Hariklia Eleftherohorinou

Pathway analysis of GWAS provides new insights into genetic susceptibility to 3 inflammatory diseases.

Although the introduction of genome-wide association studies (GWAS) have greatly increased the number of genes associated with common diseases, only a small proportion of the predicted genetic contribution has so far been elucidated. Studying the cumulative variation of polymorphisms in multiple genes acting in functional pathways may provide a complementary approach to the more common single SNP association approach in understanding genetic determinants of common disease. We developed a novel pathway-based method to assess the combined contribution of multiple genetic variants acting within canonical biological pathways and applied it to data from 14,000 UK individuals with 7 common diseases. We tested inflammatory pathways for association with Crohns disease (CD), rheumatoid arthritis (RA) and type 1 diabetes (T1D) with 4 non-inflammatory diseases as controls. Using a variable selection algorithm, we identified variants responsible for the pathway association and evaluated their use for disease prediction using a 10 fold cross-validation framework in order to calculate out-of-sample area under the Receiver Operating Curve (AUC). The generalisability of these predictive models was tested on an independent birth cohort from Northern Finland. Multiple canonical inflammatory pathways showed highly significant associations (p 10−3–10−20) with CD, T1D and RA. Variable selection identified on average a set of 205 SNPs (149 genes) for T1D, 350 SNPs (189 genes) for RA and 493 SNPs (277 genes) for CD. The pattern of polymorphisms at these SNPS were found to be highly predictive of T1D (91% AUC) and RA (85% AUC), and weakly predictive of CD (60% AUC). The predictive ability of the T1D model (without any parameter refitting) had good predictive ability (79% AUC) in the Finnish cohort. Our analysis suggests that genetic contribution to common inflammatory diseases operates through multiple genes interacting in functional pathways.

Transforming Growth Factor-β Signaling Pathway in Patients With Kawasaki Disease

Background—Transforming growth factor (TGF)-&bgr; is a multifunctional peptide that is important in T-cell activation and cardiovascular remodeling, both of which are important features of Kawasaki disease (KD). We postulated that variation in TGF-&bgr; signaling might be important in KD susceptibility and disease outcome. Methods and Results—We investigated genetic variation in 15 genes belonging to the TGF-&bgr; pathway in a total of 771 KD subjects of mainly European descent from the United States, the United Kingdom, Australia, and the Netherlands. We analyzed transcript abundance patterns using microarray and reverse transcriptase–polymerase chain reaction for these same genes, and measured TGF-&bgr;2 protein levels in plasma. Genetic variants in TGFB2, TGFBR2, and SMAD3 and their haplotypes were consistently and reproducibly associated with KD susceptibility, coronary artery aneurysm formation, aortic root dilatation, and intravenous immunoglobulin treatment response in different cohorts. A SMAD3 haplotype associated with KD susceptibility replicated in 2 independent cohorts and an intronic single nucleotide polymorphism in a separate haplotype block was also strongly associated (A/G, rs4776338) (P=0.000022; odds ratio, 1.50; 95% confidence interval, 1.25 to 1.81). Pathway analysis using all 15 genes further confirmed the importance of the TGF-&bgr; pathway in KD pathogenesis. Whole-blood transcript abundance for these genes and TGF-&bgr;2 plasma protein levels changed dynamically over the course of the illness. Conclusions—These studies suggest that genetic variation in the TGF-&bgr; pathway influences KD susceptibility, disease outcome, and response to therapy, and that aortic root and coronary artery Z scores can be used for phenotype/genotype analyses. Analysis of transcript abundance and protein levels further support the importance of this pathway in KD pathogenesis.

Pathway-driven gene stability selection of two rheumatoid arthritis GWAS identifies and validates new susceptibility genes in receptor mediated signalling pathways

Rheumatoid arthritis (RA) is the commonest chronic, systemic, inflammatory disorder affecting ∼1% of the world population. It has a strong genetic component and a growing number of associated genes have been discovered in genome-wide association studies (GWAS), which nevertheless only account for 23% of the total genetic risk. We aimed to identify additional susceptibility loci through the analysis of GWAS in the context of biological function. We bridge the gap between pathway and gene-oriented analyses of GWAS, by introducing a pathway-driven gene stability-selection methodology that identifies potential causal genes in the top-associated disease pathways that may be driving the pathway association signals. We analysed the WTCCC and the NARAC studies of ∼5000 and ∼2000 subjects, respectively. We examined 700 pathways comprising ∼8000 genes. Ranking pathways by significance revealed that the NARAC top-ranked ∼6% laid within the top 10% of WTCCC. Gene selection on those pathways identified 58 genes in WTCCC and 61 in NARAC; 21 of those were common (P(overlap)< 10(-21)), of which 16 were novel discoveries. Among the identified genes, we validated 10 known RA associations in WTCCC and 13 in NARAC, not discovered using single-SNP approaches on the same data. Gene ontology functional enrichment analysis on the identified genes showed significant over-representation of signalling activity (P< 10(-29)) in both studies. Our findings suggest a novel model of RA genetic predisposition, which involves cell-membrane receptors and genes in second messenger signalling systems, in addition to genes that regulate immune responses, which have been the focus of interest previously.

Diagnostic Test Accuracy of a 2-Transcript Host RNA Signature for Discriminating Bacterial vs Viral Infection in Febrile Children

IMPORTANCE Because clinical features do not reliably distinguish bacterial from viral infection, many children worldwide receive unnecessary antibiotic treatment, while bacterial infection is missed in others. OBJECTIVE To identify a blood RNA expression signature that distinguishes bacterial from viral infection in febrile children. DESIGN, SETTING, AND PARTICIPANTS Febrile children presenting to participating hospitals in the United Kingdom, Spain, the Netherlands, and the United States between 2009-2013 were prospectively recruited, comprising a discovery group and validation group. Each group was classified after microbiological investigation as having definite bacterial infection, definite viral infection, or indeterminate infection. RNA expression signatures distinguishing definite bacterial from viral infection were identified in the discovery group and diagnostic performance assessed in the validation group. Additional validation was undertaken in separate studies of children with meningococcal disease (n = 24) and inflammatory diseases (n = 48) and on published gene expression datasets. EXPOSURES A 2-transcript RNA expression signature distinguishing bacterial infection from viral infection was evaluated against clinical and microbiological diagnosis. MAIN OUTCOMES AND MEASURES Definite bacterial and viral infection was confirmed by culture or molecular detection of the pathogens. Performance of the RNA signature was evaluated in the definite bacterial and viral group and in the indeterminate infection group. RESULTS The discovery group of 240 children (median age, 19 months; 62% male) included 52 with definite bacterial infection, of whom 36 (69%) required intensive care, and 92 with definite viral infection, of whom 32 (35%) required intensive care. Ninety-six children had indeterminate infection. Analysis of RNA expression data identified a 38-transcript signature distinguishing bacterial from viral infection. A smaller (2-transcript) signature (FAM89A and IFI44L) was identified by removing highly correlated transcripts. When this 2-transcript signature was implemented as a disease risk score in the validation group (130 children, with 23 definite bacterial, 28 definite viral, and 79 indeterminate infections; median age, 17 months; 57% male), all 23 patients with microbiologically confirmed definite bacterial infection were classified as bacterial (sensitivity, 100% [95% CI, 100%-100%]) and 27 of 28 patients with definite viral infection were classified as viral (specificity, 96.4% [95% CI, 89.3%-100%]). When applied to additional validation datasets from patients with meningococcal and inflammatory diseases, bacterial infection was identified with a sensitivity of 91.7% (95% CI, 79.2%-100%) and 90.0% (95% CI, 70.0%-100%), respectively, and with specificity of 96.0% (95% CI, 88.0%-100%) and 95.8% (95% CI, 89.6%-100%). Of the children in the indeterminate groups, 46.3% (63/136) were classified as having bacterial infection, although 94.9% (129/136) received antibiotic treatment. CONCLUSIONS AND RELEVANCE This study provides preliminary data regarding test accuracy of a 2-transcript host RNA signature discriminating bacterial from viral infection in febrile children. Further studies are needed in diverse groups of patients to assess accuracy and clinical utility of this test in different clinical settings.

A heterotypic bystander effect for tumor cell killing after adeno-associated virus/phage–mediated, vascular-targeted suicide gene transfer

Suicide gene transfer is the most commonly used cytotoxic approach in cancer gene therapy; however, a successful suicide gene therapy depends on the generation of efficient targeted systemic gene delivery vectors. We recently reported that selective systemic delivery of suicide genes such as herpes simplex virus thymidine kinase (HSVtk) to tumor endothelial cells through a novel targeted adeno-associated virus/phage vector leads to suppression of tumor growth. This marked effect has been postulated to result primarily from the death of cancer cells by hypoxia following the targeted disruption of tumor blood vessels. Here, we investigated whether an additional mechanism of action is involved. We show that there is a heterotypic “bystander” effect between endothelial cells expressing the HSVtk suicide gene and tumor cells. Treatment of cocultures of HSVtk-transduced endothelial cells and non-HSVtk-transduced tumor cells with ganciclovir results in the death of both endothelial and tumor cells. Blocking of this effect by 18α-glycyrrhetinic acid indicates that gap junctions between endothelial and tumor cells are largely responsible for this phenomenon. Moreover, the observed bystander killing is mediated by connexins 43 and 26, which are expressed in endothelial and tumor cell types. Finally, this heterotypic bystander effect is accompanied by a suppression of tumor growth in vivo that is independent of primary gene transfer into host-derived tumor vascular endothelium. These findings add an alternative nonmutually exclusive and potentially synergistic cytotoxic mechanism to cancer gene therapy based on targeted adeno-associated virus/phage and further support the promising role of nonmalignant tumor stromal cells as therapeutic targets. [Mol Cancer Ther 2009;8(8):2383–91]

Novel association approach for variable number tandem repeats (VNTRs) identifies DOCK5 as a susceptibility gene for severe obesity.

Variable number tandem repeats (VNTRs) constitute a relatively under-examined class of genomic variants in the context of complex disease because of their sequence complexity and the challenges in assaying them. Recent large-scale genome-wide copy number variant mapping and association efforts have highlighted the need for improved methodology for association studies using these complex polymorphisms. Here we describe the in-depth investigation of a complex region on chromosome 8p21.2 encompassing the dedicator of cytokinesis 5 (DOCK5) gene. The region includes two VNTRs of complex sequence composition which flank a common 3975 bp deletion, all three of which were genotyped by polymerase chain reaction and fragment analysis in a total of 2744 subjects. We have developed a novel VNTR association method named VNTRtest, suitable for association analysis of multi-allelic loci with binary and quantitative outcomes, and have used this approach to show significant association of the DOCK5 VNTRs with childhood and adult severe obesity (P(empirical)= 8.9 × 10(-8) and P= 3.1 × 10(-3), respectively) which we estimate explains ~0.8% of the phenotypic variance. We also identified an independent association between the 3975 base pair (bp) deletion and obesity, explaining a further 0.46% of the variance (P(combined)= 1.6 × 10(-3)). Evidence for association between DOCK5 transcript levels and the 3975 bp deletion (P= 0.027) and both VNTRs (P(empirical)= 0.015) was also identified in adipose tissue from a Swedish family sample, providing support for a functional effect of the DOCK5 deletion and VNTRs. These findings highlight the potential role of DOCK5 in human obesity and illustrate a novel approach for analysis of the contribution of VNTRs to disease susceptibility through association studies.

Myocardial depressant effects of interleukin 6 in meningococcal sepsis are regulated by p38 mitogen-activated protein kinase.

Objectives:Myocardial failure, leading to inotrope-unresponsive shock, is the predominant cause of death in meningococcal and other forms of septic shock. Proinflammatory cytokines released in septic shock are known to have myocardial depressant effects. We previously showed that interleukin 6 is a major myocardial depressant factor in children with meningococcal septicemia. In the current study, we aimed to investigate the mechanisms by which interleukin 6 induces myocardial failure in meningococcal sepsis and to identify potential novel therapeutic targets. Design:Laboratory-based study. Setting:University hospital and laboratories. Patients:Children with a clinical diagnosis of meningococcal septic shock. Methods:We studied interleukin 6-induced signaling events, both in vitro using isolated rat ventricular cardiac myocytes as a model of myocardial contractility and in whole blood from children with meningococcal sepsis. Interventions:None. Measurements and Main Results:We demonstrated involvement of Janus kinase 2, phosphatidylinositol 3-kinase, Akt, and p38 mitogen-activated protein kinase in interleukin 6-induced negative inotropy in isolated cardiac myocytes. Inhibition of p38 mitogen-activated protein kinase not only reversed interleukin 6-induced myocardial depression in both rat and human myocytes, but restored inotrope responsiveness. Cardiomyocytes transduced with dominant-negative p38 mitogen-activated protein kinase showed no interleukin 6-induced myocardial depression. To investigate p38 mitogen-activated protein kinase in vivo, we profiled global RNA expression patterns in peripheral blood of children with meningococcal septicemia. Transcripts for genes mapping to the p38 mitogen-activated protein kinase pathway showed significantly altered levels of abundance with a high proportion of genes of this pathway affected. Conclusions:Our findings demonstrate an integral role of the p38 mitogen-activated protein kinase pathway in interleukin 6-mediated cardiac contractile dysfunction and inotrope insensitivity. Dysregulation of the p38 mitogen-activated protein kinase pathway in meningococcal septicemia suggests that this pathway may be an important target for novel therapies to reverse myocardial dysfunction in patients with meningococcal septic shock who are not responsive to inotropic support.

Demographic and motor features associated with the occurrence of neuropsychiatric and sleep complications of Parkinson's disease

Objective To determine whether four key neuropsychiatric and sleep related features associated with Parkinsons disease (PD) are associated with the motor handicap and demographic data. Background The growing number of recognised non-motor features of PD makes routine screening of all these symptoms impractical. Here, we investigated the hypothesis that standard demographic data and the routine assessment of motor signs is associated with the presence of dementia, psychosis, clinically probable rapid eye movement (REM) sleep behavior disorder (cpRBD) and restless legs syndrome (RLS). Methods 775 patients with PD underwent standardised assessment of motor features and the presence of dementia, psychosis, cpRBD and RLS. A stepwise feature elimination procedure with fitted logistic regression models was applied to identify which/if any combination of demographic and motor factors is associated with each of the four studied non-motor features. A within-study out-of-sample estimate of the power of the predicted values of the models was calculated using standard evaluation procedures. Results Age and Hoehn&Yahr (H&Y) stage were strongly associated with the presence of dementia (p value<0.001 for both factors in the final selected model) while a combination of age, disease duration, H&Y stage, dopamine agonists and catechol-O-methyltransferase (COMT) inhibitors was associated with the presence of psychosis. Disease duration and H&Y stage were the significant indicators of cpRBD, and the lack of significant motor asymmetry was the only significant feature associated with RLS-type symptoms but the evidence of association was weak. Conclusions Demographic and motor features routinely collected in patients with PD can estimate the occurrence of neuropsychiatric and sleep-related features of PD.

famCNV: copy number variant association for quantitative traits in families

UNLABELLED A program package to enable genome-wide association of copy number variants (CNVs) with quantitative phenotypes in families of arbitrary size and complexity. Intensity signals that act as proxies for the number of copies are modeled in a variance component framework and association with traits is assessed through formal likelihood testing. AVAILABILITY AND IMPLEMENTATION The Java package is made available at www.imperial.ac.uk/medicine/people/m.falchi/. CONTACT m.falchi@imperial.ac.uk.

Genetic variation in the SLC8A1 calcium signaling pathway is associated with susceptibility to Kawasaki disease and coronary artery abnormalities

Background—Kawasaki disease (KD) is an acute pediatric vasculitis in which host genetics influence both susceptibility to KD and the formation of coronary artery aneurysms. Variants discovered by genome-wide association studies and linkage studies only partially explain the influence of genetics on KD susceptibility. Methods and Results—To search for additional functional genetic variation, we performed pathway and gene stability analysis on a genome-wide association study data set. Pathway analysis using European genome-wide association study data identified 100 significantly associated pathways (P<5×10−4). Gene stability selection identified 116 single nucleotide polymorphisms in 26 genes that were responsible for driving the pathway associations, and gene ontology analysis demonstrated enrichment for calcium transport (P=1.05×10−4). Three single nucleotide polymorphisms in solute carrier family 8, member 1 (SLC8A1), a sodium/calcium exchanger encoding NCX1, were validated in an independent Japanese genome-wide association study data set (meta-analysis P=0.0001). Patients homozygous for the A (risk) allele of rs13017968 had higher rates of coronary artery abnormalities (P=0.029). NCX1, the protein encoded by SLC8A1, was expressed in spindle-shaped and inflammatory cells in the aneurysm wall. Increased intracellular calcium mobilization was observed in B cell lines from healthy controls carrying the risk allele. Conclusions—Pathway-based association analysis followed by gene stability selection proved to be a valuable tool for identifying risk alleles in a rare disease with complex genetics. The role of SLC8A1 polymorphisms in altering calcium flux in cells that mediate coronary artery damage in KD suggests that this pathway may be a therapeutic target and supports the study of calcineurin inhibitors in acute KD.