; LDL-cholesterol; lipids; association
6.  Phospholipid-esterified Eicosanoids Are Generated in Agonist-activated Human Platelets and Enhance Tissue Factor-dependent Thrombin Generation* 
The Journal of Biological Chemistry  2010;285(10):6891-6903.
Here, a group of specific lipids, comprising phosphatidylethanolamine (PE)- or phosphatidylcholine (PC)-esterified 12S-hydroxyeicosatetraenoic acid (12S-HETE), generated by 12-lipoxygenase was identified and characterized. 12S-HETE-PE/PCs were formed within 5 min of activation by thrombin, ionophore, or collagen. Esterified HETE levels generated in response to thrombin were 5.85 ± 1.42 (PE) or 18.35 ± 4.61 (PC), whereas free was 65.5 ± 17.6 ng/4 × 107 cells (n = 5 separate donors, mean ± S.E.). Their generation was stimulated by triggering protease-activated receptors-1 and -4 and signaling via Ca2+ mobilization secretory phospholipase A2, platelet-activating factor-acetylhydrolase, src tyrosine kinases, and protein kinase C. Stable isotope labeling showed that they form predominantly by esterification that occurs on the same time scale as free acid generation. Unlike free 12S-HETE that is secreted, esterified HETEs remain cell-associated, with HETE-PEs migrating to the outside of the plasma membrane. 12-Lipoxygenase inhibition attenuated externalization of native PE and phosphatidylserine and HETE-PEs. Platelets from a patient with the bleeding disorder, Scott syndrome, did not externalize HETE-PEs, and liposomes supplemented with HETE-PC dose-dependently enhanced tissue factor-dependent thrombin generation in vitro. This suggests a role for these novel lipids in promoting coagulation. Thus, oxidized phospholipids form by receptor/agonist mechanisms, not merely as an undesirable consequence of vascular and inflammatory disease.
doi:10.1074/jbc.M109.078428
PMCID: PMC2844139  PMID: 20061396
Eicosanoids; Eicosanoids/Arachidonic Acid; Eicosanoids/Function; Eicosanoids/Lipoxygenase Pathway; Lipid; Lipid/Phospholipid
7.  PECAM-1 expression and activity negatively regulate multiple platelet signaling pathways 
Febs Letters  2009;583(22-10):3618-3624.
Platelet endothelial cell adhesion molecule-1 (PECAM-1) inhibits platelet response to collagen and may also inhibit two other major platelet agonists ADP and thrombin although this has been less well explored. We hypothesized that the combined effect of inhibiting these three platelet activating pathways may act to significantly inhibit thrombus formation. We demonstrate a negative relationship between PECAM-1 surface expression and platelet response to cross-linked collagen related peptide (CRP-XL) and ADP, and an inhibitory effect of PECAM-1 clustering on platelet response to CRP-XL, ADP and thrombin. This combined inhibition of multiple signaling pathways results in a marked reduction in thrombus formation.
doi:10.1016/j.febslet.2009.10.037
PMCID: PMC2791847  PMID: 19850043
Platelets; PECAM-1; Collagen; ADP; Thrombin; Thrombus
8.  Thrombus Size and Doppler Embolic Signal Intensity 
Background:
Migration of thrombus through the cerebral arteries is a common cause of stroke. Thrombus emboli can be detected non-invasively using Doppler ultrasound, but even where the embolus composition is known, there is currently no method for estimating the size of an embolus based on the returned ultrasound signal. Here we report the results of in vitro experiments investigating the relationship between size and embolic signal intensity for fresh thrombus emboli with a view to estimating the sizes of thrombi detected following carotid surgery.
Method:
Thrombi were formed from whole blood using the ‘Chandler loop’ method under flow conditions similar to those associated with arterial thrombus formation in vivo. A total of 390 Doppler embolic signals were then measured from 37 pieces of thrombus circulated in a pulsatile closed-flow circuit. The dimensions of each of the thrombi were measured before and after circulation using an optical microscope. Relationships between thrombus size and embolic signal properties were then investigated using standard statistical methods with a view to size estimation of thrombi during clinical monitoring.
Results:
Although embolic signals generally became more intense with increasing thrombus size, strong oscillations due to resonance effects were observed. Pearson tests revealed strong positive correlations between embolus diameter, signal intensity and duration (r > 1 0.8, p ≤ 0.01).
Conclusions:
This study provides experimental evidence supporting theoretical predictions relating Doppler embolic signal intensity to thrombus size. In our discussion, we tentatively suggest how this information might be used to size emboli in clinical practice.
doi:10.1159/000235627
PMCID: PMC2756770  PMID: 19713699
Carotid endarterectomy; Micro-embolic signals; Thrombus formation; Transcranial Doppler ultrasound; Embolus detection
9.  Thrombus Size and Doppler Embolic Signal Intensity 
Background
Migration of thrombus through the cerebral arteries is a common cause of stroke. Thrombus emboli can be detected non-invasively using Doppler ultrasound, but even where the embolus composition is known, there is currently no method for estimating the size of an embolus based on the returned ultrasound signal. Here we report the results of in vitro experiments investigating the relationship between size and embolic signal intensity for fresh thrombus emboli with a view to estimating the sizes of thrombi detected following carotid surgery.
Method
Thrombi were formed from whole blood using the ‘Chandler loop’ method under flow conditions similar to those associated with arterial thrombus formation in vivo. A total of 390 Doppler embolic signals were then measured from 37 pieces of thrombus circulated in a pulsatile closed-flow circuit. The dimensions of each of the thrombi were measured before and after circulation using an optical microscope. Relationships between thrombus size and embolic signal properties were then investigated using standard statistical methods with a view to size estimation of thrombi during clinical monitoring.
Results
Although embolic signals generally became more intense with increasing thrombus size, strong oscillations due to resonance effects were observed. Pearson tests revealed strong positive correlations between embolus diameter, signal intensity and duration (r > 0.8, p ≤ 0.01).
Conclusions
This study provides experimental evidence supporting theoretical predictions relating Doppler embolic signal intensity to thrombus size. In our discussion, we tentatively suggest how this information might be used to size emboli in clinical practice.
doi:10.1159/000235627
PMCID: PMC2756770  PMID: 19713699
Carotid endarterectomy; Micro-embolic signals; Thrombus formation; Transcranial Doppler ultrasound; Embolus detection
10.  Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins 
Blood  2009;113(19):4754-4762.
In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.
doi:10.1182/blood-2008-06-162693
PMCID: PMC2680375  PMID: 19109564
11.  Kv1.3 is the exclusive voltage-gated K+ channel of platelets and megakaryocytes: roles in membrane potential, Ca2+ signalling and platelet count 
The Journal of Physiology  2010;588(9):1399-1406.
A delayed rectifier voltage-gated K+ channel (Kv) represents the largest ionic conductance of platelets and megakaryocytes, but is undefined at the molecular level. Quantitative RT-PCR of all known Kv α and ancillary subunits showed that only Kv1.3 (KCNA3) is substantially expressed in human platelets. Furthermore, megakaryocytes from Kv1.3−/− mice or from wild-type mice exposed to the Kv1.3 blocker margatoxin completely lacked Kv currents and displayed substantially depolarised resting membrane potentials. In human platelets, margatoxin reduced the P2X1- and thromboxaneA2 receptor-evoked [Ca2+]i increases and delayed the onset of store-operated Ca2+ influx. Megakaryocyte development was normal in Kv1.3−/− mice, but the platelet count was increased, consistent with a role of Kv1.3 in apoptosis or decreased platelet activation. We conclude that Kv1.3 forms the Kv channel of the platelet and megakaryocyte, which sets the resting membrane potential, regulates agonist-evoked Ca2+ increases and influences circulating platelet numbers.
doi:10.1113/jphysiol.2010.188136
PMCID: PMC2876798  PMID: 20308249
12.  A trans-acting locus regulates an anti-viral expression network and type 1 diabetes risk 
Nature  2010;467(7314):460-464.
Combined analyses of gene networks and DNA sequence variation can provide new insights into the aetiology of common diseases. Here, we used integrated genome-wide approaches across seven rat tissues to identify gene networks and the loci underlying their regulation. We defined an interferon regulatory factor 7 (IRF7)1-driven inflammatory network (iDIN) enriched for viral response genes, which represents a molecular biomarker for macrophages and was regulated in multiple tissues by a locus on rat chromosome 15q25. At this locus, Epstein-Barr virus induced gene 2 (Ebi2 or Gpr183), which we localised to macrophages and is known to control B lymphocyte migration2,3, regulated the iDIN. The human chromosome 13q32 locus, orthologous to rat 15q25, controlled the human equivalent of iDIN, which was conserved in monocytes. For the macrophage-associated autoimmune disease type 1 diabetes (T1D) iDIN genes were more likely to associate with T1D susceptibility than randomly selected immune response genes (P = 8.85 × 10−6). The human locus controlling the iDIN, was associated with the risk of T1D at SNP rs9585056 (P = 7.0 × 10−10, odds ratio = 1.15), which was one of five SNPs in this region associated with EBI2 expression. These data implicate IRF7 network genes and their regulatory locus in the pathogenesis of T1D.
doi:10.1038/nature09386
PMCID: PMC3657719  PMID: 20827270
13.  Genome-Wide Haplotype Analysis of Cis Expression Quantitative Trait Loci in Monocytes 
PLoS Genetics  2013;9(1):e1003240.
In order to assess whether gene expression variability could be influenced by several SNPs acting in cis, either through additive or more complex haplotype effects, a systematic genome-wide search for cis haplotype expression quantitative trait loci (eQTL) was conducted in a sample of 758 individuals, part of the Cardiogenics Transcriptomic Study, for which genome-wide monocyte expression and GWAS data were available. 19,805 RNA probes were assessed for cis haplotypic regulation through investigation of ∼2,1×109 haplotypic combinations. 2,650 probes demonstrated haplotypic p-values >104-fold smaller than the best single SNP p-value. Replication of significant haplotype effects were tested for 412 probes for which SNPs (or proxies) that defined the detected haplotypes were available in the Gutenberg Health Study composed of 1,374 individuals. At the Bonferroni correction level of 1.2×10−4 (∼0.05/412), 193 haplotypic signals replicated. 1000G imputation was then conducted, and 105 haplotypic signals still remained more informative than imputed SNPs. In-depth analysis of these 105 cis eQTL revealed that at 76 loci genetic associations were compatible with additive effects of several SNPs, while for the 29 remaining regions data could be compatible with a more complex haplotypic pattern. As 24 of the 105 cis eQTL have previously been reported to be disease-associated loci, this work highlights the need for conducting haplotype-based and 1000G imputed cis eQTL analysis before commencing functional studies at disease-associated loci.
Author Summary
In order to assess whether gene expression variability could be influenced by the presence of more than one cis-acting SNP, we have conducted a systematic genome-wide search for haplotypic cis eQTL effects in a sample of 758 individuals and replicated the findings in an independent sample of 1,374 subjects. In both studies, genome-wide monocytes expression and genotype data were available. We identified 105 genes whose monocyte expression was under the influence of multiple cis-acting SNPs. About 75% of the detected genetic effects were related to independent additive SNP effects and the last quarter due to more complex haplotype effects. Of note, 24 of the genes identified to be affected by multiple cis eSNPs have been previously reported to reside at disease-associated loci. This could suggest that such multiple locus-specific genetic effects could contribute to the susceptibility to human diseases.
doi:10.1371/journal.pgen.1003240
PMCID: PMC3561129  PMID: 23382694
14.  Powerful Identification of Cis-regulatory SNPs in Human Primary Monocytes Using Allele-Specific Gene Expression 
PLoS ONE  2012;7(12):e52260.
A large number of genome-wide association studies have been performed during the past five years to identify associations between SNPs and human complex diseases and traits. The assignment of a functional role for the identified disease-associated SNP is not straight-forward. Genome-wide expression quantitative trait locus (eQTL) analysis is frequently used as the initial step to define a function while allele-specific gene expression (ASE) analysis has not yet gained a wide-spread use in disease mapping studies. We compared the power to identify cis-acting regulatory SNPs (cis-rSNPs) by genome-wide allele-specific gene expression (ASE) analysis with that of traditional expression quantitative trait locus (eQTL) mapping. Our study included 395 healthy blood donors for whom global gene expression profiles in circulating monocytes were determined by Illumina BeadArrays. ASE was assessed in a subset of these monocytes from 188 donors by quantitative genotyping of mRNA using a genome-wide panel of SNP markers. The performance of the two methods for detecting cis-rSNPs was evaluated by comparing associations between SNP genotypes and gene expression levels in sample sets of varying size. We found that up to 8-fold more samples are required for eQTL mapping to reach the same statistical power as that obtained by ASE analysis for the same rSNPs. The performance of ASE is insensitive to SNPs with low minor allele frequencies and detects a larger number of significantly associated rSNPs using the same sample size as eQTL mapping. An unequivocal conclusion from our comparison is that ASE analysis is more sensitive for detecting cis-rSNPs than standard eQTL mapping. Our study shows the potential of ASE mapping in tissue samples and primary cells which are difficult to obtain in large numbers.
doi:10.1371/journal.pone.0052260
PMCID: PMC3530574  PMID: 23300628
16.  Comprehensive Exploration of the Effects of miRNA SNPs on Monocyte Gene Expression 
PLoS ONE  2012;7(9):e45863.
We aimed to assess whether pri-miRNA SNPs (miSNPs) could influence monocyte gene expression, either through marginal association or by interacting with polymorphisms located in 3'UTR regions (3utrSNPs). We then conducted a genome-wide search for marginal miSNPs effects and pairwise miSNPs × 3utrSNPs interactions in a sample of 1,467 individuals for which genome-wide monocyte expression and genotype data were available. Statistical associations that survived multiple testing correction were tested for replication in an independent sample of 758 individuals with both monocyte gene expression and genotype data. In both studies, the hsa-mir-1279 rs1463335 was found to modulate in cis the expression of LYZ and in trans the expression of CNTN6, CTRC, COPZ2, KRT9, LRRFIP1, NOD1, PCDHA6, ST5 and TRAF3IP2 genes, supporting the role of hsa-mir-1279 as a regulator of several genes in monocytes. In addition, we identified two robust miSNPs × 3utrSNPs interactions, one involving HLA-DPB1 rs1042448 and hsa-mir-219-1 rs107822, the second the H1F0 rs1894644 and hsa-mir-659 rs5750504, modulating the expression of the associated genes.
As some of the aforementioned genes have previously been reported to reside at disease-associated loci, our findings provide novel arguments supporting the hypothesis that the genetic variability of miRNAs could also contribute to the susceptibility to human diseases.
doi:10.1371/journal.pone.0045863
PMCID: PMC3448685  PMID: 23029284
17.  A genome-wide association study identifies two loci associated with heart failure due to dilated cardiomyopathy 
European Heart Journal  2011;32(9):1065-1076.
Aims
Dilated cardiomyopathy (DCM) is a major cause of heart failure with a high familial recurrence risk. So far, the genetics of DCM remains largely unresolved. We conducted the first genome-wide association study (GWAS) to identify loci contributing to sporadic DCM.
Methods and results
One thousand one hundred and seventy-nine DCM patients and 1108 controls contributed to the discovery phase. Pools of DNA stratified on disease status, population, age, and gender were constituted and used for testing association of DCM with 517 382 single nucleotide polymorphisms (SNPs). Three DCM-associated SNPs were confirmed by individual genotyping (P < 5.0 10−7), and two of them, rs10927875 and rs2234962, were replicated in independent samples (1165 DCM patients and 1302 controls), with P-values of 0.002 and 0.009, respectively. rs10927875 maps to a region on chromosome 1p36.13 which encompasses several genes among which HSPB7 has been formerly suggested to be implicated in DCM. The second identified locus involves rs2234962, a non-synonymous SNP (c.T757C, p. C151R) located within the sequence of BAG3 on chromosome 10q26. To assess whether coding mutations of BAG3 might cause monogenic forms of the disease, we sequenced BAG3 exons in 168 independent index cases diagnosed with familial DCM and identified four truncating and two missense mutations. Each mutation was heterozygous, present in all genotyped relatives affected by the disease and absent in a control group of 347 healthy individuals, strongly suggesting that these mutations are causing the disease.
Conclusion
This GWAS identified two loci involved in sporadic DCM, one of them probably implicates BAG3. Our results show that rare mutations in BAG3 contribute to monogenic forms of the disease, while common variant(s) in the same gene are implicated in sporadic DCM.
doi:10.1093/eurheartj/ehr105
PMCID: PMC3086901  PMID: 21459883
Dilated cardiomyopathy; Heart failure; Genome wide association study; CLCNKA; HSPB7; BAG3
18.  Inheritance of coronary artery disease in men: an analysis of the role of the Y chromosome 
Lancet  2012;379(9819):915-922.
Summary
Background
A sexual dimorphism exists in the incidence and prevalence of coronary artery disease—men are more commonly affected than are age-matched women. We explored the role of the Y chromosome in coronary artery disease in the context of this sexual inequity.
Methods
We genotyped 11 markers of the male-specific region of the Y chromosome in 3233 biologically unrelated British men from three cohorts: the British Heart Foundation Family Heart Study (BHF-FHS), West of Scotland Coronary Prevention Study (WOSCOPS), and Cardiogenics Study. On the basis of this information, each Y chromosome was tracked back into one of 13 ancient lineages defined as haplogroups. We then examined associations between common Y chromosome haplogroups and the risk of coronary artery disease in cross-sectional BHF-FHS and prospective WOSCOPS. Finally, we undertook functional analysis of Y chromosome effects on monocyte and macrophage transcriptome in British men from the Cardiogenics Study.
Findings
Of nine haplogroups identified, two (R1b1b2 and I) accounted for roughly 90% of the Y chromosome variants among British men. Carriers of haplogroup I had about a 50% higher age-adjusted risk of coronary artery disease than did men with other Y chromosome lineages in BHF-FHS (odds ratio 1·75, 95% CI 1·20–2·54, p=0·004), WOSCOPS (1·45, 1·08–1·95, p=0·012), and joint analysis of both populations (1·56, 1·24–1·97, p=0·0002). The association between haplogroup I and increased risk of coronary artery disease was independent of traditional cardiovascular and socioeconomic risk factors. Analysis of macrophage transcriptome in the Cardiogenics Study revealed that 19 molecular pathways showing strong differential expression between men with haplogroup I and other lineages of the Y chromosome were interconnected by common genes related to inflammation and immunity, and that some of them have a strong relevance to atherosclerosis.
Interpretation
The human Y chromosome is associated with risk of coronary artery disease in men of European ancestry, possibly through interactions of immunity and inflammation.
Funding
British Heart Foundation; UK National Institute for Health Research; LEW Carty Charitable Fund; National Health and Medical Research Council of Australia; European Union 6th Framework Programme; Wellcome Trust.
doi:10.1016/S0140-6736(11)61453-0
PMCID: PMC3314981  PMID: 22325189
19.  Monocyte Gene Expression Signature of Patients with Early Onset Coronary Artery Disease 
PLoS ONE  2012;7(2):e32166.
The burden of cardiovascular disease (CVD) cannot be fully addressed by therapy targeting known pathophysiological pathways. Even with stringent control of all risk factors CVD events are only diminished by half. A number of additional pathways probably play a role in the development of CVD and might serve as novel therapeutic targets. Genome wide expression studies represent a powerful tool to identify such novel pathways. We compared the expression profiles in monocytes from twenty two young male patients with premature familial CAD with those from controls matched for age, sex and smoking status, without a family history of CVD. Since all patients were on statins and aspirin treatment, potentially affecting the expression of genes in monocytes, twelve controls were subsequently treated with simvastatin and aspirin for 6 and 2 weeks, respectively.
By whole genome expression arrays six genes were identified to have differential expression in the monocytes of patients versus controls; ABCA1, ABCG1 and RGS1 were downregulated in patients, whereas ADRB2, FOLR3 and GSTM1 were upregulated. Differential expression of all genes, apart from GSTM1, was confirmed by qPCR. Aspirin and statins altered gene expression of ABCG1 and ADBR2. All finding were validated in a second group of twenty four patients and controls. Differential expression of ABCA1, RSG1 and ADBR2 was replicated. In conclusion, we identified these 3 genes to be expressed differently in CAD cases which might play a role in the pathogenesis of atherosclerotic vascular disease.
doi:10.1371/journal.pone.0032166
PMCID: PMC3283726  PMID: 22363809
20.  Integrating Genome-Wide Genetic Variations and Monocyte Expression Data Reveals Trans-Regulated Gene Modules in Humans 
PLoS Genetics  2011;7(12):e1002367.
One major expectation from the transcriptome in humans is to characterize the biological basis of associations identified by genome-wide association studies. So far, few cis expression quantitative trait loci (eQTLs) have been reliably related to disease susceptibility. Trans-regulating mechanisms may play a more prominent role in disease susceptibility. We analyzed 12,808 genes detected in at least 5% of circulating monocyte samples from a population-based sample of 1,490 European unrelated subjects. We applied a method of extraction of expression patterns—independent component analysis—to identify sets of co-regulated genes. These patterns were then related to 675,350 SNPs to identify major trans-acting regulators. We detected three genomic regions significantly associated with co-regulated gene modules. Association of these loci with multiple expression traits was replicated in Cardiogenics, an independent study in which expression profiles of monocytes were available in 758 subjects. The locus 12q13 (lead SNP rs11171739), previously identified as a type 1 diabetes locus, was associated with a pattern including two cis eQTLs, RPS26 and SUOX, and 5 trans eQTLs, one of which (MADCAM1) is a potential candidate for mediating T1D susceptibility. The locus 12q24 (lead SNP rs653178), which has demonstrated extensive disease pleiotropy, including type 1 diabetes, hypertension, and celiac disease, was associated to a pattern strongly correlating to blood pressure level. The strongest trans eQTL in this pattern was CRIP1, a known marker of cellular proliferation in cancer. The locus 12q15 (lead SNP rs11177644) was associated with a pattern driven by two cis eQTLs, LYZ and YEATS4, and including 34 trans eQTLs, several of them tumor-related genes. This study shows that a method exploiting the structure of co-expressions among genes can help identify genomic regions involved in trans regulation of sets of genes and can provide clues for understanding the mechanisms linking genome-wide association loci to disease.
Author Summary
One major expectation from the transcriptome in humans is to help characterize the biological basis of associations identified by genome-wide association studies. Here, we take advantage of recent technical and methodological advances to examine the influence of natural genetic variability on >12,000 genes expressed in the monocyte, a blood cell playing a key role in immunity-related disorders and atherosclerosis. By examining 1,490 European population-based subjects, we identify three regions of the genome reproducibly associated with specific patterns of gene expression. Two of these regions overlap genetic variants previously known to be involved in the susceptibility to type 1 diabetes, celiac disease, and hypertension. Genes whose expression is modulated by these genetic variants may act as mediators in the causal relationship linking the variability of the genome to complex disease. These findings illustrate how integration of genetic and transcriptomic data at an epidemiological scale can help decipher the genetic basis of complex diseases.
doi:10.1371/journal.pgen.1002367
PMCID: PMC3228821  PMID: 22144904
21.  A genome-wide meta-analysis identifies 22 loci associated with eight hematological parameters in the HaemGen consortium 
Soranzo, Nicole | Spector, Tim D | Mangino, Massimo | Kühnel, Brigitte | Rendon, Augusto | Teumer, Alexander | Willenborg, Christina | Wright, Benjamin | Chen, Li | Li, Mingyao | Salo, Perttu | Voight, Benjamin F | Burns, Philippa | Laskowski, Roman A | Xue, Yali | Menzel, Stephan | Altshuler, David | Bradley, John R | Bumpstead, Suzannah | Burnett, Mary-Susan | Devaney, Joseph | Döring, Angela | Elosua, Roberto | Epstein, Stephen | Erber, Wendy | Falchi, Mario | Garner, Stephen F | Ghori, Mohammed J R | Goodall, Alison H | Gwilliam, Rhian | Hakonarson, Hakon H | Hall, Alistair S | Hammond, Naomi | Hengstenberg, Christian | Illig, Thomas | König, Inke R | Knouff, Christopher W | McPherson, Ruth | Melander, Olle | Mooser, Vincent | Nauck, Matthias | Nieminen, Markku S | O’Donnell, Christopher J | Peltonen, Leena | Potter, Simon C | Prokisch, Holger | Rader, Daniel J | Rice, Catherine M | Roberts, Robert | Salomaa, Veikko | Sambrook, Jennifer | Schreiber, Stefan | Schunkert, Heribert | Schwartz, Stephen M | Serbanovic-Canic, Jovana | Sinisalo, Juha | Siscovick, David S. | Stark, Klaus | Surakka, Ida | Stephens, Jonathan | Thompson, John R | Völker, Uwe | Völzke, Henry | Watkins, Nicholas A | Wells, George A | Wichmann, H-Erich | Van Heel, David A | Tyler-Smith, Chris | Thein, Swee Lay | Kathiresan, Sekar | Perola, Markus | Reilly, Muredach P | Stewart, Alexandre F R | Erdmann, Jeanette | Samani, Nilesh J | Meisinger, Christa | Greinacher, Andreas | Deloukas, Panos | Ouwehand, Willem H | Gieger, Christian
Nature genetics  2009;41(11):1182-1190.
The number and volume of cells in the blood affect a wide range of disorders including cancer and cardiovascular, metabolic, infectious and immune conditions. We consider here the genetic variation in eight clinically relevant hematological parameters, including hemoglobin levels, red and white blood cell counts and platelet counts and volume. We describe common variants within 22 genetic loci reproducibly associated with these hematological parameters in 13,943 samples from six European population-based studies, including 6 associated with red blood cell parameters, 15 associated with platelet parameters and 1 associated with total white blood cell count. We further identified a long-range haplotype at 12q24 associated with coronary artery disease in 9,479 cases and 10,527 controls. We show that this haplotype demonstrates extensive disease pleiotropy, as it contains known risk loci for type 1 diabetes, hypertension and celiac disease and has been spread by a selective sweep specific to European and geographically nearby populations.
doi:10.1038/ng.467
PMCID: PMC3108459  PMID: 19820697
22.  A novel variant on chromosome 7q22.3 associated with mean platelet volume, counts, and function 
Blood  2009;113(16):3831-3837.
Mean platelet volume (MPV) and platelet count (PLT) are highly heritable and tightly regulated traits. We performed a genome-wide association study for MPV and identified one SNP, rs342293, as having highly significant and reproducible association with MPV (per-G allele effect 0.016 ± 0.001 log fL; P < 1.08 × 10−24) and PLT (per-G effect −4.55 ± 0.80 109/L; P < 7.19 × 10−8) in 8586 healthy subjects. Whole-genome expression analysis in the 1-MB region showed a significant association with platelet transcript levels for PIK3CG (n = 35; P = .047). The G allele at rs342293 was also associated with decreased binding of annexin V to platelets activated with collagen-related peptide (n = 84; P = .003). The region 7q22.3 identifies the first QTL influencing platelet volume, counts, and function in healthy subjects. Notably, the association signal maps to a chromosome region implicated in myeloid malignancies, indicating this site as an important regulatory site for hematopoiesis. The identification of loci regulating MPV by this and other studies will increase our insight in the processes of megakaryopoiesis and proplatelet formation, and it may aid the identification of genes that are somatically mutated in essential thrombocytosis.
doi:10.1182/blood-2008-10-184234
PMCID: PMC2714088  PMID: 19221038

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