Victoria J. Wright

A Genome-Wide Association Study Identifies Novel and Functionally Related Susceptibility Loci for Kawasaki Disease

Kawasaki disease (KD) is a pediatric vasculitis that damages the coronary arteries in 25% of untreated and approximately 5% of treated children. Epidemiologic data suggest that KD is triggered by unidentified infection(s) in genetically susceptible children. To investigate genetic determinants of KD susceptibility, we performed a genome-wide association study (GWAS) in 119 Caucasian KD cases and 135 matched controls with stringent correction for possible admixture, followed by replication in an independent cohort and subsequent fine-mapping, for a total of 893 KD cases plus population and family controls. Significant associations of 40 SNPs and six haplotypes, identifying 31 genes, were replicated in an independent cohort of 583 predominantly Caucasian KD families, with NAALADL2 (rs17531088, p combined = 1.13×10−6) and ZFHX3 (rs7199343, p combined = 2.37×10−6) most significantly associated. Sixteen associated variants with a minor allele frequency of >0.05 that lay within or close to known genes were fine-mapped with HapMap tagging SNPs in 781 KD cases, including 590 from the discovery and replication stages. Original or tagging SNPs in eight of these genes replicated the original findings, with seven genes having further significant markers in adjacent regions. In four genes (ZFHX3, NAALADL2, PPP1R14C, and TCP1), the neighboring markers were more significantly associated than the originally associated variants. Investigation of functional relationships between the eight fine-mapped genes using Ingenuity Pathway Analysis identified a single functional network (p = 10−13) containing five fine-mapped genes—LNX1, CAMK2D, ZFHX3, CSMD1, and TCP1—with functional relationships potentially related to inflammation, apoptosis, and cardiovascular pathology. Pair-wise blood transcript levels were measured during acute and convalescent KD for all fine-mapped genes, revealing a consistent trend of significantly reduced transcript levels prior to treatment. This is one of the first GWAS in an infectious disease. We have identified novel, plausible, and functionally related variants associated with KD susceptibility that may also be relevant to other cardiovascular diseases.

Genome-wide association study identifies variants in the CFH region associated with host susceptibility to meningococcal disease

Meningococcal disease is an infection caused by Neisseria meningitidis. Genetic factors contribute to host susceptibility and progression to disease, but the genes responsible for disease development are largely unknown. We report here a genome-wide association study for host susceptibility to meningococcal disease using 475 individuals with meningococcal disease (cases) and 4,703 population controls from the UK. We performed, in Western European and South European cohorts (consisting of 968 cases and 1,376 controls), two replication studies for the most significant SNPs. A cluster of complement factor SNPs replicated independently in both cohorts, including SNPs within complement factor H (CFH) (rs1065489 (p.936D<E), P = 2.2 × 10−11) and in CFH-related protein 3 (CFHR3)(rs426736, P = 4.6 × 10−13). N. meningitidis is known to evade complement-mediated killing by the binding of host CFH to the meningococcal factor H–binding protein (fHbp). Our study suggests that host genetic variation in these regulators of complement activation plays a role in determining the occurrence of invasive disease versus asymptomatic colonization by this pathogen.

Diagnosis of Childhood Tuberculosis and Host RNA Expression in Africa

BACKGROUND Improved diagnostic tests for tuberculosis in children are needed. We hypothesized that transcriptional signatures of host blood could be used to distinguish tuberculosis from other diseases in African children who either were or were not infected with the human immunodeficiency virus (HIV). METHODS The study population comprised prospective cohorts of children who were undergoing evaluation for suspected tuberculosis in South Africa (655 children), Malawi (701 children), and Kenya (1599 children). Patients were assigned to groups according to whether the diagnosis was culture-confirmed tuberculosis, culture-negative tuberculosis, diseases other than tuberculosis, or latent tuberculosis infection. Diagnostic signatures distinguishing tuberculosis from other diseases and from latent tuberculosis infection were identified from genomewide analysis of RNA expression in host blood. RESULTS We identified a 51-transcript signature distinguishing tuberculosis from other diseases in the South African and Malawian children (the discovery cohort). In the Kenyan children (the validation cohort), a risk score based on the signature for tuberculosis and for diseases other than tuberculosis showed a sensitivity of 82.9% (95% confidence interval [CI], 68.6 to 94.3) and a specificity of 83.6% (95% CI, 74.6 to 92.7) for the diagnosis of culture-confirmed tuberculosis. Among patients with cultures negative for Mycobacterium tuberculosis who were treated for tuberculosis (those with highly probable, probable, or possible cases of tuberculosis), the estimated sensitivity was 62.5 to 82.3%, 42.1 to 80.8%, and 35.3 to 79.6%, respectively, for different estimates of actual tuberculosis in the groups. In comparison, the sensitivity of the Xpert MTB/RIF assay for molecular detection of M. tuberculosis DNA in cases of culture-confirmed tuberculosis was 54.3% (95% CI, 37.1 to 68.6), and the sensitivity in highly probable, probable, or possible cases was an estimated 25.0 to 35.7%, 5.3 to 13.3%, and 0%, respectively; the specificity of the assay was 100%. CONCLUSIONS RNA expression signatures provided data that helped distinguish tuberculosis from other diseases in African children with and those without HIV infection. (Funded by the European Union Action for Diseases of Poverty Program and others).

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.

Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

Background Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch. Methodology/Principal Findings One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10−5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels. Conclusions By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system.

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.

Transcriptomic analysis reveals a global alkyl-quinolone-independent regulatory role for PqsE in facilitating the environmental adaptation of Pseudomonas aeruginosa to plant and animal hosts.

The quorum sensing (QS) system of Pseudomonas aeruginosa constitutes a sophisticated genome-wide gene regulatory network employing both N-acylhomoserine lactone and 2-alkyl-4-quinolone (AQ) signal molecules. AQ signalling utilizes 2-heptyl-3-hydroxy-4-quinolone (PQS) and its immediate precursor, 2-heptyl-4-quinolone (HHQ). AQ biosynthesis requires the first four genes of the pqsABCDE operon and while the biochemical function of pqsE is not known, it is required for the production of secondary metabolites such as pyocyanin. To gain insights into the relationship between the AQ stimulon, the PqsE stimulon and the regulatory function of PqsE, we constructed a pqsE inducible mutant (pqsEind) and compared the transcriptomes of the induced and uninduced states with a pqsA mutant. Of 158 genes exhibiting altered expression in the pqsA mutant, 51% were also affected in the pqsE mutant. Following induction of pqsE, 237 genes were differentially expressed compared with the wild-type strain. In the pqsEind strain, pqsA was highly expressed but following induction both pqsA expression and AQ biosynthesis were repressed, revealing a negative autoregulatory role for PqsE. Furthermore, pqsE was required for swarming motility and virulence in plant and animal infection models in the absence of AQs, while mature biofilm development required both pqsA and pqsE. Taken together these data reveal that PqsE is a key regulator within the QS circuitry facilitating the environmental adaptation of P. aeruginosa.

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.

Genome-wide association study of primary tooth eruption identifies pleiotropic loci associated with height and craniofacial distances

Twin and family studies indicate that the timing of primary tooth eruption is highly heritable, with estimates typically exceeding 80%. To identify variants involved in primary tooth eruption, we performed a population-based genome-wide association study of ‘age at first tooth’ and ‘number of teeth’ using 5998 and 6609 individuals, respectively, from the Avon Longitudinal Study of Parents and Children (ALSPAC) and 5403 individuals from the 1966 Northern Finland Birth Cohort (NFBC1966). We tested 2 446 724 SNPs imputed in both studies. Analyses were controlled for the effect of gestational age, sex and age of measurement. Results from the two studies were combined using fixed effects inverse variance meta-analysis. We identified a total of 15 independent loci, with 10 loci reaching genome-wide significance (P < 5 × 10−8) for ‘age at first tooth’ and 11 loci for ‘number of teeth’. Together, these associations explain 6.06% of the variation in ‘age of first tooth’ and 4.76% of the variation in ‘number of teeth’. The identified loci included eight previously unidentified loci, some containing genes known to play a role in tooth and other developmental pathways, including an SNP in the protein-coding region of BMP4 (rs17563, P = 9.080 × 10−17). Three of these loci, containing the genes HMGA2, AJUBA and ADK, also showed evidence of association with craniofacial distances, particularly those indexing facial width. Our results suggest that the genome-wide association approach is a powerful strategy for detecting variants involved in tooth eruption, and potentially craniofacial growth and more generally organ development.