Association of Tumor Necrosis Factor- Polymorphisms and Ozone-induced Change in Lung Function
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美国呼吸和危急护理医学 2005年第1期
Asthma Genetics Laboratory, Human Genetics and Infection, Inflammation and Repair Divisions, University of Southampton, Southampton, United Kingdom
Center for Pneumology and Thoracic Surgery, Hospital Grosshansdorf, Grosshansdorf
Occupational and Environmental Medicine, LMU Munich, Munich, Germany
ABSTRACT
Ozone is a major air pollutant with adverse health effects which exhibit marked inter-individual variability. In mice, regions of genetic linkage with ozone-induced lung injury include the tumor necrosis factor- (TNF), lymphotoxin- (LTA), Toll-like receptor 4 (TLR4), superoxide dismutase (SOD2), and glutathione peroxidase (GPX1) genes. We genotyped polymorphisms in these genes in 51 individuals who had undergone ozone challenge. Mean change in FEV1 with ozone challenge, as a percentage of baseline, was eC3% in TNF eC308G/A or A/A individuals, compared with eC9% in G/G individuals (p = 0.024). When considering TNF haplotypes, the smallest change in FEV1 with ozone exposure was associated with the TNF haplotype comprising LTA +252G/TNF eC1031T/TNF eC308A/TNF eC238G. This association remained statistically significant after correction for age, sex, disease, and ozone concentration (p = 0.047). SOD2 or GPX1 genotypes were not associated with lung function, and the TLR4 polymorphism was too infrequent to analyze. The results of this study support TNF as a genetic factor for susceptibility to ozone-induced changes in lung function in humans, and has potential implications for stratifying health risks of air pollution.
Key Words: air pollution polymorphism (genetics) tumor necrosis factor-
Outdoor air pollution is associated with morbidity and mortality from chronic respiratory disease (1, 2). Ozone is a gaseous air pollutant generated by sunlight from hydrocarbons and nitrogen oxides. Elevated levels of ozone are associated with increased risk of respiratory admissions (3), asthma symptoms (4), deficits in lung function growth (5), and possibly even death from asthma (6). The incidence of asthma is increased in children undertaking heavy exercise in high ozone concentrations (7). Exposure to ozone causes acute changes in lung function, neutrophil infiltration, cytokine release, and potentiation of allergen-induced bronchoconstriction (8eC11).
Although within an individual these harmful effects of ozone are reproducible, large variation in response that may be genetically determined exists among individuals. Studies of murine models of ozone exposure have identified chromosomal regions of strong genetic linkage, which contain the genes for tumor necrosis factor- (TNF) (12), lymphotoxin- (LTA) (12), Toll-like receptor 4 gene (TLR4) (13), manganese superoxide dismutase (SOD2) (12), and glutathione peroxidase (GPX1) genes (14). Given the large inter-individual variation in ozone responsiveness in humans, we hypothesized that polymorphisms in the homologous human genes influence the susceptibility to ozone. Thus, we examined the effect of polymorphisms in these genes on the change in lung function that occurred with controlled ozone challenge. Some of the results of this study have been previously reported in the form of abstracts (15eC17).
METHODS
Subjects and Phenotyping
Fifty-one nonsmoking subjects provided blood for genetic analysis. The subjects were 15 subjects with mild asthma, 25 individuals with rhinitis, and 11 healthy control subjects (individuals without asthma and without rhinitis), who had undergone inhaled ozone challenge in prospective studies at Grosshansdorf, Germany, as previously described (8eC11). Individuals with asthma had a diagnosis consistent with international guidelines (18) and displayed bronchial hyperresponsiveness, defined by less than 8 mg/ml provocative concentration of methacholine causing a fall in FEV1 of 20% or more. Subjects with asthma and subjects with rhinitis had a positive skin prick test to at least one common aeroallergen and were tested outside of the allergen season. Healthy control subjects had no respiratory symptoms. Participants had ozone exposure during intermittent exercise, consisting of 15 minutes of rest alternating with 15 minutes of exercise (11), at predefined ozone concentrations. The ozone concentrations used were 250 ppb for 3 hours (44 subjects), 200 ppb for 4 hours (4 subjects), and 400 ppb for 2 hours (3 subjects). This study was approved by the institutional ethics committees of Southampton and South West Hampshire, and the Chamber of Physicians of the State of Schleswig-Holstein. All participants gave written, informed consent.
Genotyping
Genotyping of genomic DNA was initially performed for the TNF eC308G>A, TLR4 aspartate299 glycine (Asp299Gly), SOD2 valine16alanine (Val16Ala), and GPX1 proline197leucine (Pro197Leu) polymorphisms. Genotyping of the TNF gene was extended across the promoter region and into the adjacent LTA gene, because of the positive association found with the TNF eC308G>A polymorphism. Polymerase chain reactioneCbased methods were used. Full details of the genotyping assays are described in the online supplement.
Statistical Analysis
Lung function.
For each subject, the change in FEV1 or VC from baseline to immediately after the ozone exposure was calculated as a percentage of baseline FEV1 or VC. Where an individual had multiple exposures at a certain ozone concentration, the first exposure for each individual was included for the analysis.
Genotypes.
For each genetic locus, the mean change in FEV1 or VC was compared between subjects grouped according to their genotype. The genotypes containing the minor (less common) allele (i.e., heterozygous and homozygous minor allele) were grouped, and compared with the genotype consisting of the homozygous major (more common) allele. The mean change in FEV1 or VC was compared between genotype groups by independent samples t test, or Mann-Whitney U test where appropriate.
Haplotypes (combinations of alleles).
Haplotype frequencies for the TNF and LTA genes were estimated using the expectation maximization algorithm in the software program SNPHAP (Version 1.0; David Clayton, Cambridge, UK). Linkage disequilibrium between pairs of TNF or LTA polymorphisms was calculated as Lewontin's standardized disequilibrium coefficient (D') using the Arlequin software package (University of Geneva, Geneva, Switzerland) (19). Haplotype trend regression was used to associate haplotypes with the continuous outcomes of change in FEV1 or VC (20). To control for the effects of important covariates (age, sex, disease, and concentration of ozone), a haplotype-specific score was also used, which employs a general linear model (21). A p value (two-tailed) of 0.05 was considered significant in all tests.
RESULTS
The participants (26 male, 25 female) had mean (± SD) age 29 (± 6) yr and FEV1 103 (± 13) % predicted (22). There was no difference in the degree of change in FEV1 after ozone exposure in subjects with asthma (mean eC5.4%, SD 11.4, n = 15) compared with subjects without asthma (mean eC7.1%, SD 8.8, n = 36) (p = 0.58). The distribution of ozone exposures was similar across subject groups (data not shown). The minor allele frequencies were similar to reported frequencies in whites (23) (Table 1). Genotypes at each locus were in Hardy-Weinberg equilibrium. The rare TNF eC376A allele was not identified in this cohort.
Change in lung function (FEV1 or VC) was analyzed for each genetic locus separately. The mean change in FEV1 with ozone challenge was significantly lower in TNF eC308G/A or A/A individuals combined, compared with TNF eC308G/G individuals (Table 2). The distribution of ozone exposures across these genotype groups was similar (data not shown). The difference between these TNF genotype groups remained significant even when only including 250 ppb exposures (p = 0.042, Mann-Whitney U test, n = 44). There were no differences in change in FEV1 with the TNF eC1031, LTA, GPX1, or SOD2 polymorphisms. There were too few TLR4, TNF eC376, or TNF eC238 variants to analyze statistically. There were no clear differences in mean change in VC between genotype groups (Table 3), except that the association of change in VC with TNF eC308 genotypes was of borderline significance (p = 0.029 assuming unequal variances with the t test, but p = 0.18 with the Mann-Whitney U test).
There was significant linkage disequilibrium between TNF and LTA polymorphisms (Table 4). Linkage disequilibrium was complete (or maximum) between the LTA +252 locus and TNF eC308, between TNF eC1031 and eC238, and between TNF eC308 and eC238. There was moderate to high linkage disequilibrium between LTA +252 and TNF eC1031. There was no significant linkage disequilibrium between LTA +252 and TNF eC238 and between TNF eC1031 and eC308.
Haplotype frequencies were estimated for polymorphisms in the TNF gene (together with the nearby LTA gene), and the estimated frequencies are shown in Table 5. Each haplotype refers to the order of alleles along each of the subject's two chromosomes. The most frequent haplotype was LTA +252A/TNF eC1031T/TNF eC308G/TNF eC238G (i.e., A-T-G-G), which accounted for nearly half of the haplotypes. The G-T-A-G haplotype conferred the smallest change in FEV1 with ozone exposure, and this was statistically significant even after controlling for covariates (Table 6). This haplotype was also significantly associated with change in VC with the haplotype trend regression test, although the association did not quite reach statistical significance after controlling for covariates (Table 7). The other TNF haplotypes were not associated with change in lung function.
DISCUSSION
This study extends the genetic linkage findings of ozone susceptibility in mice (12) to ozone challenges performed clinically. In the univariate analysis, the TNF eC308 locus was found to be associated with change in lung function with ozone challenge. This novel association was statistically significant for change in FEV1 (p = 0.024) and of moderate magnitude (6.2% of baseline FEV1). Linear regression modeling to obtain an R2 value showed that TNF genotype accounted for 8% of the variance in ozone-induced change in FEV1. Given the positive association and the known linkage disequilibrium between polymorphisms in the TNF gene, we extended the analysis to the haplotypes across the TNF promoter region and the adjacent LTA gene. Thus we explored whether the TNF eC308 locus alone was sufficient to explain the association, or whether combinations of alleles across the region exerted influence on the change in lung function with ozone exposure. The haplotype analysis of the TNF region revealed that the G-T-A-G haplotype was associated with the smallest change in lung function. This result was consistent with the univariate analysis, because this haplotype contains the TNF eC308A polymorphism (the "A" in G-T-A-G). The association remained statistically significant even after correcting for age, sex, disease status (e.g., asthma or rhinitis), and minor differences in ozone concentration used in some of the challenges. Thus the effect of the TNF gene was independent of these potential confounders. Overall, our results support TNF as a genetic factor for susceptibility to ozone exposure in humans.
Recent animal and human studies provide additional evidence for the role of the TNF- pathway in lung injury from ozone. In inflammation-prone mice, Kleeberger and coworkers reported protection against the inflammatory response to ozone with the administration of antieCTNF- antibody (12). Moreover, TNF receptor knockout mice demonstrate less ozone-induced lung inflammation, compared with wild-type mice, suggesting that TNF receptors mediate lung injury from ozone. Potential mechanisms for the effect of TNF polymorphisms in ozone-induced lung injury include inflammation and neural function. TNF- is a proinflammatory cytokine that has a central role in inducing neutrophil chemoattractants such as interleukin-8 and intercellular adhesion molecule-1. TNF- may also promote neutrophil apoptosis in some situations. Thus, alteration in neutrophil infiltration, due to the effect of TNF polymorphisms, could have acute effects on the airway inflammation that develops with ozone exposure. Though, at least in human subjects, ozone-induced neutrophilia appears to be dissociated from the lung function response (9), there could be links between lung function responses and TNF- pathways, based on alterations in airway neural function. Ozone is known to cause reflex inhibition of inspiratory efforts resulting in a parallel reduction of FEV1 and FVC. At the same time, it stimulates the release of substance P into the airways (24). The fact that the inhibition is reduced but not entirely blocked by airway anesthesia (25) points toward additional mechanisms involved in the breathing discomfort during ozone exposure. Irrespective of this, these data provide evidence for an involvement of neural pathways in the lung function response to ozone. Interestingly, in a mouse model, TNF- derived from airway mast cells is capable of priming sensory neurons, enhancing pulmonary hypersensitivity to inhaled stimulants (26). Thus it is conceivable that alterations in the TNF- pathway, through the presence of genetic variation, may directly influence the functional response to ozone via neural activation. Whether it also affects the extent of ozone-induced lung injury and inflammation, remains to be established.
The functional significance of individual TNF polymorphisms remains controversial. Some studies have observed increased TNF gene transcription and TNF- production with the eC308A polymorphism, and susceptibility to inflammatory conditions including asthma. The eC308A polymorphism is in linkage disequilibrium with other loci in the TNF gene which have been shown to be potentially functional, although the in vitro studies have been inconclusive in demonstrating which polymorphism is predominant in regulating TNF gene expression. In our study, further analysis showed that the TNF promoter haplotype of G-T-A-G was associated with change in lung function. Hence the true causal variation may not be at the eC308 locus alone, but could be any of the other three polymorphisms tested. It may be that the specific haplotype confers a functional change in the TNF promoter, with more than one polymorphism in the haplotype having additive effects on TNF gene expression.
Interestingly, a study of exposure to inhaled sulfur dioxide (SO2) showed that individuals with the TNF eC308G/G genotype were more responsive to this air pollutant (27). In addition, our previous work has shown that SO2 responsiveness is found in approximately 20 to 25% of subjects showing airway responsiveness to methacholine, irrespective of asthma (28). Having symptoms of asthma does seem to greatly increase the likelihood of response to SO2. There is evidence that SO2 responses are neurally mediated and there is also evidence that lung function responses to ozone exposure are neurally mediated. The fact that SO2 responsiveness needs the presence of bronchial hyperresponsiveness by no means precludes the notion that SO2 has mechanisms in common with ozone. Thus we believe that the similarity in TNF genotypes associated with both SO2 and ozone response is of scientific interest.
Alternatively, the results of the present study could be explained by linkage disequilibrium with polymorphisms in nearby genes. We calculated the linkage disequilibrium between pairs of TNF alleles using D'. It is known that D' is 1 when the observed frequency of at least one haplotype is 0. However, D' = 1 is a reliable indicator of strong disequilibrium when the absolute expected gametic frequencies are not low and the observed gametic frequencies are 0. For the loci pair of LTA +252 and TNF eC308, the absolute expected gametic frequency corresponding to the haplotype with an observed frequency of 0 was not low (12.97) and we conclude that the D' = 1 is reliable. For the loci pairs of TNF eC1031 and TNF eC238, and TNF eC238 and TNF eC308, the corresponding expected frequencies were rather low (1.59 to 1.69), and therefore D' = 1 may not be indicative of strong disequilibrium. To explore this further, we tested the null hypothesis of no disequilibrium by a Monte Carlo simulation (29, 30) (Table 4). This approach gives the 95% CI of the expected value of disequilibrium by chance. If the observed D' is outside this CI, then the D' is reliable. The results obtained confirmed that the D' values observed for the loci pairs of LTA +252 and TNF eC1031, and TNF eC1031 and TNF eC308, were reliable. On the contrary, the D' value of 1 observed for the loci pairs TNF eC1031 and TNF eC238, and TNF eC238 and TNF eC308, could be misleading. This is caused by the fact that the 2 test is very liberal (that is, the type I error is larger than the nominal significance level of = 0.05) because the frequency of one of the alleles is 0.980 (31). This result, however, does not argue against the existence of strong disequilibrium between these single nucleotide polymorphisms. A larger sample size would be necessary to have enough statistical power to estimate the actual value of D' for these cases.
Several studies have recently reported associations of ozone-related phenotypes with polymorphisms in the antioxidant genes, nicotinamide adenine dinucleotide (phosphate) reduced:quinone oxidoreductase (NQO1) and glutathione-S-transferase e? (GSTM1). The high-risk NQO1 and GSTM1 genotypes, which confer excessive production of free radicals, were associated with a greater fall in lung function and increased inflammatory markers in volunteers exposed to ozone during exercise (32, 33). In a study of nasal biopsies, GSTM1 null individuals had significant increases in the superoxide dismutase activity of their biopsies with ozone exposure, possibly as a result of accumulation of products of lipid peroxidation (34). Two genetic studies of asthma were performed in Mexico City, where ambient concentrations of ozone are high throughout the year. An epidemiologic study of asthma found that carriage of the NQO1 Serine187 polymorphism with the GSTM1 null genotype conferred a reduced risk of asthma in children (35). A pharmacogenetic study examined antioxidant supplementation as protection against the effects of ozone in children with asthma (36). Children with asthma taking placebo and who had the GSTM1 null genotype had a mean 3% fall in FEF25eC75 per 50 ppb increase in ozone concentration, whereas there was no change in lung function in GSTM1-positive children taking placebo, or in children of either genotype taking the active supplement (36). These studies of antioxidant genes (which differed from the genes we studied) support the hypothesis that certain critical polymorphisms influence the health effects of ozone exposure. Our study adds to this evidence and is the first to report associations of change in lung function during ozone challenge, with a gene involved in inflammation (TNF). In support of this, in an epidemiologic study of a subset of 1,123 children in the Children's Health Study, the TNF eC308 genotype was found to interact with ozone levels to influence the risk of asthma symptoms (37). In those communities with the lowest ozone concentrations, variant TNF genotypes were associated with a higher risk of wheezing outcomes, an effect that was not seen in the highest ozone communities (37). Thus, taken together, the results of the present study, the asthma epidemiologic study (37), and the genetic linkage studies in mice all suggest that TNF- has an important biological role in the pulmonary response to inhaled air pollutants.
Potential limitations of this study should be addressed. There was some heterogeneity in subject groups and exposures. However, the challenges were all performed using the same methods at a single center. We pooled all subjects, as we did not detect a difference in change in FEV1 among disease/control groups. This is compatible with our 10-year experience in ozone exposures; in our experimental set-up, lung function responses were, on average, very similar between groups (8eC11). Moreover, the effect of the G-T-A-G haplotype of the TNF gene on change in FEV1 was still significant even after adjusting for these and other potential confounders, using the haplotype-specific score.
The sample size of this cohort was relatively small (n = 51), and may have been underpowered to detect a difference in lung function for the TNF haplotype with the lowest frequency (G-C-A-A, 2%). Other previously published studies have found significant differences in ozone-related outcomes with half the sample size of our sample (32, 33). As we found a statistically significant difference for the TNF genotype in this study, it would seem that insufficient power (type II error) may not be so much of an issue as potential type I error is. The large variation between subjects has probably enhanced the power (through increased effect size) rather than decreasing it (in that larger responses may be more reproducible). Within our subjects, the uppermost quartile of changes in FEV1 was at eC0.2% and the lowest quartile at eC10.5%. This is a difference of eC10.3% between these quartiles, thus the spread of responses would likely be large enough to detect a 5% difference between FEV1 responses. We have not formally used power calculations here because of the wide range of genotype frequencies for the different genes and polymorphisms. This sample size is likely to be insufficient for a comprehensive analysis comparing diseases, potential types of responses (restrictive/obstructive), and other complex confounders. However, we believe that our study presents novel data which are in line with data obtained in animals, as well as data from other air pollutants (SO2), despite the differences in the choice of outcome variables.
We minimized the number of statistical analyses by performing haplotype analysis with correction for covariates. As this was an exploratory study of novel candidate genes in ozone-induced effects, we did not employ correction for multiple comparisons, to avoid missing biologically important associations. Despite the limitations of the study, it seems warranted to conclude that our data provide evidence for the eC308G>A single-nucleotide polymorphism being associated with the functional response to ozone in human subjects. As is usual in genetic association studies, these findings should be replicated in other cohorts of different genetic background. Functional ex vivo and in vitro studies should also be performed in future studies to confirm the mechanisms involved; for example, sputum TNF- during ozone exposure should be measured and correlated with TNF promoter haplotype. Such data would also facilitate study of which polymorphisms or haplotypes are potentially associated with the neutrophilic inflammatory response to ozone.
In conclusion, genetic markers such as TNF and other polymorphisms may identify individuals who are at higher risk of change in lung function from air pollution. As antioxidants have been shown to provide effective chemoprevention against ozone-induced decrements in lung function, assessing genetic risk factors could stratify those individuals, both with and without asthma, who would benefit the most from targeted prevention against the harmful effects of air pollution.
Acknowledgments
The authors are grateful to the volunteers who participated, to Petra Timm for assistance with recruiting, and to Professor Carlos Zapata for assistance with the Monte Carlo simulation in the analysis of linkage disequilibrium.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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Center for Pneumology and Thoracic Surgery, Hospital Grosshansdorf, Grosshansdorf
Occupational and Environmental Medicine, LMU Munich, Munich, Germany
ABSTRACT
Ozone is a major air pollutant with adverse health effects which exhibit marked inter-individual variability. In mice, regions of genetic linkage with ozone-induced lung injury include the tumor necrosis factor- (TNF), lymphotoxin- (LTA), Toll-like receptor 4 (TLR4), superoxide dismutase (SOD2), and glutathione peroxidase (GPX1) genes. We genotyped polymorphisms in these genes in 51 individuals who had undergone ozone challenge. Mean change in FEV1 with ozone challenge, as a percentage of baseline, was eC3% in TNF eC308G/A or A/A individuals, compared with eC9% in G/G individuals (p = 0.024). When considering TNF haplotypes, the smallest change in FEV1 with ozone exposure was associated with the TNF haplotype comprising LTA +252G/TNF eC1031T/TNF eC308A/TNF eC238G. This association remained statistically significant after correction for age, sex, disease, and ozone concentration (p = 0.047). SOD2 or GPX1 genotypes were not associated with lung function, and the TLR4 polymorphism was too infrequent to analyze. The results of this study support TNF as a genetic factor for susceptibility to ozone-induced changes in lung function in humans, and has potential implications for stratifying health risks of air pollution.
Key Words: air pollution polymorphism (genetics) tumor necrosis factor-
Outdoor air pollution is associated with morbidity and mortality from chronic respiratory disease (1, 2). Ozone is a gaseous air pollutant generated by sunlight from hydrocarbons and nitrogen oxides. Elevated levels of ozone are associated with increased risk of respiratory admissions (3), asthma symptoms (4), deficits in lung function growth (5), and possibly even death from asthma (6). The incidence of asthma is increased in children undertaking heavy exercise in high ozone concentrations (7). Exposure to ozone causes acute changes in lung function, neutrophil infiltration, cytokine release, and potentiation of allergen-induced bronchoconstriction (8eC11).
Although within an individual these harmful effects of ozone are reproducible, large variation in response that may be genetically determined exists among individuals. Studies of murine models of ozone exposure have identified chromosomal regions of strong genetic linkage, which contain the genes for tumor necrosis factor- (TNF) (12), lymphotoxin- (LTA) (12), Toll-like receptor 4 gene (TLR4) (13), manganese superoxide dismutase (SOD2) (12), and glutathione peroxidase (GPX1) genes (14). Given the large inter-individual variation in ozone responsiveness in humans, we hypothesized that polymorphisms in the homologous human genes influence the susceptibility to ozone. Thus, we examined the effect of polymorphisms in these genes on the change in lung function that occurred with controlled ozone challenge. Some of the results of this study have been previously reported in the form of abstracts (15eC17).
METHODS
Subjects and Phenotyping
Fifty-one nonsmoking subjects provided blood for genetic analysis. The subjects were 15 subjects with mild asthma, 25 individuals with rhinitis, and 11 healthy control subjects (individuals without asthma and without rhinitis), who had undergone inhaled ozone challenge in prospective studies at Grosshansdorf, Germany, as previously described (8eC11). Individuals with asthma had a diagnosis consistent with international guidelines (18) and displayed bronchial hyperresponsiveness, defined by less than 8 mg/ml provocative concentration of methacholine causing a fall in FEV1 of 20% or more. Subjects with asthma and subjects with rhinitis had a positive skin prick test to at least one common aeroallergen and were tested outside of the allergen season. Healthy control subjects had no respiratory symptoms. Participants had ozone exposure during intermittent exercise, consisting of 15 minutes of rest alternating with 15 minutes of exercise (11), at predefined ozone concentrations. The ozone concentrations used were 250 ppb for 3 hours (44 subjects), 200 ppb for 4 hours (4 subjects), and 400 ppb for 2 hours (3 subjects). This study was approved by the institutional ethics committees of Southampton and South West Hampshire, and the Chamber of Physicians of the State of Schleswig-Holstein. All participants gave written, informed consent.
Genotyping
Genotyping of genomic DNA was initially performed for the TNF eC308G>A, TLR4 aspartate299 glycine (Asp299Gly), SOD2 valine16alanine (Val16Ala), and GPX1 proline197leucine (Pro197Leu) polymorphisms. Genotyping of the TNF gene was extended across the promoter region and into the adjacent LTA gene, because of the positive association found with the TNF eC308G>A polymorphism. Polymerase chain reactioneCbased methods were used. Full details of the genotyping assays are described in the online supplement.
Statistical Analysis
Lung function.
For each subject, the change in FEV1 or VC from baseline to immediately after the ozone exposure was calculated as a percentage of baseline FEV1 or VC. Where an individual had multiple exposures at a certain ozone concentration, the first exposure for each individual was included for the analysis.
Genotypes.
For each genetic locus, the mean change in FEV1 or VC was compared between subjects grouped according to their genotype. The genotypes containing the minor (less common) allele (i.e., heterozygous and homozygous minor allele) were grouped, and compared with the genotype consisting of the homozygous major (more common) allele. The mean change in FEV1 or VC was compared between genotype groups by independent samples t test, or Mann-Whitney U test where appropriate.
Haplotypes (combinations of alleles).
Haplotype frequencies for the TNF and LTA genes were estimated using the expectation maximization algorithm in the software program SNPHAP (Version 1.0; David Clayton, Cambridge, UK). Linkage disequilibrium between pairs of TNF or LTA polymorphisms was calculated as Lewontin's standardized disequilibrium coefficient (D') using the Arlequin software package (University of Geneva, Geneva, Switzerland) (19). Haplotype trend regression was used to associate haplotypes with the continuous outcomes of change in FEV1 or VC (20). To control for the effects of important covariates (age, sex, disease, and concentration of ozone), a haplotype-specific score was also used, which employs a general linear model (21). A p value (two-tailed) of 0.05 was considered significant in all tests.
RESULTS
The participants (26 male, 25 female) had mean (± SD) age 29 (± 6) yr and FEV1 103 (± 13) % predicted (22). There was no difference in the degree of change in FEV1 after ozone exposure in subjects with asthma (mean eC5.4%, SD 11.4, n = 15) compared with subjects without asthma (mean eC7.1%, SD 8.8, n = 36) (p = 0.58). The distribution of ozone exposures was similar across subject groups (data not shown). The minor allele frequencies were similar to reported frequencies in whites (23) (Table 1). Genotypes at each locus were in Hardy-Weinberg equilibrium. The rare TNF eC376A allele was not identified in this cohort.
Change in lung function (FEV1 or VC) was analyzed for each genetic locus separately. The mean change in FEV1 with ozone challenge was significantly lower in TNF eC308G/A or A/A individuals combined, compared with TNF eC308G/G individuals (Table 2). The distribution of ozone exposures across these genotype groups was similar (data not shown). The difference between these TNF genotype groups remained significant even when only including 250 ppb exposures (p = 0.042, Mann-Whitney U test, n = 44). There were no differences in change in FEV1 with the TNF eC1031, LTA, GPX1, or SOD2 polymorphisms. There were too few TLR4, TNF eC376, or TNF eC238 variants to analyze statistically. There were no clear differences in mean change in VC between genotype groups (Table 3), except that the association of change in VC with TNF eC308 genotypes was of borderline significance (p = 0.029 assuming unequal variances with the t test, but p = 0.18 with the Mann-Whitney U test).
There was significant linkage disequilibrium between TNF and LTA polymorphisms (Table 4). Linkage disequilibrium was complete (or maximum) between the LTA +252 locus and TNF eC308, between TNF eC1031 and eC238, and between TNF eC308 and eC238. There was moderate to high linkage disequilibrium between LTA +252 and TNF eC1031. There was no significant linkage disequilibrium between LTA +252 and TNF eC238 and between TNF eC1031 and eC308.
Haplotype frequencies were estimated for polymorphisms in the TNF gene (together with the nearby LTA gene), and the estimated frequencies are shown in Table 5. Each haplotype refers to the order of alleles along each of the subject's two chromosomes. The most frequent haplotype was LTA +252A/TNF eC1031T/TNF eC308G/TNF eC238G (i.e., A-T-G-G), which accounted for nearly half of the haplotypes. The G-T-A-G haplotype conferred the smallest change in FEV1 with ozone exposure, and this was statistically significant even after controlling for covariates (Table 6). This haplotype was also significantly associated with change in VC with the haplotype trend regression test, although the association did not quite reach statistical significance after controlling for covariates (Table 7). The other TNF haplotypes were not associated with change in lung function.
DISCUSSION
This study extends the genetic linkage findings of ozone susceptibility in mice (12) to ozone challenges performed clinically. In the univariate analysis, the TNF eC308 locus was found to be associated with change in lung function with ozone challenge. This novel association was statistically significant for change in FEV1 (p = 0.024) and of moderate magnitude (6.2% of baseline FEV1). Linear regression modeling to obtain an R2 value showed that TNF genotype accounted for 8% of the variance in ozone-induced change in FEV1. Given the positive association and the known linkage disequilibrium between polymorphisms in the TNF gene, we extended the analysis to the haplotypes across the TNF promoter region and the adjacent LTA gene. Thus we explored whether the TNF eC308 locus alone was sufficient to explain the association, or whether combinations of alleles across the region exerted influence on the change in lung function with ozone exposure. The haplotype analysis of the TNF region revealed that the G-T-A-G haplotype was associated with the smallest change in lung function. This result was consistent with the univariate analysis, because this haplotype contains the TNF eC308A polymorphism (the "A" in G-T-A-G). The association remained statistically significant even after correcting for age, sex, disease status (e.g., asthma or rhinitis), and minor differences in ozone concentration used in some of the challenges. Thus the effect of the TNF gene was independent of these potential confounders. Overall, our results support TNF as a genetic factor for susceptibility to ozone exposure in humans.
Recent animal and human studies provide additional evidence for the role of the TNF- pathway in lung injury from ozone. In inflammation-prone mice, Kleeberger and coworkers reported protection against the inflammatory response to ozone with the administration of antieCTNF- antibody (12). Moreover, TNF receptor knockout mice demonstrate less ozone-induced lung inflammation, compared with wild-type mice, suggesting that TNF receptors mediate lung injury from ozone. Potential mechanisms for the effect of TNF polymorphisms in ozone-induced lung injury include inflammation and neural function. TNF- is a proinflammatory cytokine that has a central role in inducing neutrophil chemoattractants such as interleukin-8 and intercellular adhesion molecule-1. TNF- may also promote neutrophil apoptosis in some situations. Thus, alteration in neutrophil infiltration, due to the effect of TNF polymorphisms, could have acute effects on the airway inflammation that develops with ozone exposure. Though, at least in human subjects, ozone-induced neutrophilia appears to be dissociated from the lung function response (9), there could be links between lung function responses and TNF- pathways, based on alterations in airway neural function. Ozone is known to cause reflex inhibition of inspiratory efforts resulting in a parallel reduction of FEV1 and FVC. At the same time, it stimulates the release of substance P into the airways (24). The fact that the inhibition is reduced but not entirely blocked by airway anesthesia (25) points toward additional mechanisms involved in the breathing discomfort during ozone exposure. Irrespective of this, these data provide evidence for an involvement of neural pathways in the lung function response to ozone. Interestingly, in a mouse model, TNF- derived from airway mast cells is capable of priming sensory neurons, enhancing pulmonary hypersensitivity to inhaled stimulants (26). Thus it is conceivable that alterations in the TNF- pathway, through the presence of genetic variation, may directly influence the functional response to ozone via neural activation. Whether it also affects the extent of ozone-induced lung injury and inflammation, remains to be established.
The functional significance of individual TNF polymorphisms remains controversial. Some studies have observed increased TNF gene transcription and TNF- production with the eC308A polymorphism, and susceptibility to inflammatory conditions including asthma. The eC308A polymorphism is in linkage disequilibrium with other loci in the TNF gene which have been shown to be potentially functional, although the in vitro studies have been inconclusive in demonstrating which polymorphism is predominant in regulating TNF gene expression. In our study, further analysis showed that the TNF promoter haplotype of G-T-A-G was associated with change in lung function. Hence the true causal variation may not be at the eC308 locus alone, but could be any of the other three polymorphisms tested. It may be that the specific haplotype confers a functional change in the TNF promoter, with more than one polymorphism in the haplotype having additive effects on TNF gene expression.
Interestingly, a study of exposure to inhaled sulfur dioxide (SO2) showed that individuals with the TNF eC308G/G genotype were more responsive to this air pollutant (27). In addition, our previous work has shown that SO2 responsiveness is found in approximately 20 to 25% of subjects showing airway responsiveness to methacholine, irrespective of asthma (28). Having symptoms of asthma does seem to greatly increase the likelihood of response to SO2. There is evidence that SO2 responses are neurally mediated and there is also evidence that lung function responses to ozone exposure are neurally mediated. The fact that SO2 responsiveness needs the presence of bronchial hyperresponsiveness by no means precludes the notion that SO2 has mechanisms in common with ozone. Thus we believe that the similarity in TNF genotypes associated with both SO2 and ozone response is of scientific interest.
Alternatively, the results of the present study could be explained by linkage disequilibrium with polymorphisms in nearby genes. We calculated the linkage disequilibrium between pairs of TNF alleles using D'. It is known that D' is 1 when the observed frequency of at least one haplotype is 0. However, D' = 1 is a reliable indicator of strong disequilibrium when the absolute expected gametic frequencies are not low and the observed gametic frequencies are 0. For the loci pair of LTA +252 and TNF eC308, the absolute expected gametic frequency corresponding to the haplotype with an observed frequency of 0 was not low (12.97) and we conclude that the D' = 1 is reliable. For the loci pairs of TNF eC1031 and TNF eC238, and TNF eC238 and TNF eC308, the corresponding expected frequencies were rather low (1.59 to 1.69), and therefore D' = 1 may not be indicative of strong disequilibrium. To explore this further, we tested the null hypothesis of no disequilibrium by a Monte Carlo simulation (29, 30) (Table 4). This approach gives the 95% CI of the expected value of disequilibrium by chance. If the observed D' is outside this CI, then the D' is reliable. The results obtained confirmed that the D' values observed for the loci pairs of LTA +252 and TNF eC1031, and TNF eC1031 and TNF eC308, were reliable. On the contrary, the D' value of 1 observed for the loci pairs TNF eC1031 and TNF eC238, and TNF eC238 and TNF eC308, could be misleading. This is caused by the fact that the 2 test is very liberal (that is, the type I error is larger than the nominal significance level of = 0.05) because the frequency of one of the alleles is 0.980 (31). This result, however, does not argue against the existence of strong disequilibrium between these single nucleotide polymorphisms. A larger sample size would be necessary to have enough statistical power to estimate the actual value of D' for these cases.
Several studies have recently reported associations of ozone-related phenotypes with polymorphisms in the antioxidant genes, nicotinamide adenine dinucleotide (phosphate) reduced:quinone oxidoreductase (NQO1) and glutathione-S-transferase e? (GSTM1). The high-risk NQO1 and GSTM1 genotypes, which confer excessive production of free radicals, were associated with a greater fall in lung function and increased inflammatory markers in volunteers exposed to ozone during exercise (32, 33). In a study of nasal biopsies, GSTM1 null individuals had significant increases in the superoxide dismutase activity of their biopsies with ozone exposure, possibly as a result of accumulation of products of lipid peroxidation (34). Two genetic studies of asthma were performed in Mexico City, where ambient concentrations of ozone are high throughout the year. An epidemiologic study of asthma found that carriage of the NQO1 Serine187 polymorphism with the GSTM1 null genotype conferred a reduced risk of asthma in children (35). A pharmacogenetic study examined antioxidant supplementation as protection against the effects of ozone in children with asthma (36). Children with asthma taking placebo and who had the GSTM1 null genotype had a mean 3% fall in FEF25eC75 per 50 ppb increase in ozone concentration, whereas there was no change in lung function in GSTM1-positive children taking placebo, or in children of either genotype taking the active supplement (36). These studies of antioxidant genes (which differed from the genes we studied) support the hypothesis that certain critical polymorphisms influence the health effects of ozone exposure. Our study adds to this evidence and is the first to report associations of change in lung function during ozone challenge, with a gene involved in inflammation (TNF). In support of this, in an epidemiologic study of a subset of 1,123 children in the Children's Health Study, the TNF eC308 genotype was found to interact with ozone levels to influence the risk of asthma symptoms (37). In those communities with the lowest ozone concentrations, variant TNF genotypes were associated with a higher risk of wheezing outcomes, an effect that was not seen in the highest ozone communities (37). Thus, taken together, the results of the present study, the asthma epidemiologic study (37), and the genetic linkage studies in mice all suggest that TNF- has an important biological role in the pulmonary response to inhaled air pollutants.
Potential limitations of this study should be addressed. There was some heterogeneity in subject groups and exposures. However, the challenges were all performed using the same methods at a single center. We pooled all subjects, as we did not detect a difference in change in FEV1 among disease/control groups. This is compatible with our 10-year experience in ozone exposures; in our experimental set-up, lung function responses were, on average, very similar between groups (8eC11). Moreover, the effect of the G-T-A-G haplotype of the TNF gene on change in FEV1 was still significant even after adjusting for these and other potential confounders, using the haplotype-specific score.
The sample size of this cohort was relatively small (n = 51), and may have been underpowered to detect a difference in lung function for the TNF haplotype with the lowest frequency (G-C-A-A, 2%). Other previously published studies have found significant differences in ozone-related outcomes with half the sample size of our sample (32, 33). As we found a statistically significant difference for the TNF genotype in this study, it would seem that insufficient power (type II error) may not be so much of an issue as potential type I error is. The large variation between subjects has probably enhanced the power (through increased effect size) rather than decreasing it (in that larger responses may be more reproducible). Within our subjects, the uppermost quartile of changes in FEV1 was at eC0.2% and the lowest quartile at eC10.5%. This is a difference of eC10.3% between these quartiles, thus the spread of responses would likely be large enough to detect a 5% difference between FEV1 responses. We have not formally used power calculations here because of the wide range of genotype frequencies for the different genes and polymorphisms. This sample size is likely to be insufficient for a comprehensive analysis comparing diseases, potential types of responses (restrictive/obstructive), and other complex confounders. However, we believe that our study presents novel data which are in line with data obtained in animals, as well as data from other air pollutants (SO2), despite the differences in the choice of outcome variables.
We minimized the number of statistical analyses by performing haplotype analysis with correction for covariates. As this was an exploratory study of novel candidate genes in ozone-induced effects, we did not employ correction for multiple comparisons, to avoid missing biologically important associations. Despite the limitations of the study, it seems warranted to conclude that our data provide evidence for the eC308G>A single-nucleotide polymorphism being associated with the functional response to ozone in human subjects. As is usual in genetic association studies, these findings should be replicated in other cohorts of different genetic background. Functional ex vivo and in vitro studies should also be performed in future studies to confirm the mechanisms involved; for example, sputum TNF- during ozone exposure should be measured and correlated with TNF promoter haplotype. Such data would also facilitate study of which polymorphisms or haplotypes are potentially associated with the neutrophilic inflammatory response to ozone.
In conclusion, genetic markers such as TNF and other polymorphisms may identify individuals who are at higher risk of change in lung function from air pollution. As antioxidants have been shown to provide effective chemoprevention against ozone-induced decrements in lung function, assessing genetic risk factors could stratify those individuals, both with and without asthma, who would benefit the most from targeted prevention against the harmful effects of air pollution.
Acknowledgments
The authors are grateful to the volunteers who participated, to Petra Timm for assistance with recruiting, and to Professor Carlos Zapata for assistance with the Monte Carlo simulation in the analysis of linkage disequilibrium.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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