Rationale: Genomic loci are associated with FEV1 or the ratio of FEV1 to FVC in population samples, but their association with chronic obstructive pulmonary disease (COPD) has not yet been proven, nor have their combined effects on lung function and COPD been studied.

Objectives: To test association with COPD of variants at five loci (TNS1, GSTCD, HTR4, AGER, and THSD4) and to evaluate joint effects on lung function and COPD of these single-nucleotide polymorphisms (SNPs), and variants at the previously reported locus near HHIP.

Methods: By sampling from 12 population-based studies (n = 31,422), we obtained genotype data on 3,284 COPD case subjects and 17,538 control subjects for sentinel SNPs in TNS1, GSTCD, HTR4, AGER, and THSD4. In 24,648 individuals (including 2,890 COPD case subjects and 13,862 control subjects), we additionally obtained genotypes for rs12504628 near HHIP. Each allele associated with lung function decline at these six SNPs contributed to a risk score. We studied the association of the risk score to lung function and COPD.

Measurements and Main Results: Association with COPD was significant for three loci (TNS1, GSTCD, and HTR4) and the previously reported HHIP locus, and suggestive and directionally consistent for AGER and TSHD4. Compared with the baseline group (7 risk alleles), carrying 10–12 risk alleles was associated with a reduction in FEV1 (β = –72.21 ml, P = 3.90 × 10−4) and FEV1/FVC (β = –1.53%, P = 6.35 × 10−6), and with COPD (odds ratio = 1.63, P = 1.46 × 10−5).

Conclusions: Variants in TNS1, GSTCD, and HTR4 are associated with COPD. Our highest risk score category was associated with a 1.6-fold higher COPD risk than the population average score.

The aim of the current study was to evaluate primary (human bronchial epithelial cells, HBEC) and non-primary (Calu-3, BEAS-2B, BEAS-2B R1) bronchial epithelial cell culture systems as air-liquid interface- (ALI-) differentiated models for asthma research. Ability to differentiate into goblet (MUC5AC+) and ciliated (β-Tubulin IV+) cells was evaluated by confocal imaging and qPCR. Expression of tight junction/adhesion proteins (ZO-1, E-Cadherin) and development of transepithelial electrical resistance (TEER) were assessed. Primary cells showed localised MUC5AC, β-Tubulin IV, ZO-1, and E-Cadherin and developed TEER with, however, a large degree of inter- and intradonor variation. Calu-3 cells developed a more reproducible TEER and a phenotype similar to primary cells although with diffuse β-Tubulin IV staining. BEAS-2B cells did not differentiate or develop tight junctions. These data highlight the challenges in working with primary cell models and the need for careful characterisation and selection of systems to answer specific research questions.

Background

We have previously shown evidence that polymorphisms within genes controlling leukotriene B4 (LTB4) production (ALOX5AP and LTA4H) are associated with asthma susceptibility in children. Evidence also suggests a potential role of LTB4 in COPD disease mechanisms including recruitment of neutrophils to the lung. The aim of the current study was to see if these SNPs and those spanning the receptor genes for LTB4 (LTB4R1 and LTB4R2) influence baseline lung function and COPD susceptibility/severity in smokers.

Methods

Eight ALOX5AP, six LTA4H and six LTB4R single nucleotide polymorphisms (SNPs) were genotyped in a UK Smoking Cohort (n = 992). Association with baseline lung function (FEV1 and FEV1/FVC ratio) was determined by linear regression. Logistic regression was used to compare smoking controls (n = 176) with spirometry-defined COPD cases (n = 599) and to more severe COPD cases (GOLD stage 3 and 4, n = 389).

Results

No association with ALOX5AP, LTA4H or LTB4R survived correction for multiple testing. However, we showed modest association with LTA4H rs1978331C (intron 11) with increased FEV1 (p = 0.029) and with increased FEV1/FVC ratio (p = 0.020).

Conclusions

These data suggest that polymorphisms spanning ALOX5AP, LTA4H and the LTB4R locus are not major determinants of baseline lung function in smokers, but provide tentative evidence for LTA4H rs1978331C (intron 11) in determining baseline FEV1 and FEV1/FVC ratio in Caucasian Smokers in addition to our previously identified role in asthma susceptibility.

Background

The asthma-associated gene urokinase plasminogen activator receptor (uPAR) may be involved in epithelial repair and airway remodelling. These processes are not adequately targeted by existing asthma therapies. A fuller understanding of the pathways involved in remodelling may lead to development of new therapeutic opportunities. uPAR expression in the lung epithelium of normal subjects and patients with asthma was investigated and the contribution of uPAR to epithelial wound repair in vitro was studied using primary bronchial epithelial cells (NHBECs).

Methods

Bronchial biopsy sections from normal subjects and patients with asthma were immunostained for uPAR. NHBECs were used in a scratch wound model to investigate the contribution of the plasminogen pathway to repair. The pathway was targeted via blocking of the interaction between urokinase plasminogen activator (uPA) and uPAR and overexpression of uPAR. The rate of wound closure and activation of intracellular signalling pathways and matrix metalloproteinases (MMPs) were measured.

Results

uPAR expression was significantly increased in the bronchial epithelium of patients with asthma compared with controls. uPAR expression was increased during wound repair in monolayer and air-liquid interface-differentiated NHBEC models. Blocking the uPA–uPAR interaction led to attenuated wound repair via changes in Erk1/2, Akt and p38MAPK signalling. Cells engineered to have raised levels of uPAR showed attenuated repair via sequestration of uPA by soluble uPAR.

Conclusions

The uPAR pathway is required for efficient epithelial wound repair. Increased uPAR expression, as seen in the bronchial epithelium of patients with asthma, leads to attenuated wound repair which may contribute to the development and progression of airway remodelling in asthma. This pathway may therefore represent a potential novel therapeutic target for the treatment of asthma.

Pulmonary function measures are heritable traits that predict morbidity and mortality and define chronic obstructive pulmonary disease (COPD). We tested genome-wide association with forced expiratory volume in 1 s (FEV1) and the ratio of FEV1 to forced vital capacity (FVC) in the SpiroMeta consortium (n = 20,288 individuals of European ancestry). We conducted a meta-analysis of top signals with data from direct genotyping (n ≤ 32,184 additional individuals) and in silico summary association data from the CHARGE Consortium (n = 21,209) and the Health 2000 survey (n ≤ 883). We confirmed the reported locus at 4q31 and identified associations with FEV1 or FEV1/FVC and common variants at five additional loci: 2q35 in TNS1 (P = 1.11 × 10−12), 4q24 in GSTCD (2.18 × 10−23), 5q33 in HTR4 (P = 4.29 × 10−9), 6p21 in AGER (P = 3.07 × 10−15) and 15q23 in THSD4 (P = 7.24 × 10−15). mRNA analyses showed expression of TNS1, GSTCD, AGER, HTR4 and THSD4 in human lung tissue. These associations offer mechanistic insight into pulmonary function regulation and indicate potential targets for interventions to alleviate respiratory disease.

Background

We have previously identified Urokinase Plasminogen Activator Receptor (PLAUR) as an asthma susceptibility gene. In the current study we tested the hypothesis that PLAUR single nucleotide polymorphisms (SNPs) determine baseline lung function and contribute to the development of Chronic Obstructive Pulmonary Disease (COPD) in smokers.

Methods

25 PLAUR SNPs were genotyped in COPD subjects and individuals with smoking history (n = 992). Linear regression was used to determine the effects of polymorphism on baseline lung function (FEV1, FEV1/FVC) in all smokers. Genotype frequencies were compared in spirometry defined smoking controls (n = 176) versus COPD cases (n = 599) and COPD severity (GOLD stratification) using logistic regression.

Results

Five SNPs showed a significant association (p < 0.01) with baseline lung function; rs2302524(Lys220Arg) and rs2283628(intron 3) were associated with lower and higher FEV1 respectively. rs740587(-22346), rs11668247(-20040) and rs344779(-3666) in the 5'region were associated with increased FEV1/FVC ratio. rs740587 was also protective for COPD susceptibility and rs11668247 was protective for COPD severity although no allele dose relationship was apparent. Interestingly, several of these associations were driven by male smokers not females.

Conclusion

This study provides tentative evidence that the asthma associated gene PLAUR also influences baseline lung function in smokers. However the case-control analyses do not support the conclusion that PLAUR is a major COPD susceptibility gene in smokers. PLAUR is a key serine protease receptor involved in the generation of plasmin and has been implicated in airway remodelling.

Background

Expression of the urokinase plasminogen activator receptor (UPAR) has been shown to have clinical relevance in various cancers. We have recently identified UPAR as an asthma susceptibility gene and there is evidence to suggest that uPAR may be upregulated in lung diseases such as COPD and asthma. uPAR is a key receptor involved in the formation of the serine protease plasmin by interacting with uPA and has been implicated in many physiological processes including proliferation and migration. The current aim was to determine key regulatory regions and splice variants of UPAR and quantify its expression in primary human tissues and cells (including lung, bronchial epithelium (HBEC), airway smooth muscle (HASM) and peripheral cells).

Results

Using Rapid Amplification of cDNA Ends (RACE) a conserved transcription start site (-42 to -77 relative to ATG) was identified and multiple transcription factor binding sites predicted. Seven major splice variants were identified (>5% total expression), including multiple exon deletions and an alternative exon 7b (encoding a truncated, soluble, 229aa protein). Variants were differentially expressed, with a high proportion of E7b usage in lung tissue and structural cells (55–87% of transcripts), whereas classical exon 7 (encoding the GPI-linked protein) was preferentially expressed in peripheral cells (~80% of transcripts), often with exon 6 or 5+6 deletions. Real-time PCR confirmed expression of uPAR mRNA in lung, as well as airway and peripheral cell types with ~50–100 fold greater expression in peripheral cells versus airway cells and confirmed RACE data. Protein analysis confirmed expression of multiple different forms of uPAR in the same cells as well as expression of soluble uPAR in cell supernatants. The pattern of expression did not directly reflect that seen at the mRNA level, indicating that post-translational mechanisms of regulation may also play an important role.

Conclusion

We have identified multiple uPAR isoforms in the lung and immune cells and shown that expression is cell specific. These data provide a novel mechanism for uPAR regulation, as different exon splicing may determine uPAR function e.g. alternative E7b results in a soluble isoform due to the loss of the GPI anchor and exon deletions may affect uPA (ligand) and/or integrin binding and therefore influence downstream pathways. Expression of different isoforms within the lung should be taken into consideration in studies of uPAR in respiratory disease.

Background

Asthma and allergy are complex multifactorial disorders, with both genetic and environmental components determining disease expression. The use of molecular genetics holds great promise for the identification of novel drug targets for the treatment of asthma and allergy. Genome-wide linkage studies have identified a number of potential disease susceptibility loci but replication remains inconsistent. The aim of the current study was to complete a meta-analysis of data from genome-wide linkage studies of asthma and related phenotypes and provide inferences about the consistency of results and to identify novel regions for future gene discovery.

Methods

The rank based genome-scan meta-analysis (GSMA) method was used to combine linkage data for asthma and related traits; bronchial hyper-responsiveness (BHR), allergen positive skin prick test (SPT) and total serum Immunoglobulin E (IgE) from nine Caucasian asthma populations.

Results

Significant evidence for susceptibility loci was identified for quantitative traits including; BHR (989 pedigrees, n = 4,294) 2p12-q22.1, 6p22.3-p21.1 and 11q24.1-qter, allergen SPT (1,093 pedigrees, n = 4,746) 3p22.1-q22.1, 17p12-q24.3 and total IgE (729 pedigrees, n = 3,224) 5q11.2-q14.3 and 6pter-p22.3. Analysis of the asthma phenotype (1,267 pedigrees, n = 5,832) did not identify any region showing genome-wide significance.

Conclusion

This study represents the first linkage meta-analysis to determine the relative contribution of chromosomal regions to the risk of developing asthma and atopy. Several significant results were obtained for quantitative traits but not for asthma confirming the increased phenotype and genetic heterogeneity in asthma. These analyses support the contribution of regions that contain previously identified asthma susceptibility genes and provide the first evidence for susceptibility loci on 5q11.2-q14.3 and 11q24.1-qter.

Background

Airway hyper-responsiveness (AHR) is a key feature of asthma and a causal relationship between airway inflammation and AHR has been identified. The aim of the current study was to clarify the effect of proinflammatory cytokines and asthma medication on primary human airway smooth muscle (ASM) inositol phosphate (IPx) signalling and define the regulatory loci involved.

Methods

Primary Human ASM cells were isolated from explants of trachealis muscle from individuals with no history of respiratory disease. The effect of cytokine or asthma medication on histamine or bradykinin induced IPx signalling was assessed by [3H] inositol incorporation. Quantitative Real Time PCR was used to measure mRNA levels of receptors and downstream signalling components. Transcriptional mechanisms were explored using a combination of 5'Rapid Amplification of cDNA Ends (5'RACE) and promoter-reporter techniques.

Results

Treatment of Human ASM cells with IL-13, IFNγ or salmeterol for 24 hours lead to a modest augmentation of histamine induced IPx responses (144.3 +/- 9.3, 126.4 +/- 7.5 and 117.7 +/- 5.2%, p < 0.05). Similarly, TNFα, IFNγ or salmeterol treatment augmented bradykinin induced IPx responses (127.4 +/- 8.3, 128.0 +/- 8.4 and 111.7 +/- 5.0%, P < 0.05). No treatment significantly influenced sodium fluoride induced IPx responses suggesting regulation occurs at the receptor locus. Analyses of mRNA expression of components of the IPx pathway i.e. H1 Histamine Receptor (HRH1), B2 Bradykinin Receptor (BDKRB2), Gαq/11 and PLC-β1 identified that a significant induction of receptor mRNA (>2 fold) was a feature of these responses explaining the cytokine and spasmogen specificity. The HRH1 and BDKRB2 promoter regions were mapped in ASM and promoter-reporter analyses identified that salmeterol can induce HRH1 (>2 fold) and BDKRB2 (2–5 fold) transcription. The effect of cytokines on HRH1 and BDKRB2 promoter-reporter expression suggested a more complex regulation of mRNA expression involving additional loci to the core promoter.

Conclusion

Our results indicate that the spasmogen specific receptor locus may be a key site of regulation determining the magnitude of spasmogen mediated ASM IPx responses during airway inflammation or following asthma medication. These data provide further insight into the molecular basis of AHR and extend our understanding of potentially detrimental effects associated with existing therapies used in the treatment of asthma.