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 Crohn's 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

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

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

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

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

Diagnosis of Kawasaki Disease Using a Minimal Whole-Blood Gene Expression Signature.

Importance: To date, there is no diagnostic test for Kawasaki disease (KD). Diagnosis is based on clinical features shared with other febrile conditions, frequently resulting in delayed or missed treatment and an increased risk of coronary artery aneurysms. Objective: To identify a whole-blood gene expression signature that distinguishes children with KD in the first week of illness from other febrile conditions. Design, Setting, and Participants: The case-control study comprised a discovery group that included a training and test set and a validation group of children with KD or comparator febrile illness. The setting was pediatric centers in the United Kingdom, Spain, the Netherlands, and the United States. The training and test discovery group comprised 404 children with infectious and inflammatory conditions (78 KD, 84 other inflammatory diseases, and 242 bacterial or viral infections) and 55 healthy controls. The independent validation group comprised 102 patients with KD, including 72 in the first 7 days of illness, and 130 febrile controls. The study dates were March 1, 2009, to November 14, 2013, and data analysis took place from January 1, 2015, to December 31, 2017. Main Outcomes and Measures: Whole-blood gene expression was evaluated using microarrays, and minimal transcript sets distinguishing KD were identified using a novel variable selection method (parallel regularized regression model search). The ability of transcript signatures (implemented as disease risk scores) to discriminate KD cases from controls was assessed by area under the curve (AUC), sensitivity, and specificity at the optimal cut point according to the Youden index. Results: Among 404 patients in the discovery set, there were 78 with KD (median age, 27 months; 55.1% male) and 326 febrile controls (median age, 37 months; 56.4% male). Among 202 patients in the validation set, there were 72 with KD (median age, 34 months; 62.5% male) and 130 febrile controls (median age, 17 months; 56.9% male). A 13-transcript signature identified in the discovery training set distinguished KD from other infectious and inflammatory conditions in the discovery test set, with AUC of 96.2% (95% CI, 92.5%-99.9%), sensitivity of 81.7% (95% CI, 60.0%-94.8%), and specificity of 92.1% (95% CI, 84.0%-97.0%). In the validation set, the signature distinguished KD from febrile controls, with AUC of 94.6% (95% CI, 91.3%-98.0%), sensitivity of 85.9% (95% CI, 76.8%-92.6%), and specificity of 89.1% (95% CI, 83.0%-93.7%). The signature was applied to clinically defined categories of definite, highly probable, and possible KD, resulting in AUCs of 98.1% (95% CI, 94.5%-100%), 96.3% (95% CI, 93.3%-99.4%), and 70.0% (95% CI, 53.4%-86.6%), respectively, mirroring certainty of clinical diagnosis. Conclusions and Relevance: In this study, a 13-transcript blood gene expression signature distinguished KD from other febrile conditions. Diagnostic accuracy increased with certainty of clinical diagnosis. A test incorporating the 13-transcript disease risk score may enable earlier diagnosis and treatment of KD and reduce inappropriate treatment in those with other diagnoses

Childhood tuberculosis is associated with decreased abundance of T cell gene transcripts and impaired T cell function

The WHO estimates around a million children contract tuberculosis (TB) annually with over 80 000 deaths from dissemination of infection outside of the lungs. The insidious onset and association with skin test anergy suggests failure of the immune system to both recognise and respond to infection. To understand the immune mechanisms, we studied genome-wide whole blood RNA expression in children with TB meningitis (TBM). Findings were validated in a second cohort of children with TBM and pulmonary TB (PTB), and functional T-cell responses studied in a third cohort of children with TBM, other extrapulmonary TB (EPTB) and PTB. The predominant RNA transcriptional response in children with TBM was decreased abundance of multiple genes, with 140/204 (68%) of all differentially regulated genes showing reduced abundance compared to healthy controls. Findings were validated in a second cohort with concordance of the direction of differential expression in both TBM (r2 = 0.78 p = 2x10-16) and PTB patients (r2 = 0.71 p = 2x10-16) when compared to a second group of healthy controls. Although the direction of expression of these significant genes was similar in the PTB patients, the magnitude of differential transcript abundance was less in PTB than in TBM. The majority of genes were involved in activation of leucocytes (p = 2.67E-11) and T-cell receptor signalling (p = 6.56E-07). Less abundant gene expression in immune cells was associated with a functional defect in T-cell proliferation that recovered after full TB treatment (p<0.0003). Multiple genes involved in T-cell activation show decreased abundance in children with acute TB, who also have impaired functional T-cell responses. Our data suggest that childhood TB is associated with an acquired immune defect, potentially resulting in failure to contain the pathogen. Elucidation of the mechanism causing the immune paresis may identify new treatment and prevention strategies

Plasma lipid profiles discriminate bacterial from viral infection in febrile children

Fever is the most common reason that children present to Emergency Departments. Clinical signs and symptoms suggestive of bacterial infection are often non-specific, and there is no definitive test for the accurate diagnosis of infection. The 'omics' approaches to identifying biomarkers from the host-response to bacterial infection are promising. In this study, lipidomic analysis was carried out with plasma samples obtained from febrile children with confirmed bacterial infection (n = 20) and confirmed viral infection (n = 20). We show for the first time that bacterial and viral infection produces distinct profile in the host lipidome. Some species of glycerophosphoinositol, sphingomyelin, lysophosphatidylcholine and cholesterol sulfate were higher in the confirmed virus infected group, while some species of fatty acids, glycerophosphocholine, glycerophosphoserine, lactosylceramide and bilirubin were lower in the confirmed virus infected group when compared with confirmed bacterial infected group. A combination of three lipids achieved an area under the receiver operating characteristic (ROC) curve of 0.911 (95% CI 0.81 to 0.98). This pilot study demonstrates the potential of metabolic biomarkers to assist clinicians in distinguishing bacterial from viral infection in febrile children, to facilitate effective clinical management and to the limit inappropriate use of antibiotics

Identification of regulatory variants associated with genetic susceptibility to meningococcal disease.

Non-coding genetic variants play an important role in driving susceptibility to complex diseases but their characterization remains challenging. Here, we employed a novel approach to interrogate the genetic risk of such polymorphisms in a more systematic way by targeting specific regulatory regions relevant for the phenotype studied. We applied this method to meningococcal disease susceptibility, using the DNA binding pattern of RELA - a NF-kB subunit, master regulator of the response to infection - under bacterial stimuli in nasopharyngeal epithelial cells. We designed a custom panel to cover these RELA binding sites and used it for targeted sequencing in cases and controls. Variant calling and association analysis were performed followed by validation of candidate polymorphisms by genotyping in three independent cohorts. We identified two new polymorphisms, rs4823231 and rs11913168, showing signs of association with meningococcal disease susceptibility. In addition, using our genomic data as well as publicly available resources, we found evidences for these SNPs to have potential regulatory effects on ATXN10 and LIF genes respectively. The variants and related candidate genes are relevant for infectious diseases and may have important contribution for meningococcal disease pathology. Finally, we described a novel genetic association approach that could be applied to other phenotypes

Plasma lipid profiles discriminate bacterial from viral infection in febrile children

Fever is the most common reason that children present to Emergency Departments. Clinical signs and symptoms suggestive of bacterial infection are often non-specific, and there is no definitive test for the accurate diagnosis of infection. The 'omics' approaches to identifying biomarkers from the host-response to bacterial infection are promising. In this study, lipidomic analysis was carried out with plasma samples obtained from febrile children with confirmed bacterial infection (n = 20) and confirmed viral infection (n = 20). We show for the first time that bacterial and viral infection produces distinct profile in the host lipidome. Some species of glycerophosphoinositol, sphingomyelin, lysophosphatidylcholine and cholesterol sulfate were higher in the confirmed virus infected group, while some species of fatty acids, glycerophosphocholine, glycerophosphoserine, lactosylceramide and bilirubin were lower in the confirmed virus infected group when compared with confirmed bacterial infected group. A combination of three lipids achieved an area under the receiver operating characteristic (ROC) curve of 0.911 (95% CI 0.81 to 0.98). This pilot study demonstrates the potential of metabolic biomarkers to assist clinicians in distinguishing bacterial from viral infection in febrile children, to facilitate effective clinical management and to the limit inappropriate use of antibiotics

Plasma lipid profiles discriminate bacterial from viral infection in febrile children

Fever is the most common reason that children present to Emergency Departments. Clinical signs and symptoms suggestive of bacterial infection ar