Abstract
Diisocyanates are the most common cause of occupational asthma, but risk factors are not well defined. A case-control study was conducted to investigate whether genetic variants in inflammatory response genes (TNFα, IL1α, IL1β, IL1RN, IL10, TGFB1, ADAM33, ALOX-5, PTGS1, PTGS2 and NAG-1/GDF15) are associated with increased susceptibility to diisocyanate asthma (DA). These genes were selected based on their role in asthmatic inflammatory processes and previously reported associations with asthma phenotypes. The main study population consisted of 237 Caucasian French Canadians from among a larger sample of 280 diisocyanate-exposed workers in two groups: workers with specific inhalation challenge (SIC) confirmed DA (DA+, n = 95) and asymptomatic exposed workers (AW, n = 142). Genotyping was performed on genomic DNA, using a 5′ nuclease PCR assay. After adjusting for potentially confounding variables of age, smoking status and duration of exposure, the PTGS1 rs5788 and TGFB1 rs1800469 single nucleotide polymorphisms (SNP) showed a protective effect under a dominant model (OR = 0.38; 95% CI = 0.17, 0.89 and OR = 0.38; 95% CI = 0.18, 0.74, respectively) while the TNFα rs1800629 SNP was associated with an increased risk of DA (OR = 2.08; 95% CI = 1.03, 4.17). Additionally, the PTGS2 rs20417 variant showed an association with increased risk of DA in a recessive genetic model (OR = 6.40; 95% CI = 1.06, 38.75). These results suggest that genetic variations in TNFα, TGFB1, PTGS1 and PTGS2 genes contribute to DA susceptibility.
Introduction
Diisocyanates, low-molecular weight reactive chemicals used in diverse industrial processes, are one of the most frequently reported causes of occupational asthma in the work-place. Toluene diisocyanate (TDI), 4,4′-diphenylmethane diisocyanate (MDI) and hexamethylene diisocyanate (HDI) are the most commonly used monomers in industry and estimated to cause asthma in 5–15% of workers with long-term exposure (Bernstein, Citation1996; Wisnewski & Redlich, Citation2001). Diisocyanates bind selectively to human proteins and these protein conjugates act as antigens that induce airway inflammation with the release of inflammatory mediators. Of these mediators, cytokines play a critical role in asthma pathogenesis by orchestrating the allergic inflammatory response (Maestrelli et al., Citation1995). Elevated levels of both T-helper (TH)-1 and TH2 cytokines have been demonstrated in the airways of subjects with different asthma phenotypes, supporting the heterogenic nature of disease (Broide et al., Citation1992; Fisseler-Eckhoff et al., Citation2011; Maestrelli et al., Citation1995, Citation1997; Mapp et al., Citation2009; Piirila et al., Citation2008). Animal and in vitro models revealed multiple roles of cytokines in non-specific inflammatory processes as well as specific immune events in response to diisocyanate exposure (Chiung et al., Citation2010; Matheson et al., Citation2002; Swierczynska-Machura et al., Citation2012).
Although many studies investigated the association between single nucleotide polymorphisms (SNP) in pro-inflammatory cytokine genes and asthma phenotypes (Che et al., Citation2014; Padron-Morales et al., Citation2013; Panek et al., Citation2014; Savenije et al., Citation2011; Zeyrek et al., Citation2008), there is only one study that focused on DA. Beghé et al. (Citation2004) studied tumor necrosis factor-α (TNFα) A-308G polymorphism in TDI-induced asthma but found no significant difference in frequency of this variant between cases and asymptomatic exposed subjects. The interleukin-1 (IL1), IL10, TNFα and transforming growth factor-β (TGFB1) genes have not previously been studied in relation to DA susceptibility.
Other genes focused on in this study included ADAM33 (ADAM metallopeptidase domain 33), PTGS1 (prostaglandin-endoperoxide synthase-1, previously COX1), PTGS2 (previously COX2), ALOX5 (arachidonate 5-lipoxygenase) and NAG-1/GDF15 (non-steroidal anti-inflammatory drug-activated gene). ADAM33 is the first reported asthma-susceptibility gene identified by positional cloning (van Eerdewegh et al., Citation2002). It is highly expressed in epithelium and airway smooth muscle cells and plays an important role in airway remodeling (Tripathi et al., Citation2014). It has been suggested that altered ADAM33 protein function may promote inflammation through impaired or enhanced release of cytokines and growth factors (Cakebread et al., Citation2004). Cyclo-oxygenases are key enzymes that catalyze the biosynthesis of prostaglandins, which are essential mediators of inflammation in asthma (Dubois et al., Citation1998). ALOX5 is the first enzyme in the biosynthetic pathway leading to the production of leukotrienes and plays an important role in allergic and inflammatory diseases (Duroudier et al., Citation2009; Radmark et al., Citation2007). NAG-1/GDF15 is a member of the TGFβ cytokine superfamily that plays an important role in controlling inflammatory responses by reducing leukocyte recruitment and maintaining homeostasis (Zarbock & Rossaint, Citation2011). Numerous studies showed associations between genetic variants of PTGS1, PTGS2, ADAM33 and ALOX5 genes and allergic asthma, but these variations have not been studied for association with DA (Holloway et al., Citation2008; Shi et al., Citation2005, Citation2008; Song et al., Citation2013). In addition, the NAG1/GDF15 variants were not explored in asthma phenotypes.
Since the impact of a given gene on the phenotype is influenced by occupational and environmental exposures, gene–environment interaction explains phenotype variance better than two factors considered separately. Occupational asthma (OA) is an excellent model for studying gene–environment interactions since the causal agent can be identified with specific inhalation challenge (SIC) and the lag period between initial exposure and onset of sensitization and clinical symptoms can be followed. Therefore, it is important to study asthma candidate genes in OA since different occupational and environmental exposures might have a different effect on the phenotype. Based on their role in asthma pathogenesis and previously reported associations with asthma phenotypes, the present candidate gene association study was designed to investigate the role of SNP in a group of inflammatory response genes as predictors of susceptibility to DA. The identification of variations in specific genes involved in the DA process might lead to a better understanding of the underlying disease pathways and, thus, guide for the development of more effective therapeutic and preventive strategies.
Materials and methods
Study participants
The main study population consisted of 237 Caucasian French Canadians from among a larger sample of 280 diisocyanate (HDI, MDI and TDI)-exposed workers. The main study analyses were conducted on only the Caucasian French Canadians to avoid the possibility of bias due to population stratification (Heiman et al., Citation2004). Also, the numbers of subjects recruited from other non-Caucasian-French Canadian and Spanish populations were too small to independently support statistical modeling. However, identical statistical analyses were also conducted on the entire sample and the results are presented as supplementary data. Of the 237 participants, 95 workers were diagnosed with DA (DA+) based on a positive specific inhalation challenge (SIC) test, while 142 HDI-exposed asymptomatic workers (AW) served as controls. We did not perform a separate analysis on HDI-induced DA+ cases and controls since the majority of DA+ subjects were exposed to HDI and overall numbers would be too small for meaningful statistical analysis.
Symptomatic subjects were recruited from occupational pulmonary disease clinics located in Canada (Sacre Coeur Hospital, Montreal; Laval Hospital, Sainte-Foy; University Health Network, Toronto) and Spain (Fundacion Jimenez Diaz, Madrid and Hospital Vall d’Hebron, Barcelona). The subjects underwent SIC with the appropriate work-relevant diisocyanate chemicals according to previously described protocols (Malo et al., Citation1999; Sastre et al., Citation2003). A decrease in FEV1 of at least 20% from pre-challenge baseline during the early and/or late asthmatic response was defined as a positive SIC test. AW controls were recruited in Quebec (Canada) from HDI-exposed painters. Data regarding age, sex, ethnicity, smoking status, time of exposure and respiratory symptoms were collected by questionnaire. Atopy was evaluated by skin prick testing to common aeroallergens, defined by a positive reaction of at least 3 mm greater than saline control for at least one allergen. Whole blood was collected for genetic testing. All subjects gave informed consent and the study protocol is approved and renewed annually by Institutional Review Boards of the National Institute for Occupational Safety and Health and each participating institution.
Genotyping
Genomic DNA was extracted from whole blood samples using the QIAamp blood kit (QIAGEN Inc., Chatsworth, CA). Genotyping was performed on genomic DNA, using a 5′ nuclease PCR assay. Primers and probes were designed, using the Assay-by-Design™ service from Applied Biosystems (Foster City, CA). PCR amplification was performed in a volume of 25 µl containing 10 ng genomic DNA, 12.5 µl 2X Taqman® Universal Master Mix, 200 nM probe and 900 nM primer. Cycling conditions were 50 °C for 2 min and 95 °C for 10 min, followed by 50 cycles at 92 °C for 30 s and 60 °C for 1 min. Amplification was performed using a StepOnePlus™ Real-Time PCR System (Applied Biosystems). Positive and negative controls were used within each run of PCR amplification. All samples with ambiguous results were repeated, as were a random selection of 10% of all samples to ensure laboratory quality control.
Statistical analyses
Comparisons between groups on demographic variables were analyzed using chi-square tests or analysis of variance for discrete and continuous variables, respectively. Initial analyses, including tests for Hardy–Weinberg Equilibrium (HWE), allele and genotype frequencies were calculated using the Fisher’s Exact Test. Unadjusted odds ratios (OR) were calculated using contingency tables. Adjusted ORs were calculated using logistic regression while adjusting for age, exposure time (months) and smoking status (current/ex/never). Sex and atopy were not significant in any models and were not included in the final analysis. Since underlying genetic models are unknown a priori, the association between each SNP and DA status was analyzed using three genetic models. These include a dominant model (comparing homozygous wild-type genotype with variant allele-carrying genotypes), a recessive model (comparing wild-type allele-carrying genotypes with homozygous variant genotype) and an additive model. All statistical analyses were performed using SAS 9.2 (SAS Institute, Cary, NC). SNAP2 tools were used to update annotations of significant SNP according to dbSNP135 and to find proxy SNP within 500 kb based on linkage disequilibrium (LD) and physical distance (Johnson et al., Citation2008). Since SNP located in a genomic region with various transcription factor (TF) binding sites are more likely to influence transcriptional regulation, we searched functional elements overlapping with the selected SNP. RegulomeDB was used to annotate SNP with known and predicted regulatory elements (Boyle et al., Citation2012).
Results
Subject characteristics
The demographic characteristics of the study groups included in the statistical analyses are described in . The participants included in this analysis were Caucasian French-Canadians. Mean age was higher in the DA+ group than AW controls (42.4 versus 30.4 years). While the DA+ group consisted of subjects exposed to HDI, MDI and TDI (53:22:20), AW controls were exposed only to HDI in the workplace. AW controls had less duration of exposure to diisocyanates than the DA+ group (65.8 versus 144.6 months). The prevalence of smoking was significantly different between the DA+ group and AW controls (p < 0.0001). The frequency of atopy was similar between the two groups (58.9% in DA+, 53.5% in AW controls). Allele frequencies in the control population were similar to those determined in other studies involving Caucasian populations and were in HWE (data not shown). Demographic characteristics of the initial study sample are shown in Supplementary Table 1.
Table 1. Demographics of study groups.
Genotype distribution and genetic models
shows the distribution of genotypes in the study population and the p values represent the comparison of the proportions of genotypes between two groups. The frequency of the PTGS2 rs20417 SNP showed only a marginal significance between the DA+ group and AW controls (p = 0.057) in the univariate analysis.
Table 2. Distribution of genotype frequencies between the groups.
presents the results of the logistic regression model after adjusting for potentially confounding variables of age, smoking status and duration of exposure. In a dominant model, the PTGS1 rs5788 and TGFB1 rs1800469 SNP were associated with a protection against DA (OR = 0.38; 95% confidence interval (CI) = 0.17, 0.89, p = 0.026 and OR = 0.38; 95% CI = 0.18, 0.74, p = 0.005) while the TNFα rs1800629 SNP conferred an increased risk of disease (OR = 2.08; 95% CI = 1.03, 4.17, p = 0.041). On the other hand, the PTGS2 rs20417 SNP showed an association with an increased risk of DA in a recessive model (OR = 6.40; 95% CI = 1.06, 38.75, p = 0.043). This SNP also showed a trend towards significance in an additive model (OR = 5.99; 95% CI = 0.98, 36.57, p = 0.053). Results of analyses on the larger sample that included subjects from Spain and non-Caucasian French Canadians were similar to those in and , suggesting population stratification did not bias the findings (Supplementary Tables 2 and 3).
Table 3. Logistic regression model for significant variations: DA+ versus AW controls.
Regulatory information for significant associations
The four significant SNP identified from data analysis were used as inputs to the SNAP SNP Annotation and Proxy Search tools to update SNP IDs according to dbSNP135 and to find additional SNP in complete LD (r2 = 1). This led to the identification of an additional 17 correlated SNP using data from the International HapMap Project (International HapMap Consortium et al., Citation2010). The total set of 21 SNP was then used as input to the RegulomeDB web resource, which integrates data from the ENCODE projects and other data sources regarding various types of functional assays including DNaseI-seq, ChIP-seq, RNAseq and eQTL analyses (Boyle et al., Citation2012). RegulomeDB scores of all the significant and correlated SNPs are presented in Supplementary Table 4. Two SNPs (TGFB1 rs1800469 and TNFα rs1800629) were likely to affect binding and linked to expression of a gene target (MGC4093 and AIF, respectively) (RegulomeDB scores = 1b and 1f, respectively). The PTGS2 rs20417 SNP was also likely to affect binding with a RegulomeDB score of 2b. There were no regulatory data available for the PTGS1 rs5788 SNP. shows the SNP with RegulomeDB scores between 1–3 (scoring refers to available data types supporting a functional role for the variant). All SNP with putative regulatory function (RegulomeDB Score 1–6) are given in Supplementary Table 5.
Table 4. Significant and correlated SNPs with putative regulatory function (RegulomeDB Score 1–3).
Discussion
This is the first comprehensive study to explore potential associations of genetic variants in inflammatory genes with DA. The allele frequencies of 19 SNP in 11 genes were determined in a Caucasian worker population of European background. Two SNP in TNFα and TGFB1 genes were significantly associated with DA. TNFα is one of the major pro-inflammatory cytokines located within the human major histocompatibility complex (MHC). This region has been repeatedly linked to asthma (Daniels et al., Citation1996; Xu et al., Citation2001). Previous studies demonstrated increased levels of TNFα in asthmatic airways and reported an association of TNFα rs1800629 (A-308G) SNP with asthma phenotypes as well as elevated total serum IgE levels (Ricciardolo et al., Citation2013; Sharma et al., Citation2006; Shin et al., Citation2004; Witte et al., Citation2002). A recent meta-analysis involving 34 studies confirmed these findings and reported rs1800629 SNP as a risk factor for asthma (Yang et al., Citation2014). The -308A allele has been associated with increased expression levels of TNFα in stimulated human white blood cells (Wilson et al., Citation1997). In our analysis, carriage of the -308A allele was associated with an increased risk of DA.
Beghè et al. (Citation2004) investigated this SNP in TDI-induced asthma, but did not find any association. The difference between two study results could be related to differences in population genetic structure and type of diisocyanate exposure. We found that the rs1800629 SNP (RegulomeDB score, 1f) is an eQTL (expression quantitative trait loci) for AIF1 gene (Allograft Inflammatory Factor 1) and is situated in the Sp1 (specificity protein 1) binding motif. AIF1 is also located in the MHC region and is induced by cytokines. It is suspected that AIF1 is involved in the regulation of vascular smooth muscle cell growth. Sp1 is a ubiquitously expressed transcriptional activator protein that binds GC-rich regions of DNA to regulate gene transcription (Suske, Citation1999; Tan & Khachigian, Citation2009). Sp1 has been shown to be involved in the regulation of VEGF transcription that is a critical mediator of asthma pathogenesis. Clifford et al. (Citation2012) suggested that increased Sp1 binding might contribute to elevated VEGF secretion in asthmatics. Sp1 was also found to be one of the transcription factors involved in the regulation of basal and inducible 5-lipoxygenase gene transcriptions (Silverman et al., Citation1998). It is possible that the TNFα rs1800629 SNP alters Sp1 binding to this region and influences downstream biological processes.
The other significantly associated SNP (rs1800469, -509 C/T) is located in the promoter region of the TGFB1 gene. TGFB1 is an immunoregulatory cytokine that is increased in the airways of asthmatic individuals (Ohno et al., Citation1996). The rs1800469 is located within a YY1 (Ying Yang 1) consensus binding site and is known to regulate transcriptional activity (Silverman et al., Citation2004). The rs1800469 SNP has been associated with elevated gene expression and circulating TGFB1 and IgE levels (Grainger et al., Citation1999; Jacob et al., Citation2013). Increased transcriptional activity of the T allele, as compared to the C allele, has been reported (Grainger et al., Citation1999). While some studies showed an association between variant rs1800469 and allergic asthma (Li et al., Citation2007; Pulleyn et al., Citation2001; Sharma et al., Citation2009; Silverman et al., Citation2004; Wu et al., Citation2010; Yang et al., Citation2011), others reported no association with asthma or other allergies (Buckova et al., Citation2001; Hobbs et al., Citation1998). In our analysis, carriage of the T allele was associated with a protection against DA. TGFB1 is a potent suppressor of inflammation and found to be protective in acute animal models of asthma (Alcorn et al., Citation2007; Li et al., Citation2006; Nakao et al., Citation2000). On the other hand, excess TGFB1 activity was reported to induce fibrosis, pulmonary inflammation and structural remodeling (Crosby & Waters, Citation2010; McMillan et al., Citation2005). This is the first report showing an association between TGFB1 rs1800469 variant and DA. Our findings are consistent with the immunomodulatory role of TGFB1 in the asthmatic process and suggest that the same SNP could be protective or adverse depending on environmental and other genetic factors.
In line with previous observations, RegulomeDB showed evidence of regulatory function for the rs1800469 SNP (score, 1b). RegulomeDB cites rs1800469 is an eQTL for MGC4093, affects binding of POLR2A, CREBBP, CTCF and falls within the COUPTF binding motif. MGC4093 is one of the two genes (the other is MGC2055) with unknown function in the 30 kb region surrounding the TGFB1 gene. This gene lies between TGFB1 rs1800469 and rs7045 and its transcripts have been reported in various UniGene libraries of lung tissue. The genomic region including TGFB1 and MGC4093 is organized in two large haplotype blocks in Caucasians. Based on association and LD patterns and the fact that only TGFB1 spans both of these haplotype blocks, the MGC4093 has been ruled out as a modifier of other lungs diseases such as chronic obstructive pulmonary disease and cystic fibrosis. TGFB1 SNPs were proposed as a likely modifier of these diseases (Celedon et al., Citation2004; Drumm et al., Citation2005). Although these findings need to be replicated in an independent sample, our results are consistent with previous observations and suggest TGFB1 rs1800469 SNP as a likely modifier of DA.
One of the proteins affected by this SNP is CTCF (the transcription regulator CCCTC-binding factor). It is a DNA insulator protein with the ability to block enhancers from activating their target genes via chromatin remodeling. CTCF was found to be essential for the GATA3/SATB1-mediated regulation of the TH2 cytokine gene expression (Ribeiro de Almeida et al., Citation2009). Other affected protein CREBBP (CREB binding protein) is one of the transcriptional co-activators with intrinsic histone acetyltransferase (HAT) activity. HAT and histone deacetylase (HDAC) regulate chromatin acetylation and likely play a key role in induction of inflammatory genes. Several studies reported an imbalance between activities of HATs and HDAC in bronchial biopsies, alveolar macrophages and peripheral blood mononuclear cells in asthma (Cosio et al., Citation2004; Ito et al., Citation2002). POLR2A (RNA II Polypeptide A) is a DNA-dependent RNA polymerase catalyzing transcription of DNA into RNA. The rs1800469 variant may play an important role in affecting the binding of these regulatory proteins and consequently the risk of DA. The same SNP is also situated in the COUP binding motif. COUPTFs (COUP transcription factor) are transcription factors that play key roles in the development and function of the immune system (Leid et al., Citation2004). It is possible that the overlap between rs1800469 and this binding motif influence the optimal expression of particular inflammatory genes. Examining these interactions in the context of DA pathogenesis could yield additional insight into the molecular mechanisms involved.
The PTGS2 rs20417 is located within the promoter region and the -765C allele has been shown to have significantly lower promoter activity in vitro compared with the -765G allele (Papafili et al., Citation2002). PTGS2 is highly inducible by mitogenic and pro-inflammatory stimuli and involved in the regulation of inflammatory responses. Increased expression of PTGS2 is observed in the airway epithelium and submucosa of asthmatics (Sousa et al., Citation1997; Taha et al., Citation2000). While some studies reported associations of PTGS2 SNPs with asthma phenotypes (Chan et al., Citation2007; Shi et al., Citation2008; Szczeklik et al., Citation2004), others found no relationship (Shi et al., Citation2004). In our analysis, the PTGS2 rs20417 SNP conferred an increased risk of DA with an OR of 6.4 and showed evidence of regulatory function (RegulomeDB score, 2b). The rs20417 SNP has indications for binding TFAP2A (transcription factor AP-2 alpha), TFAP2C (Transcription Factor AP-2 Gamma) proteins. However, results of this interaction in the asthmatic process are unknown. This SNP is also located in the binding motifs for STAT1, E2F and EWSR1-FLI1. STAT (Signal transducer and activator of transcription) transcription factors play critical roles in regulating cell growth and differentiation, homeostasis and the immune response (Horvath, Citation2000; Ramana et al., Citation2000). E2F transcription factors are involved in the regulation of cell cycle and DNA replication in mammalian cells (Cam & Dynlacht, Citation2003). EWSR1 (EWS RNA-Binding Protein 1) acts as a transcriptional activator and plays a role in the initiation of transcription (Bertolotti et al., Citation1998). The overlap of the rs20417 SNP with these motif sites may have an impact on the regulation of gene expression.
The PTGS1 rs5788 SNP showed a protective effect under a dominant model. PTGS1 SNPs have been previously studied, but no association with asthma phenotypes (allergic and aspirin-intolerant asthma) was found (Shi et al., Citation2005). This is the first study to report an association between the PTGS1 rs5788 SNP and an asthma phenotype. No regulatory information was found for that specific SNP.
The major strengths of this study include a well-defined phenotype and examination of candidate genes based on their functional role in disease pathogenesis. We were also able to test our genetic associations while adjusting for potential independent confounding factors such as age, smoking history and exposure duration. The major limitations include small sample size due to rarity of DA, lack of information regarding the relative exposure levels of the AW and DA groups and the issue of multiple interferences. Another limitation is that the AW controls were younger and had less exposure to diisocyanates than cases. This was unintentional due to difficulty in the recruitment of age-matched workplace controls. However, the AW group had mean exposure duration of 5.5 years. It has been reported that nearly 40% and 60% of subjects exposed to isocyanates become symptomatic within, respectively, 1 year and after 5 years of exposure (Malo et al., Citation1992). Therefore, it is reasonable to expect this exposure period to be sufficient for developing DA in susceptible individuals. The results were not corrected for multiple comparisons since our analyses were based on a priori hypotheses. Instead, we reported all tests that reached the 0.05 level of significance.
In conclusion, this case-control study reports, for the first time, an association between DA and SNPs in TNFα, FB1, PTGS2 and PTGS1 genes. Further studies are warranted to confirm these findings in an independent replication cohort and to characterize the functional role of these markers in DA susceptibility.
Supplementary material available online Supplementary Tables 1–5
Supplemental Material.pdf
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This study was supported in part by an inter-agency agreement between NIOSH and NIEHS (AES12007001-1-0-6) as a collaborative National Toxicology Program research activity and NIOSH/CDC R01 OH 008795. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References
- Alcorn, J. F., Rinaldi, L. M., Jaffe, E. F., et al. 2007. TGFβ1 suppresses airway hyper-responsiveness in allergic airway disease. Am. J. Respir. Crit. Care Med. 176:974–982
- Beghe, B., Padoan, M., Moss, C. T., et al. 2004. Lack of association of HLA Class I genes and TNFα-308 polymorphism in toluene diisocyanate-induced asthma. Allergy 59:61–64
- Bernstein, J. A. 1996. Overview of diisocyanate occupational asthma. Toxicology 111:181–189
- Bertolotti, A., Melot, T., Acker, J., et al. 1998. EWS, but not EWS-FLI-1, is associated with both TFIID and RNA polymerase II: Interactions between two members of the TET family, EWS and hTAFII68, and subunits of TFIID and RNA polymerase II complexes. Mol. Cell. Biol. 18:1489–1497
- Boyle, A. P., Hong, E. L., Hariharan, M., et al. 2012. Annotation of functional variation in personal genomes using RegulomeDB. Genome Res. 22:1790–1797
- Broide, D. H., Lotz, M., Cuomo, A. J., et al. 1992. Cytokines in symptomatic asthma airways. J. Allergy Clin. Immunol. 89:958–967
- Buckova, D., Izakovicova Holla, L., Benes, P., et al. 2001. TGF−β1 gene polymorphisms. Allergy 56:1236–1237
- Cakebread, J. A., Haitchi, H. M., Holloway, J. W., et al. 2004. Role of ADAM33 in pathogenesis of asthma. Springer Semin. Immunopathol. 25:361–375
- Cam, H., and Dynlacht, B. D. 2003. Emerging roles for E2F: Beyond the G1/S transition and DNA replication. Cancer Cell 3:311–316
- Celedon, J. C., Lange, C., Raby, B. A., et al. 2004. The TGFβ1 gene is associated with chronic obstructive pulmonary disease (COPD). Human Mol. Genet. 13:1649–1656
- Chan, I. H., Tang, N. L., Leung, T. F., et al. 2007. Association of prostaglandin-endoperoxide synthase 2 gene polymorphisms with asthma and atopy in Chinese children. Allergy 62:802–809
- Che, Z., Zhu, X., Yao, C., et al. 2014. Association between the C-509T and T869C polymorphisms of TGFβ1 gene and the risk of asthma: A meta-analysis. Human Immunol. 75:141–150
- Chiung, Y. M., Kao, Y. Y., Chang, W. F., et al. 2010. Toluene diisocyanate (TDI) induces calcium elevation and interleukine (IL)-4 release - early responses upon TDI stimulation. J. Toxicol. Sci. 35:197–207
- Clifford, R. L., John, A. E., Brightling, C. E., et al. 2012. Abnormal histone methylation is responsible for increased vascular endothelial growth factor 165a secretion from airway smooth muscle cells in asthma. J. Immunol. 189:819–831
- Cosio, B. G., Mann, B., Ito, K., et al. 2004. Histone acetylase and deacetylase activity in alveolar macrophages and blood mononocytes in asthma. Am. J. Respir. Crit. Care Med. 170:141–147
- Crosby, L. M., and Waters, C. M. 2010. Epithelial repair mechanisms in the lung. Am. J. Physiol. 298:L715–731
- Daniels, S. E., Bhattacharrya, S., James, A., et al. 1996. A genome-wide search for quantitative trait loci underlying asthma. Nature 383:247–250
- Drumm, M. L., Konstan, M. W., Schluchter, M. D., et al. 2005. Genetic modifiers of lung disease in cystic fibrosis. New Engl. J. Med. 353:1443–1453
- Dubois, R. N., Abramson, S. B., Crofford, L., et al. 1998. Cyclooxygenase in biology and disease. FASEB J. 12:1063–1073
- Duroudier, N. P., Tulah, A. S., and Sayers, I. 2009. Leukotriene pathway genetics and pharmaco-genetics in allergy. Allergy 64:823–839
- Fisseler-Eckhoff, A., Bartsch, H., Zinsky, R., et al. 2011. Environmental isocyanate-induced asthma: Morphologic and pathogenetic aspects of an increasing occupational disease. Int. J. Environ. Res. Publ. Health 8:3672–3687
- Grainger, D. J., Heathcote, K., Chiano, M., et al. 1999. Genetic control of the circulating concentration of TGFβ1. Human Mol. Genet. 8:93–97
- Heiman, G. A., Hodge, S. E., Gorroochurn, P., et al. 2004. Effect of population stratification on case-control association studies. I. Elevation in false positive rates and comparison to confounding risk ratios (a simulation study). Hum. Hered. 58:30–39
- Hobbs, K., Negri, J., Klinnert, M., et al. 1998. IL-10 and TGFβ promoter polymorphisms in allergies and asthma. Am. J. Respir. Crit. Care Med. 158:1958–1962
- Holloway, J. W., Barton, S. J., Holgate, S. T., et al. 2008. The role of LTA4H and ALOX5AP polymorphism in asthma and allergy susceptibility. Allergy 63:1046–1053
- Horvath, C. M. 2000. STAT proteins and transcriptional responses to extracellular signals. Trends Biochem. Sci. 25:496–502
- International HapMap Consortium, Altshuler, D. M., Gibbs, R. A., et al. 2010. Integrating common and rare genetic variation in diverse human populations. Nature 467:52–58
- Ito, K., Caramori, G., Lim, S., et al. 2002. Expression and activity of histone deacetylases in human asthmatic airways. Am. J. Respir. Crit. Care Med. 166:392–396
- Jacob, C. M., Pastorino, A. C., Okay, T. S., et al. 2013. IL-10 and TGFβ1 gene polymorphisms in persistent IgE-mediated cow's milk allergy. Clinics (Sao Paulo) 68:1004–1009
- Johnson, A. D., Handsaker, R. E., Pulit, S. L., et al. 2008. SNAP: A web-based tool for identification and annotation of proxy SNP using HapMap. Bioinformatics 24:2938–2939
- Leid, M., Ishmael, J. E., Avram, D., et al. 2004. CTIP1 and CTIP2 are differentially expressed during mouse embryogenesis. Gene Expr. Patterns 4:733–739
- Li, H., Romieu, I., Wu, H., et al. 2007. Genetic polymorphisms in TGFβ1 and childhood asthma and atopy. Human Genet. 121:529–538
- Li, M. O., Wan, Y. Y., Sanjabi, S., et al. 2006. TGFβ regulation of immune responses. Annu. Rev. Immunol. 24:99–146
- Maestrelli, P., di Stefano, A., Occari, P., et al. 1995. Cytokines in the airway mucosa of subjects with asthma induced by toluene diisocyanate. Am. J. Respir. Crit. Care Med. 151:607–612
- Maestrelli, P., Occari, P., Turato, G., et al. 1997. Expression of IL-4 and IL-5 proteins in asthma induced by toluene diisocyanate (TDI). Clin. Exp. Allergy 27:1292–1298
- Malo, J. L., Ghezzo, H., D'Aquino, C., et al. 1992. Natural history of occupational asthma: Relevance of type of agent and other factors in the rate of development of symptoms in affected subjects. J. Allergy Clin. Immunol. 90:937–944
- Malo, J. L., Ghezzo, H., and Elie, R. 1999. Occupational asthma caused by isocyanates: Patterns of asthmatic reactions to increasing day-to-day doses. Am. J. Respir. Crit. Care Med. 159:1879–1883
- Mapp, C. E., Ferrazzoni, S., Rizzo, R., et al. 2009. Soluble human leucocyte antigen-G and IL-10 levels in isocyanate-induced asthma. Clin. Exp. Allergy 39:812–819
- Matheson, J. M., Lemus, R., Lange, R. W., et al. 2002. Role of TNF in toluene diisocyanate asthma. Am. J. Respir. Cell Mol. Biol. 27:396–405
- McMillan, S. J., Xanthou, G., and Lloyd, C. M. 2005. Manipulation of allergen-induced airway re-modeling by treatment with anti-TGFβ antibody: Effect on Smad signaling pathway. J. Immunol. 174:5774–5780
- Nakao, A., Miike, S., Hatano, M., et al. 2000. Blockade of TGFβ/Smad signaling in T-cells by over-expression of Smad7 enhances antigen-induced airway inflammation and airway reactivity. J. Exp. Med. 192:151–158
- Ohno, I., Nitta, Y., Yamauchi, K., et al. 1996. TGFβ1 gene expression by eosinophils in asthmatic airway inflammation. Am. J. Respir. Cell Mol. Biol. 15:404–409
- Padron-Morales, J., Sanz, C., Davila, I., et al. 2013. Polymorphisms of the IL12B, IL1B, and TNFA genes and susceptibility to asthma. J. Invest. Allergol. Clin. Immunol. 23:487–494
- Panek, M., Pietras, T., Fabijan, A., et al. 2014. Identification and association of the single nucleotide polymorphisms, C-509T, C+466T and T+869C, of the TGFβ1 gene in patients with asthma and their influence on the mRNA expression level of TGFβ1. Int. J. Mol. Med. 34:975–986
- Papafili, A., Hill, M. R., Brull, D. J., et al. 2002. Common promoter variant in cyclooxygenase-2 represses gene expression: Evidence of role in acute-phase inflammatory response. Arterioscler. Thromb. Vasc. Biol. 22:1631–1636
- Piirila, P. L., Meuronen, A., Majuri, M. L., et al. 2008. Inflammation and functional outcome in diisocyanate-induced asthma after cessation of exposure. Allergy 63:583–591
- Pulleyn, L. J., Newton, R., Adcock, I. M., et al. 2001. TGFβ1 allele association with asthma severity. Human Genet. 109:623–627
- Radmark, O., Werz, O., Steinhilber, D., et al. 2007. 5-Lipoxygenase: Regulation of expression and enzyme activity. Trends Biochem. Sci. 32:332–341
- Ramana, C. V., Chatterjee-Kishore, M., Nguyen, H., et al. 2000. Complex roles of Stat1 in regulating gene expression. Oncogene 19:2619–2627
- Ribeiro de Almeida, C., Heath, H., Krpic, S., et al. 2009. Critical role for the transcription regulator CCCTC-binding factor in control of TH2 cytokine expression. J. Immunol. 182:999–1010
- Ricciardolo, F. L., Sorbello, V., Silvestri, M., et al. 2013. TNFα, IL-4Rα, and IL-4 polymorphisms in mild to severe asthma from Italian Caucasians. Int. J. Immunopathol. Pharmacol. 26:75–84
- Sastre, J., Fernandez-Nieto, M., Novalbos, A., et al. 2003. Need for monitoring non-specific bronchial hyper-responsiveness before and after isocyanate inhalation challenge. Chest 123:1276–1279
- Savenije, O. E., Kerkhof, M., Reijmerink, N. E., et al. 2011. IL-1 receptor-like 1 polymorphisms are associated with serum IL1RL1-a, eosinophils, and asthma in childhood. J. Allergy Clin. Immunol. 127:750–756
- Sharma, S., Raby, B. A., Hunninghake, G. M., et al. 2009. Variants in TGFβ1, dust mite exposure, and disease severity in children with asthma. Am. J. Respir. Crit. Care Med. 179:356–362
- Sharma, S., Sharma, A., Kumar, S., et al. 2006. Association of TNF haplotypes with asthma, serum IgE levels, and correlation with serum TNFα levels. Am. J. Respir. Cell Mol. Biol. 35:488–495
- Shi, J., Misso, N. L., Duffy, D. L., et al. 2004. A functional polymorphism in the promoter region of the COX2 gene is not associated with asthma and atopy in an Australian population. Clin. Exp. Allergy 34:1714–1718
- Shi, J., Misso, N. L., Duffy, D. L., et al. 2005. COX1 gene polymorphisms in patients with different asthma phenotypes and atopy. Eur. Respir. J. 26:249–256
- Shi, J., Misso, N. L., Kedda, M. A., et al. 2008. COX2 gene polymorphisms in an Australian population: Association of the -1195G > A promoter polymorphism with mild asthma. Clin. Exp. Allergy 38:913–920
- Shin, H. D., Park, B. L., Kim, L. H., et al. 2004. Association of TNF polymorphisms with asthma and serum total IgE. Human Mol. Genet. 13:397–403
- Silverman, E. S., Du, J., De Sanctis, G. T., et al. 1998. Egr-1 and Sp1 interact functionally with the 5-lipoxygenase promoter and its naturally occurring mutants. Am. J. Respir. Cell Mol. Biol. 19:316–323
- Silverman, E. S., Palmer, L. J., Subramaniam, V., et al. 2004. TGFβ1 promoter polymorphism C-509T is associated with asthma. Am. J. Respir. Crit. Care Med. 169:214–219
- Song, G. G., Kim, J. H., and Lee, Y. H. 2013. Association between ADAM33 S2 and ST + 4 polymorphisms and susceptibility to asthma: A meta-analysis. Gene 524:72–78
- Sousa, A., Pfister, R., Christie, P. E., et al. 1997. Enhanced expression of COX2 in asthmatic airways and its cellular distribution in aspirin-sensitive asthma. Thorax 52:940–945
- Suske, G. 1999. The Sp-family of transcription factors. Gene 238:291–300
- Swierczynska-Machura, D., Walusiak-Skorupa, J., Nowakowska-Swirta, E., et al. 2012. Immunological determinants in a murine model of toluene diisocyanate-induced asthma. Int. J. Occup. Med. Environ. Health 25:492–498
- Szczeklik, W., Sanak, M., and Szczeklik, A. 2004. Functional effects and gender association of COX2 gene polymorphism G-765C in bronchial asthma. J. Allergy Clin. Immunol. 114:248–253
- Taha, R., Olivenstein, R., Utsumi, T., et al. 2000. Prostaglandin H synthase 2 expression in airway cells from patients with asthma and chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 161:636–640
- Tan, N. Y., and Khachigian, L. M. 2009. Sp1 phosphorylation and its regulation of gene transcription. Mol. Cell. Biol. 29:2483–2488
- Tripathi, P., Awasthi, S., and Gao, P. 2014. ADAM metallopeptidase domain 33 (ADAM33): A promising target for asthma. Med. Inflamm. 2014:572025
- van Eerdewegh, P., Little, R. D., Dupuis, J., et al. 2002. Association of the ADAM33 gene with asthma and bronchial hyper-responsiveness. Nature 418:426–430
- Wilson, C. S., Seatter, S. C., Rodriguez, J. L., et al. 1997. In vivo endotoxin tolerance: Impaired LPS-stimulated TNF release of monocytes from patients with sepsis, but not SIRS. J. Surg. Res. 69:101–106
- Wisnewski, A. V., and Redlich, C. A. 2001. Recent developments in diisocyanate asthma. Curr. Opin. Allergy Clin. Immunol. 1:169–175
- Witte, J. S., Palmer, L. J., O’Connor, R. D., et al. 2002. Relation between tumour necrosis factor polymorphism TNFα-308 and risk of asthma. Eur. J. Human Genet. 10:82–85
- Wu, H., Romieu, I., Shi, M., et al. 2010. Evaluation of candidate genes in a genome-wide association study of childhood asthma in Mexicans. J. Allergy Clin. Immunol. 125:321–327 e313
- Xu, J., Meyers, D. A., Ober, C., et al. 2001. Genomewide screen and identification of gene-gene interactions for asthma-susceptibility loci in three U.S. populations: Collaborative study on the genetics of asthma. Am. J. Human Genet. 68:1437–1446
- Yang, G., Chen, J., Xu, F., et al. 2014. Association between TNFα rs1800629 polymorphism and risk of asthma: A meta-analysis. PLoS One 9:e99962
- Yang, X. X., Li, F. X., Wu, Y. S., et al. 2011. Association of TGFβ1, IL-4, and IL-13 gene polymorphisms with asthma in a Chinese population. Asian Pacif. J. Allergy Immunol. 29:273–277
- Zarbock, A., and Rossaint, J. 2011. Regulating inflammation: ADAM8 - a new player in the game. Eur. J. Immunol. 41:3419–3422
- Zeyrek, D., Demir, E., Alpman, A., et al. 2008. Association of IL-1β and IL-1 receptor anta-gonist gene polymorphisms in Turkish children with atopic asthma. Allergy Asthma Proc. 29:468–474