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1.  Reactome - a curated knowledgebase of biological pathways: megakaryocytes and platelets 
The platelet field is undergoing a radical transformation from reductionist simplification to large scale integration. Following the era of simplification whereby biological processes were dissected at the molecular and atomic level, new technologies have now generated an overwhelming flow of information that can only be comprehended in an integrated approach. High throughput analyses of transcription and translation in megakaryocytes and platelets, individual analyses of membranes and secretory granules, the clustering of pathways for platelet activation and inhibition in signalosomes all add to a complexity that requires platforms for knowledge accumulation. Here we introduce Reactome, a curated knowledgebase of biological pathways with extensive coverage of pathways relevant to megakaryocytes, platelets and haemostasis. This resource is compared with other data resources for platelets, e.g. the Platelet Web.
PMCID: PMC3578965  PMID: 22985186
2.  Comparison of Methods for Competitive Tests of Pathway Analysis 
PLoS ONE  2012;7(7):e41018.
It has been suggested that pathway analysis can complement single-SNP analysis in exploring genomewide association data. Pathway analysis incorporates the available biological knowledge of genes and SNPs and is expected to improve the chances of revealing the underlying genetic architecture of complex traits. Methods for pathway analysis can be classified as competitive (enrichment) or self-contained (association) according to the hypothesis tested. Although association tests are statistically more powerful than enrichment tests they can be difficult to calibrate because biases in analysis accumulate across multiple SNPs or genes. Furthermore, enrichment tests can be more scientifically relevant than association tests, as they detect pathways with relatively more evidence for association than the remaining genes. Here we show how some well known association tests can be simply adapted to test for enrichment, and compare their performance to some established enrichment tests. We propose versions of the Adaptive Rank Truncated Product (ARTP), Tail Strength Measure and Fisher’s combination of p-values for testing the enrichment null hypothesis. We compare the behaviour of these proposed methods with the established Hypergeometric Test and Gene-Set Enrichment Analysis (GSEA). The results of the simulation study show that the modified version of the ARTP method has generally the best performance across the situations considered. The methods were also applied for finding enriched pathways for body mass index (BMI) and platelet function phenotypes. The pathway analysis of BMI identified the Vasoactive Intestinal Peptide pathway as significantly associated with BMI. This pathway has been previously reported as associated with BMI and the risk of obesity. The ARTP method was the method that identified the largest number of enriched pathways across all tested pathway databases and phenotypes. The simulation and data application results are in agreement with previous work on association tests and suggests that the ARTP should be preferred for both enrichment and association testing.
PMCID: PMC3409204  PMID: 22859961
3.  Inherited Variation in Vitamin D Genes Is Associated With Predisposition to Autoimmune Disease Type 1 Diabetes 
Diabetes  2011;60(5):1624-1631.
Vitamin D deficiency (25-hydroxyvitamin D [25(OH)D] <50 nmol/L) is commonly reported in both children and adults worldwide, and growing evidence indicates that vitamin D deficiency is associated with many extraskeletal chronic disorders, including the autoimmune diseases type 1 diabetes and multiple sclerosis.
We measured 25(OH)D concentrations in 720 case and 2,610 control plasma samples and genotyped single nucleotide polymorphisms from seven vitamin D metabolism genes in 8,517 case, 10,438 control, and 1,933 family samples. We tested genetic variants influencing 25(OH)D metabolism for an association with both circulating 25(OH)D concentrations and disease status.
Type 1 diabetic patients have lower circulating levels of 25(OH)D than similarly aged subjects from the British population. Only 4.3 and 18.6% of type 1 diabetic patients reached optimal levels (≥75 nmol/L) of 25(OH)D for bone health in the winter and summer, respectively. We replicated the associations of four vitamin D metabolism genes (GC, DHCR7, CYP2R1, and CYP24A1) with 25(OH)D in control subjects. In addition to the previously reported association between type 1 diabetes and CYP27B1 (P = 1.4 × 10−4), we obtained consistent evidence of type 1 diabetes being associated with DHCR7 (P = 1.2 × 10−3) and CYP2R1 (P = 3.0 × 10−3).
Circulating levels of 25(OH)D in children and adolescents with type 1 diabetes vary seasonally and are under the same genetic control as in the general population but are much lower. Three key 25(OH)D metabolism genes show consistent evidence of association with type 1 diabetes risk, indicating a genetic etiological role for vitamin D deficiency in type 1 diabetes.
PMCID: PMC3292339  PMID: 21441443
4.  Image-based characterization of thrombus formation in time-lapse DIC microscopy 
Medical Image Analysis  2012;16(4):915-931.
Graphical abstract
► Automatized characterization of thrombus formation in time-lapse microscopy. ► Novel energy model for segmentation of multiple dynamic regions. ► Novel algorithm for the joint segmentation of thrombus and aortic regions. ► Exhaustive validation on synthetic and real microscopic data.
The characterization of thrombus formation in time-lapse DIC microscopy is of increased interest for identifying genes which account for atherothrombosis and coronary artery diseases (CADs). In particular, we are interested in large-scale studies on zebrafish, which result in large amount of data, and require automatic processing. In this work, we present an image-based solution for the automatized extraction of parameters quantifying the temporal development of thrombotic plugs. Our system is based on the joint segmentation of thrombotic and aortic regions over time. This task is made difficult by the low contrast and the high dynamic conditions observed in vivo DIC microscopic scenes. Our key idea is to perform this segmentation by distinguishing the different motion patterns in image time series rather than by solving standard image segmentation tasks in each image frame. Thus, we are able to compensate for the poor imaging conditions. We model motion patterns by energies based on the idea of dynamic textures, and regularize the model by two prior energies on the shape of the aortic region and on the topological relationship between the thrombus and the aorta. We demonstrate the performance of our segmentation algorithm by qualitative and quantitative experiments on synthetic examples as well as on real in vivo microscopic sequences.
PMCID: PMC3740235  PMID: 22482997
Time-lapse microscopy; DIC microscopy; Motion-segmentation; Dynamic texture; Tracking
5.  Inheritance of coronary artery disease in men: an analysis of the role of the Y chromosome 
Lancet  2012;379(9819):915-922.
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.
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.
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.
The human Y chromosome is associated with risk of coronary artery disease in men of European ancestry, possibly through interactions of immunity and inflammation.
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.
PMCID: PMC3314981  PMID: 22325189
6.  Genome-wide Analysis of Simultaneous GATA1/2, RUNX1, FLI1, and SCL Binding in Megakaryocytes Identifies Hematopoietic Regulators 
Developmental Cell  2011;20(5):597-609.
Hematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for the five key hematopoietic transcription factors—GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL—in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes.
► Genome-wide TF binding analysis in primary human megakaryocyte ► Regulatory network reconstruction from TF, epigenetic, and expression data ► Simultaneous binding of five TFs marks hematopoietic regulators ► Five-factor binding reveals eight genes essential for zebrafish blood development
PMCID: PMC3145975  PMID: 21571218
7.  Cell-specific protein phenotypes for the autoimmune locus IL2RA using a genotype-selectable human bioresource 
Nature genetics  2009;41(9):1011-1015.
Genome-wide association (GWA) studies have identified over 300 regions associated with more than 70 common diseases1. However, identifying causal genes within an associated region remains a major challenge1,2. One approach to resolving causal genes is through the dissection of gene-phenotype correlations. Here we use polychromatic flow cytometry to show that differences in surface expression of interleukin-2 (IL-2) receptor alpha-chain (IL-2RA, or CD25) protein are restricted to particular immune cell types and correlate with several haplotypes in the IL2RA region that have previously been associated to the autoimmune diseases type 1 diabetes (T1D) and multiple sclerosis2-4. We confirm our strongest gene-phenotype correlation at the RNA level by allele-specific expression (ASE). We also define key parameters for the design and implementation of post-GWA gene-phenotype investigations, and demonstrate the usefulness of a large bioresource of genotype-selectable normal donors from whom fresh, primary cells can be analyzed.
PMCID: PMC2749506  PMID: 19701192
8.  Adiposity-Related Heterogeneity in Patterns of Type 2 Diabetes Susceptibility Observed in Genome-Wide Association Data 
Diabetes  2009;58(2):505-510.
OBJECTIVE—This study examined how differences in the BMI distribution of type 2 diabetic case subjects affected genome-wide patterns of type 2 diabetes association and considered the implications for the etiological heterogeneity of type 2 diabetes.
RESEARCH DESIGN AND METHODS—We reanalyzed data from the Wellcome Trust Case Control Consortium genome-wide association scan (1,924 case subjects, 2,938 control subjects: 393,453 single-nucleotide polymorphisms [SNPs]) after stratifying case subjects (into “obese” and “nonobese”) according to median BMI (30.2 kg/m2). Replication of signals in which alternative case-ascertainment strategies generated marked effect size heterogeneity in type 2 diabetes association signal was sought in additional samples.
RESULTS—In the “obese-type 2 diabetes” scan, FTO variants had the strongest type 2 diabetes effect (rs8050136: relative risk [RR] 1.49 [95% CI 1.34–1.66], P = 1.3 × 10−13), with only weak evidence for TCF7L2 (rs7901695 RR 1.21 [1.09–1.35], P = 0.001). This situation was reversed in the “nonobese” scan, with FTO association undetectable (RR 1.07 [0.97–1.19], P = 0.19) and TCF7L2 predominant (RR 1.53 [1.37–1.71], P = 1.3 × 10−14). These patterns, confirmed by replication, generated strong combined evidence for between-stratum effect size heterogeneity (FTO: PDIFF = 1.4 × 10−7; TCF7L2: PDIFF = 4.0 × 10−6). Other signals displaying evidence of effect size heterogeneity in the genome-wide analyses (on chromosomes 3, 12, 15, and 18) did not replicate. Analysis of the current list of type 2 diabetes susceptibility variants revealed nominal evidence for effect size heterogeneity for the SLC30A8 locus alone (RRobese 1.08 [1.01–1.15]; RRnonobese 1.18 [1.10–1.27]: PDIFF = 0.04).
CONCLUSIONS—This study demonstrates the impact of differences in case ascertainment on the power to detect and replicate genetic associations in genome-wide association studies. These data reinforce the notion that there is substantial etiological heterogeneity within type 2 diabetes.
PMCID: PMC2628627  PMID: 19056611
9.  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.
PMCID: PMC2791847  PMID: 19850043
Platelets; PECAM-1; Collagen; ADP; Thrombin; Thrombus
10.  Identification of Tspan9 as a novel platelet tetraspanin and the collagen receptor GPVI as a component of tetraspanin microdomains 
Biochemical Journal  2008;417(Pt 1):391-400.
Platelets are essential for wound healing and inflammatory processes, but can also play a deleterious role by causing heart attack and stroke. Normal platelet activation is dependent on tetraspanins, a superfamily of glycoproteins that function as ‘organisers’ of cell membranes by recruiting other receptors and signalling proteins into tetraspanin-enriched microdomains. However, our understanding of how tetraspanin microdomains regulate platelets is hindered by the fact that only four of the 33 mammalian tetraspanins have been identified in platelets. This is because of a lack of antibodies to most tetraspanins and difficulties in measuring mRNA, due to low levels in this anucleate cell. To identify potentially platelet-expressed tetraspanins, mRNA was measured in their nucleated progenitor cell, the megakaryocyte, using serial analysis of gene expression and DNA microarrays. Amongst 19 tetraspanins identified in megakaryocytes, Tspan9, a previously uncharacterized tetraspanin, was relatively specific to these cells. Through generating the first Tspan9 antibodies, Tspan9 expression was found to be tightly regulated in platelets. The relative levels of CD9, CD151, Tspan9 and CD63 were 100, 14, 6 and 2 respectively. Since CD9 was expressed at 49000 cell surface copies per platelet, this suggested a copy number of 2800 Tspan9 molecules. Finally, Tspan9 was shown to be a component of tetraspanin microdomains that included the collagen receptor GPVI (glycoprotein VI) and integrin α6β1, but not the von Willebrand receptor GPIbα or the integrins αIIbβ3 or α2β1. These findings suggest a role for Tspan9 in regulating platelet function in concert with other platelet tetraspanins and their associated proteins.
PMCID: PMC2652832  PMID: 18795891
glycoprotein VI (GPVI); megakaryocyte; membrane microdomain; platelet; tetraspanin; Tspan9; DIC, differential interference contrast; ECL, enhanced chemiluminescence; GP, glycoprotein; HEK-293T cells, HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40); PNGase F, peptide N-glycosidase F; RT–PCR, reverse transcription–PCR; SAGE, serial analysis of gene expression
11.  Developing recombinant HPA-1a–specific antibodies with abrogated Fcγ receptor binding for the treatment of fetomaternal alloimmune thrombocytopenia 
The Journal of Clinical Investigation  2008;118(8):2929-2938.
Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal generation of antibodies specific for paternal platelet antigens and can lead to fetal intracranial hemorrhage. A SNP in the gene encoding integrin β3 causes a clinically important maternal-paternal antigenic difference; Leu33 generates the human platelet antigen 1a (HPA-1a), whereas Pro33 generates HPA-1b. As a potential treatment to prevent fetal intracranial hemorrhage in HPA-1a alloimmunized pregnancies, we generated an antibody that blocks the binding of maternal HPA-1a–specific antibodies to fetal HPA-1a1b platelets by combining a high-affinity human HPA-1a–specific scFv (B2) with an IgG1 constant region modified to minimize Fcγ receptor–dependent platelet destruction (G1Δnab). B2G1Δnab saturated HPA-1a+ platelets and substantially inhibited binding of clinical HPA-1a–specific sera to HPA-1a+ platelets. The response of monocytes to B2G1Δnab-sensitized platelets was substantially less than their response to unmodified B2G1, as measured by chemiluminescence. In addition, B2G1Δnab inhibited chemiluminescence induced by B2G1 and HPA-1a–specific sera. In a chimeric mouse model, B2G1 and polyclonal Ig preparations from clinical HPA-1a–specific sera reduced circulating HPA-1a+ platelets, concomitant with transient thrombocytopenia. As the Δnab constant region is uninformative in mice, F(ab′)2 B2G1 was used as a proof of principle blocking antibody and prevented the in vivo platelet destruction seen with B2G1 and polyclonal HPA-1a–specific antibodies. These results provide rationale for human clinical studies.
PMCID: PMC2483683  PMID: 18654666
12.  Platelet genomics and proteomics in human health and disease 
Journal of Clinical Investigation  2005;115(12):3370-3377.
Proteomic and genomic technologies provide powerful tools for characterizing the multitude of events that occur in the anucleate platelet. These technologies are beginning to define the complete platelet transcriptome and proteome as well as the protein-protein interactions critical for platelet function. The integration of these results provides the opportunity to identify those proteins involved in discrete facets of platelet function. Here we summarize the findings of platelet proteome and transcriptome studies and their application to diseases of platelet function.
PMCID: PMC1297260  PMID: 16322782
13.  Mutation in KERA Identified by Linkage Analysis and Targeted Resequencing in a Pedigree with Premature Atherosclerosis 
PLoS ONE  2014;9(5):e98289.
Genetic factors explain a proportion of the inter-individual variation in the risk for atherosclerotic events, but the genetic basis of atherosclerosis and atherothrombosis in families with Mendelian forms of premature atherosclerosis is incompletely understood. We set out to unravel the molecular pathology in a large kindred with an autosomal dominant inherited form of premature atherosclerosis.
Methods and Results
Parametric linkage analysis was performed in a pedigree comprising 4 generations, of which a total of 11 members suffered from premature vascular events. A parametric LOD-score of 3.31 was observed for a 4.4 Mb interval on chromosome 12. Upon sequencing, a non-synonymous variant in KERA (c.920C>G; p.Ser307Cys) was identified. The variant was absent from nearly 28,000 individuals, including 2,571 patients with premature atherosclerosis. KERA, a proteoglycan protein, was expressed in lipid-rich areas of human atherosclerotic lesions, but not in healthy arterial specimens. Moreover, KERA expression in plaques was significantly associated with plaque size in a carotid-collar Apoe−/− mice (r2 = 0.69; p<0.0001).
A rare variant in KERA was identified in a large kindred with premature atherosclerosis. The identification of KERA in atherosclerotic plaque specimen in humans and mice lends support to its potential role in atherosclerosis.
PMCID: PMC4039470  PMID: 24879339
14.  Single Nucleotide Variants in the Protein C Pathway and Mortality in Dialysis Patients 
PLoS ONE  2014;9(5):e97251.
The protein C pathway plays an important role in the maintenance of endothelial barrier function and in the inflammatory and coagulant processes that are characteristic of patients on dialysis. We investigated whether common single nucleotide variants (SNV) in genes encoding protein C pathway components were associated with all-cause 5 years mortality risk in dialysis patients.
Single nucleotides variants in the factor V gene (F5 rs6025; factor V Leiden), the thrombomodulin gene (THBD rs1042580), the protein C gene (PROC rs1799808 and 1799809) and the endothelial protein C receptor gene (PROCR rs867186, rs2069951, and rs2069952) were genotyped in 1070 dialysis patients from the NEtherlands COoperative Study on the Adequacy of Dialysis (NECOSAD) cohort) and in 1243 dialysis patients from the German 4D cohort.
Factor V Leiden was associated with a 1.5-fold (95% CI 1.1–1.9) increased 5-year all-cause mortality risk and carriers of the AG/GG genotypes of the PROC rs1799809 had a 1.2-fold (95% CI 1.0–1.4) increased 5-year all-cause mortality risk. The other SNVs in THBD, PROC, and PROCR were not associated with 5-years mortality.
Our study suggests that factor V Leiden and PROC rs1799809 contributes to an increased mortality risk in dialysis patients.
PMCID: PMC4016291  PMID: 24816905
15.  The Human Phenotype Ontology project: linking molecular biology and disease through phenotype data 
Nucleic Acids Research  2013;42(D1):D966-D974.
The Human Phenotype Ontology (HPO) project, available at, provides a structured, comprehensive and well-defined set of 10,088 classes (terms) describing human phenotypic abnormalities and 13,326 subclass relations between the HPO classes. In addition we have developed logical definitions for 46% of all HPO classes using terms from ontologies for anatomy, cell types, function, embryology, pathology and other domains. This allows interoperability with several resources, especially those containing phenotype information on model organisms such as mouse and zebrafish. Here we describe the updated HPO database, which provides annotations of 7,278 human hereditary syndromes listed in OMIM, Orphanet and DECIPHER to classes of the HPO. Various meta-attributes such as frequency, references and negations are associated with each annotation. Several large-scale projects worldwide utilize the HPO for describing phenotype information in their datasets. We have therefore generated equivalence mappings to other phenotype vocabularies such as LDDB, Orphanet, MedDRA, UMLS and phenoDB, allowing integration of existing datasets and interoperability with multiple biomedical resources. We have created various ways to access the HPO database content using flat files, a MySQL database, and Web-based tools. All data and documentation on the HPO project can be found online.
PMCID: PMC3965098  PMID: 24217912
16.  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.
PMCID: PMC3657719  PMID: 20827270
17.  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.
PMCID: PMC3561129  PMID: 23382694
18.  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.
PMCID: PMC3530574  PMID: 23300628
20.  Inheritance of low-frequency regulatory SNPs and a rare null mutation in exon-junction complex subunit RBM8A causes TAR 
Nature genetics  2012;44(4):435-S2.
The exon-junction complex (EJC) performs essential RNA processing tasks1-5. Here, we describe the first human disorder, Thrombocytopenia with Absent Radii6 (TAR), caused by deficiency in one of the four EJC subunits. A compound inheritance mechanism of a rare null allele and one of two low-frequency SNPs in the regulatory regions of RBM8A, encoding the Y14 subunit of EJC, causes TAR. We found that this mechanism explained 53 of 55 cases (P<5×10−228) with the rare congenital malformation syndrome. Fifty-one of those 53 carried a previously associated7 submicroscopic deletion of 1q21.1; two carried a truncation or frameshift null mutation in RBM8A. We show that the two regulatory SNPs result in reduction of RBM8A transcription in vitro and that Y14 expression is reduced in platelets from TAR cases. Our data implicate Y14 insufficiency, and presumably EJC defect, as the cause of TAR syndrome.
PMCID: PMC3428915  PMID: 22366785
21.  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.
PMCID: PMC3448685  PMID: 23029284
22.  Exome sequencing identifies NBEAL2 as the causative gene for Gray Platelet Syndrome 
Nature genetics  2011;43(8):735-737.
Gray platelet syndrome (GPS) is a predominantly recessive platelet disorder characterized by a mild thrombocytopenia with large platelets and a paucity of α-granules; these abnormalities cause mostly moderate but in rare cases severe bleeding. We sequenced the exomes of four unrelated cases and identified as the causative gene NBEAL2, a gene with previously unknown function but a member of a gene family involved in granule development. Silencing of nbeal2 in zebrafish abrogated thrombocyte formation.
PMCID: PMC3428934  PMID: 21765411
23.  Homocysteine and Coronary Heart Disease: Meta-analysis of MTHFR Case-Control Studies, Avoiding Publication Bias 
PLoS Medicine  2012;9(2):e1001177.
Robert Clarke and colleagues conduct a meta-analysis of unpublished datasets to examine the causal relationship between elevation of homocysteine levels in the blood and the risk of coronary heart disease. Their data suggest that an increase in homocysteine levels is not likely to result in an increase in risk of coronary heart disease.
Moderately elevated blood levels of homocysteine are weakly correlated with coronary heart disease (CHD) risk, but causality remains uncertain. When folate levels are low, the TT genotype of the common C677T polymorphism (rs1801133) of the methylene tetrahydrofolate reductase gene (MTHFR) appreciably increases homocysteine levels, so “Mendelian randomization” studies using this variant as an instrumental variable could help test causality.
Methods and Findings
Nineteen unpublished datasets were obtained (total 48,175 CHD cases and 67,961 controls) in which multiple genetic variants had been measured, including MTHFR C677T. These datasets did not include measurements of blood homocysteine, but homocysteine levels would be expected to be about 20% higher with TT than with CC genotype in the populations studied. In meta-analyses of these unpublished datasets, the case-control CHD odds ratio (OR) and 95% CI comparing TT versus CC homozygotes was 1.02 (0.98–1.07; p = 0.28) overall, and 1.01 (0.95–1.07) in unsupplemented low-folate populations. By contrast, in a slightly updated meta-analysis of the 86 published studies (28,617 CHD cases and 41,857 controls), the OR was 1.15 (1.09–1.21), significantly discrepant (p = 0.001) with the OR in the unpublished datasets. Within the meta-analysis of published studies, the OR was 1.12 (1.04–1.21) in the 14 larger studies (those with variance of log OR<0.05; total 13,119 cases) and 1.18 (1.09–1.28) in the 72 smaller ones (total 15,498 cases).
The CI for the overall result from large unpublished datasets shows lifelong moderate homocysteine elevation has little or no effect on CHD. The discrepant overall result from previously published studies reflects publication bias or methodological problems.
Please see later in the article for the Editors' Summary
Editors' Summary
Coronary heart disease (CHD) is the leading cause of death among adults in developed countries. With age, fatty deposits (atherosclerotic plaques) coat the walls of the coronary arteries, the blood vessels that supply the heart with oxygen and nutrients. The resultant restriction of the heart's blood supply causes shortness of breath, angina (chest pains that are usually relieved by rest), and sometimes fatal heart attacks. Many established risk factors for CHD, including smoking, physical inactivity, being overweight, and eating a fat-rich diet, can be modified by lifestyle changes. Another possible modifiable risk factor for CHD is a high blood level of the amino acid homocysteine. Methylene tetrahydofolate reductase, which is encoded by the MTHFR gene, uses folate to break down and remove homocysteine so fortification of cereals with folate can reduce population homocysteine blood levels. Pooled results from prospective observational studies that have looked for an association between homocysteine levels and later development of CHD suggest that the reduction in homocysteine levels that can be achieved by folate supplementation is associated with an 11% lower CHD risk.
Why Was This Study Done?
Prospective observational studies cannot prove that high homocysteine levels cause CHD because of confounding, the potential presence of other unknown shared characteristics that really cause CHD. However, an approach called “Mendelian randomization” can test whether high blood homocysteine causes CHD. A common genetic variant of the MTHFR gene—the C677T polymorphism—reduces MTHFR efficiency so TT homozygotes (individuals in whom both copies of the MTHFR gene have the nucleotide thymine at position 677; the human genome contains two copies of most genes) have 25% higher blood homocysteine levels than CC homozygotes. In meta-analyses (statistical pooling of the results of several studies) of published Mendelian randomized studies, TT homozygotes have a higher CHD risk than CC homozygotes. Because gene variants are inherited randomly, they are not subject to confounding, so this result suggests that high blood homocysteine causes CHD. But what if only Mendelian randomization studies that found an association have been published? Such publication bias would affect this aggregate result. Here, the researchers investigate the association of the MTHFR C677T polymorphism with CHD in unpublished datasets that have analyzed this polymorphism incidentally during other genetic studies.
What Did the Researchers Do and Find?
The researchers obtained 19 unpublished datasets that contained data on the MTHFR C677T polymorphism in thousands of people with and without CHD. Meta-analysis of these datasets indicates that the excess CHD risk in TT homozygotes compared to CC homozygotes was 2% (much lower than predicted from the prospective observational studies), a nonsignificant difference (that is, it could have occurred by chance). When the probable folate status of the study populations (based on when national folic acid fortification legislation came into effect) was taken into account, there was still no evidence that TT homozygotes had an excess CHD risk. By contrast, in an updated meta-analysis of 86 published studies of the association of the polymorphism with CHD, the excess CHD risk in TT homozygotes compared to CC homozygotes was 15%. Finally, in a meta-analysis of randomized trials on the use of vitamin B supplements for homocysteine reduction, folate supplementation had no significant effect on the 5-year incidence of CHD.
What Do These Findings Mean?
These analyses of unpublished datasets are consistent with lifelong moderate elevation of homocysteine levels having no significant effect on CHD risk. In other words, these findings indicate that circulating homocysteine levels within the normal range are not causally related to CHD risk. The meta-analysis of the randomized trials of folate supplementation also supports this conclusion. So why is there a discrepancy between these findings and those of meta-analyses of published Mendelian randomization studies? The discrepancy is too large to be dismissed as a chance finding, suggest the researchers, but could be the result of publication bias—some studies might have been prioritized for publication because of the positive nature of their results whereas the unpublished datasets used in this study would not have been affected by any failure to publish null results. Overall, these findings reveal a serious example of publication bias and argue against the use of folate supplements as a means of reducing CHD risk.
Additional Information
Please access these Web sites via the online version of this summary at
The American Heart Association provides information about CHD and tips on keeping the heart healthy; it also provides information on homocysteine, folic acid, and CHD, general information on supplements and heart health, and personal stories about CHD
The UK National Health Service Choices website provides information about CHD, including personal stories about CHD
Information is available from the British Heart Foundation on heart disease and keeping the heart healthy
The US National Heart Lung and Blood Institute also provides information on CHD (in English and Spanish)
MedlinePlus provides links to many other sources of information on CHD (in English and Spanish)
Wikipedia has a page on Mendelian randomization (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC3283559  PMID: 22363213
24.  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.
PMCID: PMC3283726  PMID: 22363809
25.  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.
PMCID: PMC3228821  PMID: 22144904

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