Primary osteoarthritis chondrocyte map of chromatin conformation reveals novel candidate effector genes.

OBJECTIVES: Osteoarthritis is a complex disease with a huge public health burden. Genome-wide association studies (GWAS) have identified hundreds of osteoarthritis-associated sequence variants, but the effector genes underpinning these signals remain largely elusive. Understanding chromosome organisation in three-dimensional (3D) space is essential for identifying long-range contacts between distant genomic features (e.g., between genes and regulatory elements), in a tissue-specific manner. Here, we generate the first whole genome chromosome conformation analysis (Hi-C) map of primary osteoarthritis chondrocytes and identify novel candidate effector genes for the disease. METHODS: Primary chondrocytes collected from 8 patients with knee osteoarthritis underwent Hi-C analysis to link chromosomal structure to genomic sequence. The identified loops were then combined with osteoarthritis GWAS results and epigenomic data from primary knee osteoarthritis chondrocytes to identify variants involved in gene regulation via enhancer-promoter interactions. RESULTS: We identified 345 genetic variants residing within chromatin loop anchors that are associated with 77 osteoarthritis GWAS signals. Ten of these variants reside directly in enhancer regions of 10 newly described active enhancer-promoter loops, identified with multiomics analysis of publicly available chromatin immunoprecipitation sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) data from primary knee chondrocyte cells, pointing to two new candidate effector genes SPRY4 and PAPPA (pregnancy-associated plasma protein A) as well as further support for the gene SLC44A2 known to be involved in osteoarthritis. For example, PAPPA is directly associated with the turnover of insulin-like growth factor 1 (IGF-1) proteins, and IGF-1 is an important factor in the repair of damaged chondrocytes. CONCLUSIONS: We have constructed the first Hi-C map of primary human chondrocytes and have made it available as a resource for the scientific community. By integrating 3D genomics with large-scale genetic association and epigenetic data, we identify novel candidate effector genes for osteoarthritis, which enhance our understanding of disease and can serve as putative high-value novel drug targets.

Identification of differences in CD4+ T-cell gene expression between people with asthma and healthy controls.

Functional enrichment analysis of genome-wide association study (GWAS)-summary statistics has suggested that CD4+ T-cells play an important role in asthma pathogenesis. Despite this, CD4+ T-cells are under-represented in asthma transcriptome studies. To fill the gap, 3'-RNA-Seq was used to generate gene expression data on CD4+ T-cells (isolated within 2 h from collection) from peripheral blood from participants with well-controlled asthma (n = 32) and healthy controls (n = 11). Weighted Gene Co-expression Network Analysis (WGCNA) was used to identify sets of co-expressed genes (modules) associated with the asthma phenotype. We identified three modules associated with asthma, which are strongly enriched for GWAS-identified asthma genes, antigen processing/presentation and immune response to viral infections. Through integration of publicly available eQTL and GWAS summary statistics (colocalisation), and protein-protein interaction (PPI) data, we identified PTPRC, a potential druggable target, as a putative master regulator of the asthma gene-expression profiles. Using a co-expression network approach, with integration of external genetic and PPI data, we showed that CD4+ T-cells from peripheral blood from asthmatics have different expression profiles, albeit small in magnitude, compared to healthy controls, for sets of genes involved in immune response to viral infections (upregulated) and antigen processing/presentation (downregulated).

Comparative phenotype of circulating versus tissue immune cells in human lung and blood compartments during health and disease.

The lung is a dynamic mucosal surface constantly exposed to a variety of immunological challenges including harmless environmental antigens, pollutants, and potentially invasive microorganisms. Dysregulation of the immune system at this crucial site is associated with a range of chronic inflammatory conditions including asthma and Chronic Pulmonary Obstructive Disease (COPD). However, due to its relative inaccessibility, our fundamental understanding of the human lung immune compartment is limited. To address this, we performed flow cytometric immune phenotyping of human lung tissue and matched blood samples that were isolated from 115 donors undergoing lung tissue resection. We provide detailed characterization of the lung mononuclear phagocyte and T cell compartments, demonstrating clear phenotypic differences between lung tissue cells and those in peripheral circulation. Additionally, we show that CD103 expression demarcates pulmonary T cells that have undergone recent TCR and IL-7R signalling. Unexpectedly, we discovered that the immune landscape from asthmatic or COPD donors was broadly comparable to controls. Our data provide a much-needed expansion of our understanding of the pulmonary immune compartment in both health and disease.

A meta-analysis of genome-wide association studies of childhood wheezing phenotypes identifies ANXA1 as a susceptibility locus for persistent wheezing.

Background: Many genes associated with asthma explain only a fraction of its heritability. Most genome-wide association studies (GWASs) used a broad definition of 'doctor-diagnosed asthma', thereby diluting genetic signals by not considering asthma heterogeneity. The objective of our study was to identify genetic associates of childhood wheezing phenotypes. Methods: We conducted a novel multivariate GWAS meta-analysis of wheezing phenotypes jointly derived using unbiased analysis of data collected from birth to 18 years in 9568 individuals from five UK birth cohorts. Results: Forty-four independent SNPs were associated with early-onset persistent, 25 with pre-school remitting, 33 with mid-childhood remitting, and 32 with late-onset wheeze. We identified a novel locus on chr9q21.13 (close to annexin 1 [ANXA1], p<6.7 × 10-9), associated exclusively with early-onset persistent wheeze. We identified rs75260654 as the most likely causative single nucleotide polymorphism (SNP) using Promoter Capture Hi-C loops, and then showed that the risk allele (T) confers a reduction in ANXA1 expression. Finally, in a murine model of house dust mite (HDM)-induced allergic airway disease, we demonstrated that anxa1 protein expression increased and anxa1 mRNA was significantly induced in lung tissue following HDM exposure. Using anxa1-/- deficient mice, we showed that loss of anxa1 results in heightened airway hyperreactivity and Th2 inflammation upon allergen challenge. Conclusions: Targeting this pathway in persistent disease may represent an exciting therapeutic prospect. Funding: UK Medical Research Council Programme Grant MR/S025340/1 and the Wellcome Trust Strategic Award (108818/15/Z) provided most of the funding for this study.

Three-quarters of children hospitalized for wheezing or asthma symptoms are preschool-aged. Some will continue to experience breathing difficulties through childhood and adulthood. Others will undergo a complete resolution of their symptoms by the time they reach elementary school. The varied trajectories of young children with wheezing suggest that it is not a single disease. There are likely different genetic or environmental causes. Despite these differences, wheezing treatments for young children are 'one size fits all.' Studying the genetic underpinnings of wheezing may lead to more customized treatment options. Granell et al. studied the genetic architecture of different patterns of wheezing from infancy to adolescence. To do so, they used machine learning technology to analyze the genomes of 9,568 individuals, who participated in five studies in the United Kingdom from birth to age 18. The experiments found a new genetic variation in the ANXA1 gene linked with persistent wheezing starting in early childhood. By comparing mice with and without this gene, Granell et al. showed that the protein encoded by ANXA1 controls inflammation in the lungs in response to allergens. Animals lacking the protein develop worse lung inflammation after exposure to dust mite allergens. Identifying a new gene linked to a specific subtype of wheezing might help scientists develop better strategies to diagnose, treat, and prevent asthma. More studies are needed on the role of the protein encoded by ANXA1 in reducing allergen-triggered lung inflammation to determine if this protein or therapies that boost its production may offer relief for chronic lung inflammation.

Identification of Mechanisms by Which Genetic Susceptibility Loci Influence Systemic Sclerosis Risk Using Functional Genomics in Primary T Cells and Monocytes.

OBJECTIVE: Systemic sclerosis (SSc) is a complex autoimmune disease with a strong genetic component. However, most of the genes associated with the disease are still unknown because associated variants affect mostly noncoding intergenic elements of the genome. We used functional genomics to translate the genetic findings into a better understanding of the disease. METHODS: Promoter capture Hi-C and RNA-sequencing experiments were performed in CD4+ T cells and CD14+ monocytes from 10 SSc patients and 5 healthy controls to link SSc-associated variants with their target genes, followed by differential expression and differential interaction analyses between cell types. RESULTS: We linked SSc-associated loci to 39 new potential target genes and confirmed 7 previously known SSc-associated genes. We highlight novel causal genes, such as CXCR5, as the most probable candidate gene for the DDX6 locus. Some previously known SSc-associated genes, such as IRF8, STAT4, and CD247, showed cell type-specific interactions. We also identified 15 potential drug targets already in use in other similar immune-mediated diseases that could be repurposed for SSc treatment. Furthermore, we observed that interactions were directly correlated with the expression of important genes implicated in cell type-specific pathways and found evidence that chromatin conformation is associated with genotype. CONCLUSION: Our study revealed potential causal genes for SSc-associated loci, some of them acting in a cell type-specific manner, suggesting novel biologic mechanisms that might mediate SSc pathogenesis.

Chromatin Looping Links Target Genes with Genetic Risk Loci for Dermatological Traits.

Chromatin looping between regulatory elements and gene promoters presents a potential mechanism whereby disease risk variants affect their target genes. In this study, we use H3K27ac HiChIP, a method for assaying the active chromatin interactome in two cell lines: keratinocytes and skin lymphoma-derived CD8+ T cells. We integrate public datasets for a lymphoblastoid cell line and primary CD4+ T cells and identify gene targets at risk loci for skin-related disorders. Interacting genes enrich for pathways of known importance in each trait, such as cytokine response (psoriatic arthritis and psoriasis) and replicative senescence (melanoma). We show examples of how our analysis can inform changes in the current understanding of multiple psoriasis-associated risk loci. For example, the variant rs10794648, which is generally assigned to IFNLR1, was linked to GRHL3, a gene essential in skin repair and development, in our dataset. Our findings, therefore, indicate a renewed importance of skin-related factors in the risk of disease.

Phenotypic and functional translation of IL33 genetics in asthma.

BACKGROUND: Asthma is a complex disease with multiple phenotypes that may differ in disease pathobiology and treatment response. IL33 single nucleotide polymorphisms (SNPs) have been reproducibly associated with asthma. IL33 levels are elevated in sputum and bronchial biopsies of patients with asthma. The functional consequences of IL33 asthma SNPs remain unknown. OBJECTIVE: This study sought to determine whether IL33 SNPs associate with asthma-related phenotypes and with IL33 expression in lung or bronchial epithelium. This study investigated the effect of increased IL33 expression on human bronchial epithelial cell (HBEC) function. METHODS: Association between IL33 SNPs (Chr9: 5,815,786-6,657,983) and asthma phenotypes (Lifelines/DAG [Dutch Asthma GWAS]/GASP [Genetics of Asthma Severity & Phenotypes] cohorts) and between SNPs and expression (lung tissue, bronchial brushes, HBECs) was done using regression modeling. Lentiviral overexpression was used to study IL33 effects on HBECs. RESULTS: We found that 161 SNPs spanning the IL33 region associated with 1 or more asthma phenotypes after correction for multiple testing. We report a main independent signal tagged by rs992969 associating with blood eosinophil levels, asthma, and eosinophilic asthma. A second, independent signal tagged by rs4008366 presented modest association with eosinophilic asthma. Neither signal associated with FEV1, FEV1/forced vital capacity, atopy, and age of asthma onset. The 2 IL33 signals are expression quantitative loci in bronchial brushes and cultured HBECs, but not in lung tissue. IL33 overexpression in vitro resulted in reduced viability and reactive oxygen species-capturing of HBECs, without influencing epithelial cell count, metabolic activity, or barrier function. CONCLUSIONS: We identify IL33 as an epithelial susceptibility gene for eosinophilia and asthma, provide mechanistic insight, and implicate targeting of the IL33 pathway specifically in eosinophilic asthma.

Mapping DNA interaction landscapes in psoriasis susceptibility loci highlights KLF4 as a target gene in 9q31.

BACKGROUND: Genome-wide association studies (GWAS) have uncovered many genetic risk loci for psoriasis, yet many remain uncharacterised in terms of the causal gene and their biological mechanism in disease. This is largely a result of the findings that over 90% of GWAS variants map outside of protein-coding DNA and instead are enriched in cell type- and stimulation-specific gene regulatory regions. RESULTS: Here, we use a disease-focused Capture Hi-C (CHi-C) experiment to link psoriasis-associated variants with their target genes in psoriasis-relevant cell lines (HaCaT keratinocytes and My-La CD8+ T cells). We confirm previously assigned genes, suggest novel candidates and provide evidence for complexity at psoriasis GWAS loci. For one locus, uniquely, we combine further epigenomic evidence to demonstrate how a psoriasis-associated region forms a functional interaction with the distant (> 500 kb) KLF4 gene. This interaction occurs between the gene and active enhancers in HaCaT cells, but not in My-La cells. We go on to investigate this long-distance interaction further with Cas9 fusion protein-mediated chromatin modification (CRISPR activation) coupled with RNA-seq, demonstrating how activation of the psoriasis-associated enhancer upregulates KLF4 and its downstream targets, relevant to skin cells and apoptosis. CONCLUSIONS: This approach utilises multiple functional genomic techniques to follow up GWAS-associated variants implicating relevant cell types and causal genes in each locus; these are vital next steps for the translation of genetic findings into clinical benefit.

Phenotypic and functional translation of IL1RL1 locus polymorphisms in lung tissue and asthmatic airway epithelium.

The IL1RL1 (ST2) gene locus is robustly associated with asthma; however, the contribution of single nucleotide polymorphisms (SNPs) in this locus to specific asthma subtypes and the functional mechanisms underlying these associations remain to be defined. We tested for association between IL1RL1 region SNPs and characteristics of asthma as defined by clinical and immunological measures and addressed functional effects of these genetic variants in lung tissue and airway epithelium. Utilizing 4 independent cohorts (Lifelines, Dutch Asthma GWAS [DAG], Genetics of Asthma Severity and Phenotypes [GASP], and Manchester Asthma and Allergy Study [MAAS]) and resequencing data, we identified 3 key signals associated with asthma features. Investigations in lung tissue and primary bronchial epithelial cells identified context-dependent relationships between the signals and IL1RL1 mRNA and soluble protein expression. This was also observed for asthma-associated IL1RL1 nonsynonymous coding TIR domain SNPs. Bronchial epithelial cell cultures from asthma patients, exposed to exacerbation-relevant stimulations, revealed modulatory effects for all 4 signals on IL1RL1 mRNA and/or protein expression, suggesting SNP-environment interactions. The IL1RL1 TIR signaling domain haplotype affected IL-33-driven NF-κB signaling, while not interfering with TLR signaling. In summary, we identify that IL1RL1 genetic signals potentially contribute to severe and eosinophilic phenotypes in asthma, as well as provide initial mechanistic insight, including genetic regulation of IL1RL1 isoform expression and receptor signaling.

Moderate-to-severe asthma in individuals of European ancestry: a genome-wide association study.

BACKGROUND: Few genetic studies that focus on moderate-to-severe asthma exist. We aimed to identity novel genetic variants associated with moderate-to-severe asthma, see whether previously identified genetic variants for all types of asthma contribute to moderate-to-severe asthma, and provide novel mechanistic insights using expression analyses in patients with asthma. METHODS: In this genome-wide association study, we used a two-stage case-control design. In stage 1, we genotyped patient-level data from two UK cohorts (the Genetics of Asthma Severity and Phenotypes [GASP] initiative and the Unbiased BIOmarkers in PREDiction of respiratory disease outcomes [U-BIOPRED] project) and used data from the UK Biobank to collect patient-level genomic data for cases and controls of European ancestry in a 1:5 ratio. Cases were defined as having moderate-to-severe asthma if they were taking appropriate medication or had been diagnosed by a doctor. Controls were defined as not having asthma, rhinitis, eczema, allergy, emphysema, or chronic bronchitis as diagnosed by a doctor. For stage 2, an independent cohort of cases and controls (1:5) was selected from the UK Biobank only, with no overlap with stage 1 samples. In stage 1 we undertook a genome-wide association study of moderate-to-severe asthma, and in stage 2 we followed up independent variants that reached the significance threshold of p less than 1 × 10-6 in stage 1. We set genome-wide significance at p less than 5 × 10-8. For novel signals, we investigated their effect on all types of asthma (mild, moderate, and severe). For all signals meeting genome-wide significance, we investigated their effect on gene expression in patients with asthma and controls. FINDINGS: We included 5135 cases and 25 675 controls for stage 1, and 5414 cases and 21 471 controls for stage 2. We identified 24 genome-wide significant signals of association with moderate-to-severe asthma, including several signals in innate or adaptive immune-response genes. Three novel signals were identified: rs10905284 in GATA3 (coded allele A, odds ratio [OR] 0·90, 95% CI 0·88-0·93; p=1·76 × 10-10), rs11603634 in the MUC5AC region (coded allele G, OR 1·09, 1·06-1·12; p=2·32 × 10-8), and rs560026225 near KIAA1109 (coded allele GATT, OR 1·12, 1·08-1·16; p=3·06 × 10-9). The MUC5AC signal was not associated with asthma when analyses included mild asthma. The rs11603634 G allele was associated with increased expression of MUC5AC mRNA in bronchial epithelial brush samples via proxy SNP rs11602802; (p=2·50 × 10-5) and MUC5AC mRNA was increased in bronchial epithelial samples from patients with severe asthma (in two independent analyses, p=0·039 and p=0·022). INTERPRETATION: We found substantial shared genetic architecture between mild and moderate-to-severe asthma. We also report for the first time genetic variants associated with the risk of developing moderate-to-severe asthma that regulate mucin production. Finally, we identify candidate causal genes in these loci and provide increased insight into this difficult to treat population. FUNDING: Asthma UK, AirPROM, U-BIOPRED, UK Medical Research Council, and Rosetrees Trust.

Epistasis between FLG and IL4R Genes on the Risk of Allergic Sensitization: Results from Two Population-Based Birth Cohort Studies.

Immune-specific genes as well as genes responsible for the formation and integrity of the epidermal barrier have been implicated in the pathogeneses of allergic sensitization. This study sought to determine whether an epistatic effect (gene-gene interaction) between genetic variants within interleukin 4 receptor (IL4R) and filaggrin (FLG) genes predispose to the development of allergic sensitization. Data from two birth cohort studies were analyzed, namely the Isle of Wight (IOW; n = 1,456) and the Manchester Asthma and Allergy Study (MAAS; n = 1,058). In the IOW study, one interaction term (IL4R rs3024676 × FLG variants) showed statistical significance (interaction term: P = 0.003). To illustrate the observed epistasis, stratified analyses were performed, which showed that FLG variants were associated with allergic sensitization only among IL4R rs3024676 homozygotes (OR, 1.97; 95% CI, 1.27-3.05; P = 0.003). In contrast, FLG variants effect was masked among IL4R rs3024676 heterozygotes (OR, 0.53; 95% CI, 0.22-1.32; P = 0.175). Similar results were demonstrated in the MAAS study. Epistasis between immune (IL4R) and skin (FLG) regulatory genes exist in the pathogenesis of allergic sensitization. Hence, genetic susceptibility towards defective epidermal barrier and deviated immune responses could work together in the development of allergic sensitization.

Peanut allergy: effect of environmental peanut exposure in children with filaggrin loss-of-function mutations.

BACKGROUND: Filaggrin (FLG) loss-of-function mutations lead to an impaired skin barrier associated with peanut allergy. Household peanut consumption is associated with peanut allergy, and peanut allergen in household dust correlates with household peanut consumption. OBJECTIVE: We sought to determine whether environmental peanut exposure increases the odds of peanut allergy and whether FLG mutations modulate these odds. METHODS: Exposure to peanut antigen in dust within the first year of life was measured in a population-based birth cohort. Peanut sensitization and peanut allergy (defined by using oral food challenges or component-resolved diagnostics [CRD]) were assessed at 8 and 11 years. Genotyping was performed for 6 FLG mutations. RESULTS: After adjustment for infantile atopic dermatitis and preceding egg skin prick test (SPT) sensitization, we found a strong and significant interaction between natural log (ln [loge]) peanut dust levels and FLG mutations on peanut sensitization and peanut allergy. Among children with FLG mutations, for each ln unit increase in the house dust peanut protein level, there was a more than 6-fold increased odds of peanut SPT sensitization, CRD sensitization, or both in children at ages 8 years, 11 years, or both and a greater than 3-fold increased odds of peanut allergy compared with odds seen in children with wild-type FLG. There was no significant effect of exposure in children without FLG mutations. In children carrying an FLG mutation, the threshold level for peanut SPT sensitization was 0.92 µg of peanut protein per gram (95% CI, 0.70-1.22 µg/g), that for CRD sensitization was 1.03 µg/g (95% CI, 0.90-1.82 µg/g), and that for peanut allergy was 1.17 µg/g (95% CI, 0.01-163.83 µg/g). CONCLUSION: Early-life environmental peanut exposure is associated with an increased risk of peanut sensitization and allergy in children who carry an FLG mutation. These data support the hypothesis that peanut allergy develops through transcutaneous sensitization in children with an impaired skin barrier.

Genetic variants in endotoxin signalling pathway, domestic endotoxin exposure and asthma exacerbations.

BACKGROUND: We investigated the interaction between genetic variants in endotoxin signalling pathway and domestic endotoxin exposure in relation to asthma presence, and amongst children with asthma, we explored the association of these genetic variants and endotoxin exposure with hospital admissions due to asthma exacerbations. METHODS: In a case-control study, we analysed data from 824 children (417 asthmatics, 407 controls; age 5-18 yr). Amongst asthmatics, we extracted data on hospitalization for asthma exacerbation from medical records. Endotoxin exposure was measured in dust samples collected from homes. We included 26 single-nucleotide polymorphisms (SNPs) in the final analysis (5 CD14, 7LY96 and 14 TLR4). RESULTS: Two variants remained significantly associated with hospital admissions with asthma exacerbations after correction for multiple testing: for CD14 SNP rs5744455, carriers of T allele had decreased risk of repeated hospital admissions compared with homozygotes for C allele [OR (95% CI), 0.42 (0.25-0.88), p = 0.01, False Discovery Rate (FDR) p = 0.02]; for LY96 SNP rs17226566, C-allele carriers were at a lower risk of hospital admissions compared with T-allele homozygotes [0.59 (0.38-0.90), p = 0.01, FDR p = 0.04]. We observed two interactions between SNPs in CD14 and LY96 with environmental endotoxin exposure in relation to hospital admissions due to asthma exacerbation which remained significant after correction for multiple testing (CD14 SNPs rs2915863 and LY96 SNP rs17226566). CONCLUSION: Amongst children with asthma, genetic variants in CD14 and LY96 may increase the risk of hospital admissions with acute exacerbations. Polymorphisms in endotoxin pathway interact with domestic endotoxin exposure in further modification of the risk of hospitalization.

Interaction between glutathione S-transferase variants, maternal smoking and childhood wheezing changes with age.

BACKGROUND: Maternal smoking increases the risk of respiratory symptoms in children. Glutathione S-transferases (GSTs) detoxify xenobiotics from tobacco smoke, and functional polymorphism in GST gene(s) could predispose children to the detrimental effects of maternal smoking. Our objective was to investigate interactions between GST variants and maternal smoking in relation to the development of wheezing during childhood and whether any such interaction changes with time. METHODS: In a population-based birth cohort, we assessed maternal smoking and current wheeze at five time points during the first 11 yr of life. DNA was genotyped for GSTP1, GSTM1 and GSTT1 (n = 807). Longitudinal analyses were performed using generalized estimating equations. RESULTS: During early childhood, children whose mothers smoked were more likely to wheeze, with the strongest association observed at age 3 yr (p = 0.006). In a longitudinal model, children with GSTP1 AA and AG genotypes had significantly higher risk of wheeze compared with GG homozygotes. We observed a significant interaction between GSTP1 and maternal smoking where the risk of infantile wheezing was significantly increased in AA homozygotes, but only if their mothers smoked (OR 2.59, [1.08-6.21], p(int) = 0.03). Furthermore, amongst AA carriers, there was a significant interaction between child's age and maternal smoking, with the effect of maternal smoking on the risk of wheeze significantly diminishing with age (p(int) = 0.05); no such findings were observed for GSTM1 and GSTT1. CONCLUSIONS: Children with AA genotype for GSTP1 are at increased risk of early-life wheezing if their mothers smoke, but the effect of maternal smoking on wheezing diminishes with time.

17q12-21 and asthma: interactions with early-life environmental exposures.

BACKGROUND: 17q12-21 polymorphisms are associated with asthma presence and severity across different populations. OBJECTIVE: To extensively investigate the genes in this region among Croatian schoolchildren in a case-control study, taking account of early-life environmental exposures. METHODS: We included 423 children with asthma and 414 controls aged 5 to 18 years. Fifty-one haplotype tagging single-nucleotide polymorphisms (SNPs) were genotyped (GSDMA, GSDMB, ORMDL3, IKZF3, ZPBP2, and TOP2). Data on exposure to smoking and furry pet ownership were collected using a validated questionnaire. Information on severe asthma exacerbations with hospital admission were retrieved from hospital notes. All patients underwent spirometry. RESULTS: We found 2 SNPs (1 novel rs9635726 in IKZF3) to be associated with asthma. Among children with asthma, 4 SNPs (in ZPBP2, GSDMB, and GSDMA) were associated with hospital admissions and 8 SNPs with lung function. One SNP (rs9635726) remained significantly associated with a predicted forced expiratory volume in 1 second after false discovery rate correction. Nine markers across 5 genes showed interaction with early-life environmental tobacco smoke (ETS) exposure in relation to asthma and 2 with furry pet ownership. Among children with asthma, we observed significant interactions between early-life ETS exposure and 3 SNPs for lung function and among early-life ETS exposure, 3 SNPs (in ORMDL3 and GSDMA), and hospital admission with asthma exacerbation. Three SNPs (in ORMDL3) interacted with current furry pet ownership in relation to hospital admissions for asthma exacerbation. CONCLUSION: Our results indicate that several genes in the 17q12-21 region may be associated with asthma. This study confirms that environmental exposures may need to be included into the genetic association studies.

Genetic analysis of the Trichuris muris-induced model of colitis reveals QTL overlap and a novel gene cluster for establishing colonic inflammation.

BACKGROUND: Genetic susceptibility to colonic inflammation is poorly defined at the gene level. Although Genome Wide Association studies (GWAS) have identified loci in the human genome which confer susceptibility to Inflammatory Bowel Disease (Crohn's and Ulcerative Colitis), it is not clear if precise loci exist which confer susceptibility to inflammation at specific locations within the gut e.g. small versus large intestine. Susceptibility loci for colitis in particular have been defined in the mouse, although specific candidate genes have not been identified to date. We have previously shown that infection with Trichuris muris (T. muris) induces chronic colitis in susceptible mouse strains with clinical, histological, and immunological homology to human colonic Crohn's disease. We performed an integrative analysis of colitis susceptibility, using an F2 inter-cross of resistant (BALB/c) and susceptible (AKR) mice following T. muris infection. Quantitative Trait Loci (QTL), polymorphic and expression data were analysed alongside in silico workflow analyses to discover novel candidate genes central to the development and biology of chronic colitis. RESULTS: 7 autosomal QTL regions were associated with the establishment of chronic colitis following infection. 144 QTL genes had parental strain SNPs and significant gene expression changes in chronic colitis (expression fold-change ≥ +/-1.4). The T. muris QTL on chromosome 3 (Tm3) mapped to published QTL in 3 unrelated experimental models of colitis and contained 33 significantly transcribed polymorphic genes. Phenotypic pathway analysis, text mining and time-course qPCR replication highlighted several potential cis-QTL candidate genes in colitis susceptibility, including FcgR1, Ptpn22, RORc, and Vav3. CONCLUSION: Genetic susceptibility to induced colonic mucosal inflammation in the mouse is conserved at Tm3 and overlays Cdcs1.1. Genes central to the maintenance of intestinal homeostasis reside within this locus, implicating several candidates in susceptibility to colonic inflammation. Combined methodology incorporating genetic, transcriptional and pathway data allowed identification of biologically relevant candidate genes, with Vav3 newly implicated as a colitis susceptibility gene of functional relevance.

Asthma severity, polymorphisms in 20p13 and their interaction with tobacco smoke exposure.

BACKGROUND: We investigated the association between genetic variation in chromosomal region 20p13-p12 (ADAM33 and flanking genes ATRN, GFRA4, SIGLEC1 and HSPA12B) and asthma. Amongst asthmatics, we then investigated the association between genetic variants and asthma severity. We evaluated the effect of environmental tobacco smoke (ETS) exposure in the context of genetic variants. METHODS: In a case-control study, we recruited 423 asthmatic children and 414 non-asthmatic controls (age 5-18 yr). Amongst asthmatics, we measured lung function and extracted data on hospitalisation for asthma exacerbation from medical records. Early-life ETS exposure was assessed by questionnaire. We included 85 single-nucleotide polymorphisms (SNPs) in the analysis. RESULTS: Seventeen SNPs were significantly associated with asthma; one (rs41534847 in ADAM33) remained significant after correction for multiple testing. Thirty-six SNPs were significantly associated with lung function, of which 15 (six ARTN, three ADAM33, five SIGLEC1 and one HSPA12B) remained significant after correction. We observed a significant interaction between 23 SNPs and early-life ETS exposure in relation to lung function measures. For example, for rs512625 in ADAM33, there was significant interaction with ETS exposure in relation to hospitalisations (p(int)  = 0.02) and lung function (p(int)  = 0.03); G-allele homozygotes had a 9.15-fold [95% CI 2.28-36.89] higher risk of being hospitalized and had significantly poorer lung function if exposed to ETS, with no effect of ETS exposure amongst A-allele carriers. CONCLUSION: We demonstrated several novel significant interactions between polymorphisms in 20p13-p12 and early-life ETS exposure with asthma presence and, amongst asthmatics, a significant association with the severity of their disease.

Genetic variation in vascular endothelial growth factor-a and lung function.

RATIONALE: Given the role of vascular endothelial growth factor (VEGF) in lung development, we hypothesized that polymorphisms in VEGF-A may be associated with lung function. OBJECTIVES: The current study was designed to assess the role of genetic variants in VEGF-A as determinants of airway function from infancy through early adulthood. METHODS: Association between five single-nucleotide polymorphisms (SNPs) in VEGF-A and lung function were assessed longitudinally in two unselected birth cohorts and cross-sectionally among infants. Replication with two SNPs was conducted in adults and children with asthma. We investigated the functionality of the SNP most consistently associated with lung function (rs3025028) using Western blotting to measure the ratio of plasma VEGF-A(165b)/panVEGF-A(165) among homozygotes. MEASUREMENTS AND MAIN RESULTS: In two populations in infancy, C-allele homozygotes of rs3025028 had significantly higher VmaxFRC, forced expiratory flow(50), and forced expiratory flow(25-75) compared with other genotype groups. Among preschool children (age 3 yr), C allele of rs3025028 was associated with significantly higher specific airway conductance, with similar findings observed for lung function in school-age children. For FEV(1)/FVC ratio similar findings were observed among adolescents and young adults (birth cohort), and then replicated in adults and schoolchildren with asthma (cross-sectional studies). For rs3025038, plasma VEGF-A(165b)/panVEGF-A(165) was significantly higher among CC versus GG homozygotes (P ≤ 0.02) at birth, in school-age children, and in adults. CONCLUSIONS: We report significant associations between VEGF-A SNP rs3025028 and parameters of airway function measured throughout childhood, with the effect persisting into adulthood. We propose that the mechanism may be mediated through the ratios of active and inhibitory isoforms of VEGF-A(165), which may be determined by alternative splicing.

Effect of day care attendance on sensitization and atopic wheezing differs by Toll-like receptor 2 genotype in 2 population-based birth cohort studies.

BACKGROUND: Variation in the Toll-like receptor 2 gene (TLR2/-16934) is associated with allergic diseases among farmers' children but not among children not living on farms. OBJECTIVE: To test the hypothesis that the same genetic variant conferring protection in the farming environment is associated with reduced risk of developing allergic phenotypes among urban children attending day care in early life. METHODS: In 2 population-based birth cohorts (Manchester, United Kingdom, Manchester Asthma and Allergy Study [MAAS]; Tucson, Ariz, Tucson Infant Immune Study [IIS]), participants were recruited prenatally and followed prospectively (MAAS: 3, 5, 8 and 11 years; IIS: 1, 2, 3 and 5 years). We assessed allergic sensitization and atopic wheezing at each follow-up. RESULTS: A total of 727 children participated in Manchester and 263 in Tucson. We found no significant associations between TLR2/-16934 and sensitization and atopic wheeze in either cohort. However, a different pattern emerged when we explored the interaction between TLR2/-16934 and day care attendance on these outcomes. We found a significant interaction between day care and TLR2/-16934 on the development of sensitization in the longitudinal model in MAAS in that children carrying the T allele who attended day care were less likely to be sensitized than those who did not attend day care, whereas among AA homozygotes, the association tended to be in the opposite direction. In a longitudinal model in IIS, we found a significant interaction between day care attendance and TLR2/-16934 on the development atopic wheezing. Significant interactions between TLR2/-16934 and day care were maintained when adjusting for socioeconomic status. CONCLUSION: The effect of day care on sensitization and atopic wheezing may differ among children with different variants of the TLR2 gene.