[Show abstract][Hide abstract] ABSTRACT: COPD is a complex chronic disease with poorly understood pathogenesis. Integrative genomic approaches have the potential to elucidate the biological networks underlying COPD and lung function. We recently combined genome-wide genotyping and gene expression in 1111 human lung specimens to map expression quantitative trait loci (eQTL).
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress is involved in the pathogenesis of airway obstruction in α1-antitrypsin deficient patients. This may result in a shortening of telomere length, resulting in cellular senescence. To test whether telomere length differs in α1-antitrypsin deficient patients compared with controls, we measured telomere length in DNA from peripheral blood cells of 217 α1-antitrypsin deficient patients and 217 control COPD patients. We also tested for differences in telomere length between DNA from blood and DNA from lung tissue in a subset of 51 controls. We found that telomere length in the blood was significantly longer in α1-antitrypsin deficient COPD patients compared with control COPD patients (p = 1×10-29). Telomere length was not related to lung function in α1-antitrypsin deficient patients (p = 0.3122) or in COPD controls (p = 0.1430). Although mean telomere length was significantly shorter in the blood when compared with the lungs (p = 0.0078), telomere length was correlated between the two tissue types (p = 0.0122). Our results indicate that telomere length is better preserved in α1-antitrypsin deficient COPD patients than in non-deficient patients. In addition, measurement of telomere length in the blood may be a suitable surrogate for measurement in the lung.
PLoS ONE 01/2014; 9(4):e95600. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Due to the pleiotropic effects of nitric oxide (NO) within the lungs, it is likely that NO is a significant factor in the pathogenesis of chronic obstructive pulmonary disease (COPD). The aim of this study was to test for association between single nucleotide polymorphisms (SNPs) in three NO synthase (NOS) genes and lung function, as well as to examine gene expression and protein levels in relation to the genetic variation.
One SNP in each NOS gene (neuronal NOS (NOS1), inducible NOS (NOS2), and endothelial NOS (NOS3)) was genotyped in the Lung Health Study (LHS) and correlated with lung function. One SNP (rs1800779) was also analyzed for association with COPD and lung function in four COPD case--control populations. Lung tissue expression of NOS3 mRNA and protein was tested in individuals of known genotype for rs1800779. Immunohistochemistry of lung tissue was used to localize NOS3 expression.
For the NOS3 rs1800779 SNP, the baseline forced expiratory volume in one second in the LHS was significantly higher in the combined AG + GG genotypic groups compared with the AA genotypic group. Gene expression and protein levels in lung tissue were significantly lower in subjects with the AG + GG genotypes than in AA subjects. NOS3 protein was expressed in the airway epithelium and subjects with the AA genotype demonstrated higher NOS3 expression compared with AG and GG individuals. However, we were not able to replicate the associations with COPD or lung function in the other COPD study groups.
Variants in the NOS genes were not associated with lung function or COPD status. However, the G allele of rs1800779 resulted in a decrease of NOS3 gene expression and protein levels and this has implications for the numerous disease states that have been associated with this polymorphism.
BMC Pulmonary Medicine 11/2013; 13(1):64. · 2.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several infrequent genetic polymorphisms in the SERPINA1 gene are known to substantially reduce concentration of alpha1-antitrypsin (AAT) in the blood. Since low AAT serum levels fail to protect pulmonary tissue from enzymatic degradation, these polymorphisms also increase the risk for early onset chronic obstructive pulmonary disease (COPD). The role of more common SERPINA1 single nucleotide polymorphisms (SNPs) in respiratory health remains poorly understood. We present here an agnostic investigation of genetic determinants of circulating AAT levels in a general population sample by performing a genome-wide association study (GWAS) in 1392 individuals of the SAPALDIA cohort. Five common SNPs, defined by showing minor allele frequencies (MAFs) >5%, reached genome-wide significance, all located in the SERPINA gene cluster at 14q32.13. The top-ranking genotyped SNP rs4905179 was associated with an estimated effect of β = -0.068 g/L per minor allele (P = 1.20*10(-12)). But denser SERPINA1 locus genotyping in 5569 participants with subsequent stepwise conditional analysis, as well as exon-sequencing in a subsample (N = 410), suggested that AAT serum level is causally determined at this locus by rare (MAF<1%) and low-frequent (MAF 1-5%) variants only, in particular by the well-documented protein inhibitor S and Z (PI S, PI Z) variants. Replication of the association of rs4905179 with AAT serum levels in the Copenhagen City Heart Study (N = 8273) was successful (P<0.0001), as was the replication of its synthetic nature (the effect disappeared after adjusting for PI S and Z, P = 0.57). Extending the analysis to lung function revealed a more complex situation. Only in individuals with severely compromised pulmonary health (N = 397), associations of common SNPs at this locus with lung function were driven by rarer PI S or Z variants. Overall, our meta-analysis of lung function in ever-smokers does not support a functional role of common SNPs in the SERPINA gene cluster in the general population.
[Show abstract][Hide abstract] ABSTRACT: Alveolar macrophages play an important role in chronic obstructive pulmonary disease (COPD) via production of matrix metalloproteinases (MMPs) and cathepsins as well as their inhibitors, tissue inhibitors of metalloproteinases (TIMPs) and cystatin C (CST3). We hypothesized that expression levels of these molecules by alveolar macrophages at baseline and after stimulation would be influenced by genotype and associated with COPD phenotypes.Quantitative PCR and enzyme-linked immunosorbent assays/gelatin zymography were used to investigate expression levels of mRNA and protein, respectively. The relationships of expression with genotype, pulmonary function and emphysema were analysed.The results showed that basal expression level of MMP12 mRNA was inversely related to DL,CO/VA and to FEV1/FVC after correction for multiple comparisons. The expression level of MMP12 protein stimulated with LPS was also inversely related to DL,CO/VA and was positively related to the extent of emphysema. The basal expression of MMP1 mRNA was positively correlated with the extent of emphysema. Cathepsin L protein level was positively associated with FEV1% predicted.We conclude that increased MMP12 and MMP1 expression may play a role in the pathogenesis of emphysema. Cathepsin L and MMP9 may be involved in the development of airflow limitation.
European Respiratory Journal 07/2013; · 6.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Respiratory diseases are the most frequent chronic illnesses in babies and children. Although a vigorous innate immune system is critical for maintaining lung health, a balanced response is essential to minimize damaging inflammation. We investigated the functional and clinical impact of human genetic variants in the promoter of NFKBIA, which encodes IκBα, the major negative regulator of NF-κB. In this study, we quantified the functional impact of NFKBIA promoter polymorphisms (rs3138053, rs2233406, and rs2233409) on promoter-driven protein expression, allele-specific and total NFKBIA mRNA expression, IκBα protein expression, and TLR responsiveness; mapped innate immune regulatory networks active during respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia; and genotyped and analyzed independent cohorts of children with respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia. Genetic variants in the promoter of NFKBIA influenced NFKBIA gene expression, IκBα protein expression, and TLR-mediated inflammatory responses. Using a systems biology approach, we demonstrated that NFKBIA/IκBα is a central hub in transcriptional responses of prevalent childhood lung diseases, including respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia. Finally, by examining independent pediatric lung disease cohorts, we established that this immunologically relevant genetic variation in the promoter of NFKBIA is associated with differential susceptibility to severe bronchiolitis following infection with respiratory syncytial virus, airway hyperresponsiveness, and severe bronchopulmonary dysplasia. These data highlight the importance of negative innate immune regulators, such as NFKBIA, in pediatric lung disease and begin to unravel common aspects in the genetic predisposition to bronchopulmonary dysplasia, bronchiolitis, and childhood asthma.
The Journal of Immunology 03/2013; · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peanut allergy (PA) is a common and serious food allergy and its prevalence has increased in the past decade. Although there is strong evidence of inheritance, the genetic causes of this disease are not well understood. Previously, a large-scale genome-wide association study described an association between human leukocyte antigen (HLA)-DQB1 and asthma; the aim of this study was to evaluate the association between HLA-DQB1 and PA. Genotypic and allelic profiles were established for 311 Caucasian members of a well-described Canadian group of children with PA and 226 Caucasian controls. Firth's logistic regression analyses showed associations between HLA-DQB1 alleles and PA for DQB1*02 (P=1.1 × 10(-8), odds ratio (OR)=0.09 (CI=0.03-0.23)) and DQB1*06:03P alleles (P=2.1 × 10(-2), OR=2.82 (CI=1.48-5.45)). This study of HLA in PA demonstrates specific association between two allelic groups of the HLA-DQB1 gene (DQB1*02 and DQB1*06:03P) and PA, highlighting its possible role in the development of this disease.European Journal of Human Genetics advance online publication, 27 February 2013; doi:10.1038/ejhg.2013.13.
European journal of human genetics: EJHG 02/2013; · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human beings come in all shapes and sizes. Heterogeneity makes life interesting, but leads to inter-individual variation in disease susceptibility and response to therapy. One major health challenge is to develop "personalised medicine"; therapeutic interventions tailored to an individual to ensure optimal treatment of disease. Asthma is a heterogeneous disease with several different phenotypes triggered by multiple gene-environment interactions. Inhaled corticosteroids and β2-agonists have been the mainstay asthma therapies for 30 years, but they are not effective in all patients, while high costs and side-effects also drive the need for better targeted treatment of asthma. Pharmacogenetics is the study of variations in the genetic code for proteins in signaling pathways targeted by pharmacological therapies. Biomarkers are biological markers obtained from patients that can aid in asthma diagnosis, prediction of treatment response, and monitoring of disease control. This review presents a broad discussion of the use of genetic profiling and biomarkers to better diagnose, monitor, and tailor the treatment of asthmatics. We also discuss possible future developments in personalised medicine, including the construction of artificially engineered airway tissues containing a patient's own cells for use as personalised drug-testing tools.
Current Allergy and Asthma Reports 12/2012; · 2.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genome-wide association studies (GWAS) have identified loci reproducibly associated with pulmonary diseases; however, the molecular mechanism underlying these associations are largely unknown. The objectives of this study were to discover genetic variants affecting gene expression in human lung tissue, to refine susceptibility loci for asthma identified in GWAS studies, and to use the genetics of gene expression and network analyses to find key molecular drivers of asthma. We performed a genome-wide search for expression quantitative trait loci (eQTL) in 1,111 human lung samples. The lung eQTL dataset was then used to inform asthma genetic studies reported in the literature. The top ranked lung eQTLs were integrated with the GWAS on asthma reported by the GABRIEL consortium to generate a Bayesian gene expression network for discovery of novel molecular pathways underpinning asthma. We detected 17,178 cis- and 593 trans- lung eQTLs, which can be used to explore the functional consequences of loci associated with lung diseases and traits. Some strong eQTLs are also asthma susceptibility loci. For example, rs3859192 on chr17q21 is robustly associated with the mRNA levels of GSDMA (P = 3.55×10(-151)). The genetic-gene expression network identified the SOCS3 pathway as one of the key drivers of asthma. The eQTLs and gene networks identified in this study are powerful tools for elucidating the causal mechanisms underlying pulmonary disease. This data resource offers much-needed support to pinpoint the causal genes and characterize the molecular function of gene variants associated with lung diseases.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: The innate immune system is essential for host survival because of its ability to recognize invading pathogens and mount defensive responses. OBJECTIVES: We sought to identify genetic associations of innate immunity genes with atopy and asthma and interactions with early viral infections (first 12 months of life) in a high-risk birth cohort. METHODS: Three Canadian family-based studies and 1 Australian population-based case-control study (n = 5565) were used to investigate associations of 321 single nucleotide polymorphisms (SNPs) in 26 innate immunity genes with atopy, asthma, atopic asthma, and airway hyperresponsiveness. Interactions between innate immunity genes and early viral exposure to 3 common viruses (parainfluenza, respiratory syncytial virus, and picornavirus) were examined in the Canadian Asthma Primary Prevention Study by using both an affected-only family-based transmission disequilibrium test and case-control methods. RESULTS: In a joint analysis of all 4 cohorts, IL-1 receptor 2 (IL1R2) and Toll-like receptor 1 (TLR1) SNPs were associated with atopy after correction for multiple comparisons. In addition, an NFKBIA SNP was associated with atopic asthma. Six SNPs (rs1519309 [TLR3], rs740044 [ILIR2], rs4543123 [TLR1], rs5741812 [LBP], rs917998 [IL18RAP], and rs3136641 [NFKBIB]) were significant (P < .05, confirmed with 30,000 permutations) in both the combined analysis of main genetic effects and SNP-virus interaction analyses in both case-control and family-based methods. The TLR1 variant (rs4543123) was associated with both multiple viruses (respiratory syncytial virus and parainfluenza virus) and multiple phenotypes. CONCLUSION: We have identified novel susceptibility genes for asthma and related traits and interactions between these genes and early-life viral infections.
The Journal of allergy and clinical immunology 10/2012; · 12.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Prior studies have demonstrated that the distal 1.5 kb of the MMP-1 promoter is fundamental in directing the induction of the MMP-1 gene by cigarette smoke. METHODS: To characterize the genetic variants in the MMP-1 cigarette smoke-responsive element, deep re-sequencing of this element was performed on DNA samples from participants in the Lung Health Study. Furthermore, evidence of Sp1 binding to the MMP-1 promoter was assessed using chromatin immunoprecipitation assays and the influence of cigarette smoke exposure on this interaction was evaluated in cultured human small airway epithelial cells. RESULTS: Ten polymorphisms (four novel) were detected in the cigarette smoke-responsive element. Chromatin immunoprecipitation assays to assess the protein-DNA interactions at Sp1 sites in the MMP-1 promoter showed increased binding to the Sp1 sites in the cigarette smoke-responsive element in small airway epithelial cells treated with cigarette smoke extract. In contrast, a Sp1 site outside of the element exhibited the opposite effect. None of the polymorphisms were more prevalent in the fast decliners versus the slow decliners (fast decliners = mean -4.14% decline in FEV1 % predicted per year vs. slow decliners = mean +1.07%/year). CONCLUSIONS: Sequencing analyses identified four novel polymorphisms within the cigarette smoke-responsive element of the MMP-1 promoter. This study identifies functional activity within the cigarette smoke-responsive element that is influenced by cigarette smoke exposure and examines this region of the promoter within a small patient population.
Respiratory research 09/2012; 13(1):79. · 3.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Accelerated lung function decline is a key COPD phenotype; however, its genetic control remains largely unknown. We performed a genome-wide association study using the Illumina Human660W-Quad v.1_A BeadChip. Generalized estimation equations were used to assess genetic contributions to lung function decline over a 5-year period in 4,048 European American Lung Health Study participants with largely mild COPD. Genotype imputation was performed using reference HapMap II data. To validate regions meeting genome-wide significance, replication of top SNPs was attempted in independent cohorts. Three genes (TMEM26, ANK3 and FOXA1) within the regions of interest were selected for tissue expression studies using immunohistochemistry. Two intergenic SNPs (rs10761570, rs7911302) on chromosome 10 and one SNP on chromosome 14 (rs177852) met genome-wide significance after Bonferroni. Further support for the chromosome 10 region was obtained by imputation, the most significantly associated imputed SNPs (rs10761571, rs7896712) being flanked by observed markers rs10761570 and rs7911302. Results were not replicated in four general population cohorts or a smaller cohort of subjects with moderate to severe COPD; however, we show novel expression of genes near regions of significantly associated SNPS, including TMEM26 and FOXA1 in airway epithelium and lung parenchyma, and ANK3 in alveolar macrophages. Levels of expression were associated with lung function and COPD status. We identified two novel regions associated with lung function decline in mild COPD. Genes within these regions were expressed in relevant lung cells and their expression related to airflow limitation suggesting they may represent novel candidate genes for COPD susceptibility.
[Show abstract][Hide abstract] ABSTRACT: An oxidant-antioxidant imbalance in the lung contributes to the development of chronic obstructive pulmonary disease (COPD) that is caused by a complex interaction of genetic and environmental risk factors. Nuclear erythroid 2-related factor 2 (NFE2L2 or NRF2) is a critical molecule in the lung's defense mechanism against oxidants. We investigated whether polymorphisms in the NFE2L2 pathway affected the rate of decline of lung function in smokers from the Lung Health Study (LHS)(n = 547) and in a replication set, the Vlagtwedde-Vlaardingen cohort (n = 533). We selected polymorphisms in NFE2L2 in genes that positively or negatively regulate NFE2L2 transcriptional activity and in genes that are regulated by NFE2L2. Polymorphisms in 11 genes were significantly associated with rate of lung function decline in the LHS. One of these polymorphisms, rs11085735 in the KEAP1 gene, was previously shown to be associated with the level of lung function in the Vlagtwedde-Vlaardingen cohort but not with decline of lung function. Of the 23 associated polymorphisms in the LHS, only rs634534 in the FOSL1 gene showed a significant association in the Vlagtwedde-Vlaardingen cohort with rate of lung function decline, but the direction of the association was not consistent with that in the LHS. In summary, despite finding several nominally significant polymorphisms in the LHS, none of these associations were replicated in the Vlagtwedde-Vlaardingen cohort, indicating lack of effect of polymorphisms in the NFE2L2 pathway on the rate of decline of lung function.
[Show abstract][Hide abstract] ABSTRACT: Some have suggested that chronic obstructive pulmonary disease (COPD) is a disease of accelerated aging. Aging is characterized by shortening of telomeres. The relationship of telomere length to important clinical outcomes such as mortality, disease progression and cancer in COPD is unknown. Using quantitative polymerase chain reaction (qPCR), we measured telomere length of peripheral leukocytes in 4,271 subjects with mild to moderate COPD who participated in the Lung Health Study (LHS). The subjects were followed for approximately 7.5 years during which time their vital status, FEV(1) and smoking status were ascertained. Using multiple regression methods, we determined the relationship of telomere length to cancer and total mortality in these subjects. We also measured telomere length in healthy "mid-life" volunteers and patients with more severe COPD. The LHS subjects had significantly shorter telomeres than those of healthy "mid-life" volunteers (p<.001). Compared to individuals in the 4(th) quartile of relative telomere length (i.e. longest telomere group), the remaining participants had significantly higher risk of cancer mortality (Hazard ratio, HR, 1.48; p = 0.0324) and total mortality (HR, 1.29; p = 0.0425). Smoking status did not make a significant difference in peripheral blood cells telomere length. In conclusion, COPD patients have short leukocyte telomeres, which are in turn associated increased risk of total and cancer mortality. Accelerated aging is of particular relevance to cancer mortality in COPD.
PLoS ONE 01/2012; 7(4):e35567. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: No optimal housekeeping genes (HKGs) have been identified for CD4(+) T cells from non-depressive asthmatic and depressive asthmatic adults for normalizing quantitative real-time PCR (qPCR) assays. The aim of present study was to select appropriate HKGs for gene expression analysis in purified CD4(+) T cells from these asthmatics.
Three groups of subjects (Non-depressive asthmatic, NDA, n = 10, Depressive asthmatic, DA, n = 11, and Healthy control, HC, n = 10 respectively) were studied. qPCR for 9 potential HKGs, namely RNA, 28S ribosomal 1 (RN28S1), ribosomal protein, large, P0 (RPLP0), actin, beta (ACTB), cyclophilin A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase 1 (PGK1), beta-2-microglobulin (B2M), glucuronidase, beta (GUSB) and ribosomal protein L13a (RPL13A), was performed. Then the data were analyzed with three different applications namely BestKeeper, geNorm, and NormFinder.
The analysis of gene expression data identified B2M and RPLP0 as the most stable reference genes and showed that the level of PPIA was significantly different among subjects of three groups when the two best HKGs identified were applied. Post-hoc analysis by Student-Newman-Keuls correction shows that depressive asthmatics and non-depressive asthmatics exhibited lower expression level of PPIA than healthy controls (p<0.05).
B2M and RPLP0 were identified as the most optimal HKGs in gene expression studies involving human blood CD4(+) T cells derived from normal, depressive asthmatics and non-depressive asthmatics. The suitability of using the PPIA gene as the HKG for such studies was questioned due to its low expression in asthmatics.
PLoS ONE 01/2012; 7(10):e48367. · 3.53 Impact Factor