Forced vital capacity (FVC), a spirometric measure of pulmonary function, reflects lung volume and is used to diagnose and monitor lung diseases. We performed genome-wide association study meta-analysis of FVC in 52,253 individuals from 26 studies and followed up the top associations in 32,917 additional individuals of European ancestry. We found six new regions associated at genome-wide significance (P < 5 × 10−8) with FVC in or near EFEMP1, BMP6, MIR-129-2/HSD17B12, PRDM11, WWOX, and KCNJ2. Two (GSTCD and PTCH1) loci previously associated with spirometric measures were related to FVC. Newly implicated regions were followed-up in samples of African American, Korean, Chinese, and Hispanic individuals. We detected transcripts for all six newly implicated genes in human lung tissue. The new loci may inform mechanisms involved in lung development and pathogenesis of restrictive lung disease.
Protocadherin-1 (PCDH1) is a novel susceptibility gene for airway hyperresponsiveness, first identified in families exposed to cigarette smoke and is expressed in bronchial epithelial cells. Here, we asked how mouse Pcdh1 expression is regulated in lung structural cells in vivo under physiological conditions, and in both short-term cigarette smoke exposure models characterized by airway inflammation and hyperresponsiveness and chronic cigarette smoke exposure models. Pcdh1 gene-structure was investigated by Rapid Amplification of cDNA Ends. Pcdh1 mRNA and protein expression was investigated by qRT-PCR, western blotting using isoform-specific antibodies. We observed 87% conservation of the Pcdh1 nucleotide sequence, and 96% conservation of the Pcdh1 protein sequence between men and mice. We identified a novel Pcdh1 isoform encoding only the intracellular signalling motifs. Cigarette smoke exposure for 4 consecutive days markedly reduced Pcdh1 mRNA expression in lung tissue (3 to 4-fold), while neutrophilia and airway hyperresponsiveness was induced. Moreover, Pcdh1 mRNA expression in lung tissue was reduced already 6 hours after an acute cigarette-smoke exposure in mice. Chronic exposure to cigarette smoke induced loss of Pcdh1 protein in lung tissue after 2 months, while Pcdh1 protein levels were no longer reduced after 9 months of cigarette smoke exposure. We conclude that Pcdh1 is highly homologous to human PCDH1, encodes two transmembrane proteins and one intracellular protein, and is regulated by cigarette smoke exposure in vivo.
Mesenchyme-derived airway cell populations including airway smooth muscle (ASM) cells, fibroblasts and myofibroblasts play key roles in the pathogenesis of airway inflammation and remodeling. Phenotypic and functional characterisation of these cell populations are confounded by their heterogeneity in vitro. It is unclear which mechanisms underlie the creation of these different sub-populations.
The study objectives were to investigate whether ASM cells are capable of clonal expansion and if so (i) what proportion possess this capability and (ii) do clonal populations exhibit variation in terms of morphology, phenotype, proliferation rates and pro-relaxant or pro-contractile signaling pathways.
Early passage human ASM cells were subjected to single-cell cloning and their doubling time was recorded. Immunocytochemistry was performed to assess localization and levels of markers previously reported to be specifically associated with smooth muscle or fibroblasts. Finally functional assays were used to reveal differences between clonal populations specifically assessing mitogen-induced proliferation and pro-relaxant and pro-contractile signaling pathways.
Our studies provide evidence that a high proportion (58%) of single cells present within early passage human ASM cell cultures have the potential to create expanded cell populations. Despite being clonally-originated, morphological heterogeneity was still evident within these clonal populations as assessed by the range in expression of markers associated with smooth muscle cells. Functional diversity was observed between clonal populations with 10 μM isoproterenol-induced cyclic AMP responses ranging from 1.4 - 5.4 fold cf basal and bradykinin-induced inositol phosphate from 1.8 - 5.2 fold cf basal.
In summary we show for the first time that primary human ASM cells are capable of clonal expansion and that the resulting clonal populations themselves exhibit phenotypic plasticity.
Human airway smooth muscle; Clonal expansion; Phenotype; Plasticity; Remodeling
Chronic obstructive pulmonary disease (COPD) is a leading cause of global morbidity and mortality and, whilst smoking remains the single most important risk factor, COPD risk is heritable. Of 26 independent genomic regions showing association with lung function in genome-wide association studies, eleven have been reported to show association with airflow obstruction. Although the main risk factor for COPD is smoking, some individuals are observed to have a high forced expired volume in 1 second (FEV1) despite many years of heavy smoking. We hypothesised that these “resistant smokers” may harbour variants which protect against lung function decline caused by smoking and provide insight into the genetic determinants of lung health. We undertook whole exome re-sequencing of 100 heavy smokers who had healthy lung function given their age, sex, height and smoking history and applied three complementary approaches to explore the genetic architecture of smoking resistance. Firstly, we identified novel functional variants in the “resistant smokers” and looked for enrichment of these novel variants within biological pathways. Secondly, we undertook association testing of all exonic variants individually with two independent control sets. Thirdly, we undertook gene-based association testing of all exonic variants. Our strongest signal of association with smoking resistance for a non-synonymous SNP was for rs10859974 (P = 2.34×10−4) in CCDC38, a gene which has previously been reported to show association with FEV1/FVC, and we demonstrate moderate expression of CCDC38 in bronchial epithelial cells. We identified an enrichment of novel putatively functional variants in genes related to cilia structure and function in resistant smokers. Ciliary function abnormalities are known to be associated with both smoking and reduced mucociliary clearance in patients with COPD. We suggest that genetic influences on the development or function of cilia in the bronchial epithelium may affect growth of cilia or the extent of damage caused by tobacco smoke.
Very large genome-wide association studies in general population cohorts have successfully identified at least 26 genes or gene regions associated with lung function and a number of these also show association with chronic obstructive pulmonary disease (COPD). However, these findings explain a small proportion of the heritability of lung function. Although the main risk factor for COPD is smoking, some individuals have normal or good lung function despite many years of heavy smoking. We hypothesised that studying these individuals might tell us more about the genetics of lung health. Re-sequencing of exomes, where all of the variation in the protein-coding portion of the genome can be measured, is a recent approach for the study of low frequency and rare variants. We undertook re-sequencing of the exomes of “resistant smokers” and used publicly available exome data for comparisons. Our findings implicate CCDC38, a gene which has previously shown association with lung function in the general population, and genes involved in cilia structure and lung function as having a role in resistance to smoking.
The soluble cleaved urokinase plasminogen activator receptor (scuPAR) is a circulating protein detected in multiple diseases, including various cancers, cardiovascular disease, and kidney disease, where elevated levels of scuPAR have been associated with worsening prognosis and increased disease aggressiveness. We aimed to identify novel genetic and biomolecular mechanisms regulating scuPAR levels. Elevated serum scuPAR levels were identified in asthma (n=514) and chronic obstructive pulmonary disease (COPD; n=219) cohorts when compared to controls (n=96). In these cohorts, a genome-wide association study of serum scuPAR levels identified a human plasma kallikrein gene (KLKB1) promoter polymorphism (rs4253238) associated with serum scuPAR levels in a control/asthma population (P=1.17×10−7), which was also observed in a COPD population (combined P=5.04×10−12). Using a fluorescent assay, we demonstrated that serum KLKB1 enzymatic activity was driven by rs4253238 and is inverse to scuPAR levels. Biochemical analysis identified that KLKB1 cleaves scuPAR and negates scuPAR's effects on primary human bronchial epithelial cells (HBECs) in vitro. Chymotrypsin was used as a proproteolytic control, while basal HBECs were used as a control to define scuPAR-driven effects. In summary, we reveal a novel post-translational regulatory mechanism for scuPAR using a hypothesis-free approach with implications for multiple human diseases.—Portelli, M. A., Siedlinski, M., Stewart, C. E., Postma, D. S., Nieuwenhuis, M. A., Vonk, J. M., Nurnberg, P., Altmuller, J., Moffatt, M. F., Wardlaw, A. J., Parker, S. G., Connolly, M. J., Koppelman, G. H., Sayers, I. Genome-wide protein QTL mapping identifies human plasma kallikrein as a post-translational regulator of serum uPAR levels.
GWAS; proteolysis; respiratory disease; HBECs; cellular proliferation and wound repair
Lung function measures are heritable, predict mortality and are relevant in diagnosis of chronic obstructive pulmonary disease (COPD). COPD and asthma are diseases of the airways with major public health impacts and each have a heritable component. Genome-wide association studies of SNPs have revealed novel genetic associations with both diseases but only account for a small proportion of the heritability. Complex copy number variation may account for some of the missing heritability. A well-characterised genomic region of complex copy number variation contains beta-defensin genes (DEFB103, DEFB104 and DEFB4), which have a role in the innate immune response. Previous studies have implicated these and related genes as being associated with asthma or COPD. We hypothesised that copy number variation of these genes may play a role in lung function in the general population and in COPD and asthma risk. We undertook copy number typing of this locus in 1149 adult and 689 children using a paralogue ratio test and investigated association with COPD, asthma and lung function. Replication of findings was assessed in a larger independent sample of COPD cases and smoking controls. We found evidence for an association of beta-defensin copy number with COPD in the adult cohort (OR = 1.4, 95%CI:1.02–1.92, P = 0.039) but this finding, and findings from a previous study, were not replicated in a larger follow-up sample(OR = 0.89, 95%CI:0.72–1.07, P = 0.217). No robust evidence of association with asthma in children was observed. We found no evidence for association between beta-defensin copy number and lung function in the general populations. Our findings suggest that previous reports of association of beta-defensin copy number with COPD should be viewed with caution. Suboptimal measurement of copy number can lead to spurious associations. Further beta-defensin copy number measurement in larger sample sizes of COPD cases and children with asthma are needed.
Asthma genome-wide association studies (GWAS) have identified several asthma susceptibility genes with confidence; however the relative contribution of these genetic variants or single nucleotide polymorphisms (SNPs) to clinical endpoints (as opposed to disease diagnosis) remains largely unknown. Thus the aim of this study was to firstly bridge this gap in knowledge and secondly investigate whether these SNPs or those that are in linkage disequilibrium are likely to be functional candidates with respect to regulation of gene expression, using reported data from the ENCODE project.
Eleven of the key SNPs identified in eight loci from recent asthma GWAS were evaluated for association with asthma and clinical outcomes, including percent predicted FEV1, bronchial hyperresponsiveness (BHR) to methacholine, severity defined by British Thoracic Society steps and positive response to skin prick test, using the family based association test additive model in a well characterised UK cohort consisting of 370 families with at least two asthmatic children.
GSDMB SNP rs2305480 (Ser311Pro) was associated with asthma diagnosis (p = 8.9×10-4), BHR (p = 8.2×10-4) and severity (p = 1.5×10-4) with supporting evidence from a second GSDMB SNP rs11078927 (intronic). SNPs evaluated in IL33, IL18R1, IL1RL1, SMAD3, IL2RB, PDE4D, CRB1 and RAD50 did not show association with any phenotype tested when corrected for multiple testing. Analysis using ENCODE data provides further insight into the functional relevance of these SNPs.
Our results provide further support for the role of GSDMB SNPs in determining multiple asthma related phenotypes in childhood asthma including associations with lung function and disease severity.
Asthma; ENCODE; eQTL; GWAS; Clinical endpoints
Genome-Wide Association Study (GWAS) meta-analyses have identified a strong association signal for lung function, which maps to a region on 4q24 containing two oppositely transcribed genes: glutathione S-transferase, C-terminal domain containing (GSTCD) and integrator complex subunit 12 (INTS12). Both genes were found to be expressed in a range of human airway cell types. The promoter regions and transcription start sites were determined in mRNA from human lung and a novel splice variant was identified for each gene. We obtained the following evidence for GSTCD and INTS12 co-regulation and expression: (i) correlated mRNA expression was observed both via Q-PCR and in a lung expression quantitative trait loci (eQTL) study, (ii) induction of both GSTCD and INTS12 mRNA expression in human airway smooth muscle cells was seen in response to TGFβ1, (iii) a lung eQTL study revealed that both GSTCD and INTS12 mRNA levels positively correlate with percent predicted FEV1, and (iv) FEV1 GWAS associated SNPs in 4q24 were found to act as an eQTL for INTS12 in a number of tissues. In fixed sections of human lung tissue, GSTCD protein expression was ubiquitous, whereas INTS12 expression was predominantly in epithelial cells and pneumocytes. During human fetal lung development, GSTCD protein expression was observed to be highest at the earlier pseudoglandular stage (10-12 weeks) compared with the later canalicular stage (17-19 weeks), whereas INTS12 expression levels did not alter throughout these stages. Knowledge of the transcriptional and translational regulation and expression of GSTCD and INTS12 provides important insights into the potential role of these genes in determining lung function. Future work is warranted to fully define the functions of INTS12 and GSTCD.
Meta-analyses of genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) spanning the 5-hydroxytryptamine receptor 4 (5-HT4R) gene (HTR4) associated with lung function. The aims of this study were to i) investigate the expression profile of HTR4 in adult and fetal lung tissue and cultured airway cells, ii) further define HTR4 gene structure and iii) explore the potential functional implications of key SNPs using a bioinformatic approach.
Following reverse transcription (RT)-PCR in human brain, 5′ rapid amplification of cDNA ends (5′ RACE) was used to examine the exonic structure of HTR4 at the 5′ end. Quantitative (Q)-PCR was used to quantify HTR4 mRNA expression in total RNA from cultured airway cells and whole lung tissue. Publically available gene microarray data on fetal samples of estimated gestational age 7–22 weeks were mined for HTR4 expression. Immunohistochemistry (IHC; in adult and fetal lung tissue) and a radioligand binding assay (in cultured airway cells) were used to analyze 5HT4R protein expression.
IHC in adult lung, irrespective of the presence of chronic obstructive pulmonary disease (COPD), suggested low level expression of 5-HT4R protein, which was most prominent in alveolar pneumocytes. There was evidence of differential 5-HT4R protein levels during gestation in fetal lung, which was also evident in gene expression microarray data. HTR4 mRNA expression, assessed by Q-PCR, was <0.5% relative to brain in total adult lung tissue and in human airway smooth muscle (HASM) and bronchial epithelial cells (HBEC) derived from adult donors. Radioligand binding experiments also indicated that HBEC and HASM cells did not express a significant 5-HT4R population. 5′ RACE in brain identified a novel N-terminal variant, containing an extended N-terminal sequence. The functional significance of key HTR4 SNPs was investigated using the encyclopedia of DNA elements consortium (ENCODE) dataset. These analyses identified multiple alterations in regulatory motifs for transcription factors implicated in lung development, including Foxp1.
Taken together, these data suggest a role for HTR4 in lung development, which may at least in part explain the genetic association with lung function.
5-hydroxytryptamine; HTR4; 5-HT4R; Splice variant; Lung development; COPD; GPCR
Polymorphisms spanning genes involved in the production of leukotriene B4 (LTB4) e.g. ALOX5AP and LTA4H are associated with asthma susceptibility, suggesting a role for LTB4 in disease. The contribution of LTB4receptor polymorphism is currently unknown. The aim of this study was to characterise the genes for the two pivotal LTB4 receptors, LTB4R1 and LTB4R2 in lung tissue and determine if polymorphisms spanning these genes are associated with asthma and disease severity.
Rapid amplification of cDNA ends (RACE) was used to characterise the LTB4R1 and LTB4R2 gene structure in lung. The LTB4R1/2 locus on chromosome 14q11.2 was screened for polymorphic variation. Six LTB4R single nucleotide polymorphisms (SNPs) were genotyped in 370 Caucasian asthma families and 299 Adult Asthma Individuals (n=1877 total) and were evaluated for association with asthma and severity (BTS) outcome measures using Family Based Association Test, linear regression and chi square.
LTB4R1 has complex mRNA arrangement including multiple 5′-untranslated exons, suggesting additional levels of regulation. Three potential promoter regions across the LTB4R1/2 locus were identified with some airway cell specificity. 22 SNPs (MAF>0.01) were validated across the LTB4R locus in the Caucasian population. LTB4R1 and LTB4R2 SNPs were not associated with asthma susceptibility, FEV1 or severity.
LTB4R1 and LTB4R2 shows splice variation in the 5′-untranslated region and multiple promoter regions. The functional significance of this is yet to be determined. Both receptor genes were shown to be polymorphic. LTB4R polymorphisms do not appear to be susceptibility markers for the development of asthma in Caucasian subjects.
Association; Asthma; Family based association test; Leukotriene; Leukotriene B4 receptor; RACE; Severity
Pulmonary function measures reflect respiratory health and predict mortality, and are used in the diagnosis of chronic obstructive pulmonary disease (COPD). We tested genome-wide association with the forced expiratory volume in 1 second (FEV1) and the ratio of FEV1 to forced vital capacity (FVC) in 48,201 individuals of European ancestry, with follow-up of top associations in up to an additional 46,411 individuals. We identified new regions showing association (combined P<5×10−8) with pulmonary function, in or near MFAP2, TGFB2, HDAC4, RARB, MECOM (EVI1), SPATA9, ARMC2, NCR3, ZKSCAN3, CDC123, C10orf11, LRP1, CCDC38, MMP15, CFDP1, and KCNE2. Identification of these 16 new loci may provide insight into the molecular mechanisms regulating pulmonary function and into molecular targets for future therapy to alleviate reduced lung function.
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.
FEV1; FVC; genome-wide association study; modeling risk
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.
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.
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).
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).
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.
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).
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.
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.
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.
Asthma; urokinase plasminogen activator receptor; bronchial epithelial cells; wound repair; airway epithelium; asthma genetics; COPD mechanisms; allergic lung disease; asthma pharmacology; COPD exacerbations; COPD pathology; COPD pharmacology; allergic lung disease; asthma genetics
Lung function measures are heritable traits that predict population morbidity and mortality and are essential for the diagnosis of chronic obstructive pulmonary disease (COPD). Variations in many genes have been reported to affect these traits, but attempts at replication have provided conflicting results. Recently, we undertook a meta-analysis of Genome Wide Association Study (GWAS) results for lung function measures in 20,288 individuals from the general population (the SpiroMeta consortium).
To comprehensively analyse previously reported genetic associations with lung function measures, and to investigate whether single nucleotide polymorphisms (SNPs) in these genomic regions are associated with lung function in a large population sample.
We analysed association for SNPs tagging 130 genes and 48 intergenic regions (+/−10 kb), after conducting a systematic review of the literature in the PubMed database for genetic association studies reporting lung function associations.
The analysis included 16,936 genotyped and imputed SNPs. No loci showed overall significant association for FEV1 or FEV1/FVC traits using a carefully defined significance threshold of 1.3×10−5. The most significant loci associated with FEV1 include SNPs tagging MACROD2 (P = 6.81×10−5), CNTN5 (P = 4.37×10−4), and TRPV4 (P = 1.58×10−3). Among ever-smokers, SERPINA1 showed the most significant association with FEV1 (P = 8.41×10−5), followed by PDE4D (P = 1.22×10−4). The strongest association with FEV1/FVC ratio was observed with ABCC1 (P = 4.38×10−4), and ESR1 (P = 5.42×10−4) among ever-smokers.
Polymorphisms spanning previously associated lung function genes did not show strong evidence for association with lung function measures in the SpiroMeta consortium population. Common SERPINA1 polymorphisms may affect FEV1 among smokers in the general population.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Asthma; Bacterial Infection