Published online August 1, 2006
PEDIATRICS
Vol. 118
No. 2
August 2006, pp.
710-716
(doi:10.1542/peds.2005-3030)
Glutathione S-Transferase M1 and P1 Genotype, Passive Smoking, and Peak Expiratory Flow in Asthma
Colin N.A. Palmer, PhDa,
Alex S.F. Doney, PhDb,
Simon P. Lee, MSca,
Inez Murrie, MScc,
Tahmina Ismail, MScc,
Donald F. Macgregor, FRCPCHc,d and
Somnath Mukhopadhyay, MD, PhD, FRCPHc
a Population Pharmacogenetics Group, Biomedical Research Centre
b Division of Medicine and Therapeutics, University of Dundee, Ninewells Hospital and Medical School, Dundee, Scotland, United Kingdom
c Children's Asthma and Allergy Research Unit, University of Dundee
d Directorate of Pediatrics, National Health Service Tayside, Ninewells Hospital, Dundee and Perth Royal Infirmary, Perth, Scotland, United Kingdom
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ABSTRACT
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OBJECTIVES. Our purpose with this work was to assess the contribution of glutathione S-transferase gene variants to asthma susceptibility and pulmonary function in relation to tobacco smoke exposure in the home.
METHODS. Young individuals with asthma (age: 3–21 years; n = 504) were recruited through primary and secondary care throughout Tayside, Scotland (BREATHE Study). Spirometry was obtained on 407 individuals. Binary logistic regression and general linear modeling were used to explore phenotypic characteristics by genotype and tobacco smoke exposure status in younger children (3–12 years; n = 384) and teenagers and young adults (13–21 years; n = 120).
RESULTS. Three- to 12-year-olds with asthma, null for the GSTM1 gene or homozygous for the GSTP1Val105 allele, were overrepresented in the group exposed to environmental tobacco smoke. No differences in lung function values could be detected in this group. In contrast, 13- to 21-year-olds with the GSTM1-null genotype or homozygous for the GSTP1Val105 allele from smoking households were more likely to have a substantially lower percentage of predicted peak expiratory flow rates than those from nonsmoking households (83% vs 98%).
CONCLUSIONS. Three- to 12-year-olds who are null for GSTM1 or homozygous for the GSTP1Val105 allele are more susceptible to asthma associated with environmental tobacco smoke exposure than those with more intact glutathione S-transferase status. In the 13- to 21-year-olds, GSTM1-null status interacts with environmental tobacco smoke exposure to substantially reduce peak expiratory flow rate. The environmental tobacco smoke effect in GSTM1-null children with asthma could be cumulative over time, resulting in detrimental effects on peak expiratory flow rate in 13- to 21-year-olds with asthma.
Key Words: asthma • child • glutathione S-transferase • smoking • pulmonary function • peak expiratory flow
Abbreviations: GST—glutathione S-transferase • ETS—environmental tobacco smoke • PEFR—peak expiratory flow rate • FEV1—forced expiratory volume in 1 second • FVC—forced vital capacity • OR—odds ratio • CI—confidence interval
Genes encoding the glutathione S-transferases (GSTs) have been implicated in various aspects of immune responses in the pulmonary and cardiovascular systems and have been reported recently to modulate asthma susceptibility.1–4 A number of common variants of the GST family have been described. The most commonly studied variants are the complete deletions of the GSTM1 and GSTT1 genes, which are present in the homozygous null forms at 50% and 20% of the white populations, respectively, and also a variant of GSTP1, which results in an isoleucine to valine change at codon 105. Individuals homozygous for Val105 constitute 
10% of the white population. In GSTM1-null children of school age, in utero exposure to smoking is associated with an increased prevalence of early onset asthma, asthma with current symptoms, persistent asthma, lifetime history of wheezing, wheezing with exercise, wheezing requiring medication, and emergency department visits in the past year, in contrast to children with the GSTM1+ genotype.2 GSTM1, GSTP1, and GSTT1-null variant status are also associated with diminished annual growth rates for pulmonary function in normal children.3
In a pooled analyses of 21 surveys of children and teenagers exploring the effects of passive smoking on the respiratory tract, the overall percentage reduction in expiratory flows in those exposed to parental smoking compared with those not exposed was in the order of 1% to 5%.5 There are differences in the natural history of asthma between children and adolescents; for example, asthma prevalence is significantly higher for children 
13 years of age, in comparison with children below the age of 13 years, particularly within Scotland,6 whereas the practical issues relating to the day-to-day management of asthma differ between children and teenagers.7 Recent evidence suggests that teenagers are routinely exposed to environmental tobacco smoke (ETS) at home.8,9 Thus, 13- to 21-year-olds may be at a greater overall risk from long-term exposure to tobacco smoke than 3-to 12-year-olds, possibly because of a higher cumulative dose of oxidants from tobacco smoke, leading to greater overall damage over time.
The observed variability and the relatively small overall effect5 of passive smoking on lung function may, thus, be explained, at least in part, by 2 factors: first, the coexistence of different GST genotype-stratified populations with varying degrees of susceptibility to pulmonary damage from tobacco smoke-derived oxidants; second, by larger cumulative doses of oxidants to the respiratory system in teenagers compared with children, resulting from longer periods of exposure. In addition, a genetic component to susceptibility to a tobacco smoke-induced impairment of lung function is likely to be more evident in children and teenagers with asthma compared with similar groups with no asthma.
We explored these questions specifically in an asthmatic population by studying the prevalence of GST variants and their role on pulmonary function in tobacco smoke-exposed versus unexposed individuals stratified as 2 groups: 3- to 12-year-old children and 13- to 21-year-old teenagers and young adults attending primary and secondary care clinics in Scotland.
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METHODS
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We collected demographic and clinical details, including pulmonary function measurements (peak expiratory flow rate [PEFR], forced expiratory volume in 1 second [FEV1], and forced vital capacity [FVC]) and DNA for GST genotyping in 504 white children, teenagers, and young adults (3–21 years) with physician-diagnosed asthma attending primary and secondary clinics in 10 primary care practices and a secondary care asthma clinic in Tayside, Scotland, in 2004–2005 (The BREATHE Study; Table 1). PEFR, FEV1, and FVC were measured using a portable spirometer (Micromed, Rochester, United Kingdom). All of the pulmonary function data were collected at a single visit. A minimum of 3 results within 10% of each other was recorded, and the result with the highest FEV1 was analyzed. The participants were not suffering from asthma exacerbations or other acute illnesses at the time of the measurement of pulmonary function. The lung function test results were expressed as a percentage of that predicted using the data of Rosenthal et al.10
We asked participants and/or parents regarding family smoking in the home environment, defined as ETS exposure in this study. The study was approved by the Tayside Committee on Medical Research Ethics. Informed consent was provided by the patient and, where relevant, the parent/guardian. All of the clinical information was recorded according to a standard data set. Samples were collected by mouthwash for DNA extraction and genotyping. Each participating individual was assigned a unique code number and remained anonymous for our study. We did not distinguish between maternal and paternal smoking for the purposes of analysis nor did we seek to define the effect of smoking in public places. All of the participants reported that they were living with their family (single or both parents with or without siblings). The participants were specifically asked about active smoking as part of the questionnaire. Only 5 of the participants admitted to active smoking, 3 in the 13- to 21-year-olds and 2 in the 3- to 12-year-olds. All of these individuals were also exposed to ETS. We performed the analysis with and without the actively smoking individuals (n = 5). No differences were obtained with these analyses, and the data including these individuals is shown.
Genotyping for GSTIle105Val polymorphisms was done by TaqMan-based allelic discrimination assays4 and the genotyping of the GSTM1 and GSTT1 deletions was by real-time polymerase chain reaction.1 The a priori hypothesis that GST variation exerts different effects on pulmonary function in 3- to 12-year-olds versus 13- to 21-year-olds6–9 was tested by analyzing the data separately for each of the 2 age-defined groups. All of the statistical analysis was conducted using SPSS 11 (SPSS Inc, Chicago, IL) and Instat for Macintosh version 4 (Graphpad, San Diego, CA). Binary logistic regression and univariate analysis of variance (SPSS) was used to determine differences, and significance was assessed at P < .05.
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RESULTS
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The population characteristics are typical of children and young adults with well-controlled asthma in the United Kingdom (Table 1).11 Both the GSTM1-null genotype and the GSTP1 Val105/Val105 were individually overrepresented in 3- to 12-year-olds with asthma exposed to ETS (Table 2), although only GSTP1 was significant in the total population (odds ratio [OR]: 1.93; 95% confidence interval [CI]: 1.11–3.37; P = .021). The associations were more marked in the 3- to 12-year-olds, with a model including both GSTM1 and GSTP1 best describing the at-risk group (OR: 1.83; 95% CI: 1.14–1.93; P = .013; Table 2). No significant distortion of the GSTT1-null frequencies was observed in the ETS-exposed group. When assessing the overall effect of ETS exposure, we found that PEFR measures were lower in the ETS-exposed group (Fig 1 A). However, there were no significant differences in FEV1 or FVC in the ETS-exposed group. In addition, there was a strong effect of age on the observed PEFR values with the younger age group showing lower PEFR (P = .002); in addition, the age groups showed differential sensitivity of PEFR to ETS exposure (Fig 1 B) with the younger age group only showing 2.5% drop (P = .238) in PEFR in individuals exposed to ETS, whereas a more substantial (7.4%) decrease in PEFR was observed in the older age group (P = .023). Accordingly, pulmonary function was analyzed by GST genotype and ETS exposure in both age groups (Tables 3 through 5 ). Very little evidence was found for any gene effect of GSTs on FEV1 or FVC, with only a modest association of GSTM1-null genotype with FEV1 in the 3- to 12-year-olds unexposed to ETS. This observation was not supported by any significant interaction terms with ETS exposure or age (P = .134). In contrast, we observed a 15% reduction in the percentage of predicted PEFR in GSTM1-null versus GSTM1+ 13- to 21-year-old participants exposed to ETS (P = .01). This was supported by a strongly significant interaction term between GSTM1-null status and ETS exposure on PEFR in this group but not in the 3- to 12-year-olds (P = .003; Table 5). In addition, the 3-way interaction term among age group, ETS exposure, and GSTM1 genotype was significant (P = .012). The GSTP1 Val105/Val105 individuals also demonstrated a reduction in PEFR in the older ETS exposed group, but this did not achieve significance because of the small numbers in this group (Table 5). Using an identical genetic model to that used in the analysis of gene frequencies by ETS exposure for the 3- to 12-year-olds, which included both the GSTM1 and GSTP1 Val105/Val105 genotypes as the risk group, a highly significant model for ETS exposure in the 13- to 21-year-old asthmatics was obtained (Fig 2). In this case the 3-way interaction term among age group, ETS exposure, and GSTM1/GSTP1 dual genotype was also highly significant (P = .006). This GSTM1/GSTP1 dual model was not associated with either FEV1 or FVC in any of the subgroups tested.
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TABLE 2 GSTP1 Val105/Val105 and GSTM1-Null Genotypes Are Overrepresented in 3- to 12-Year-Olds With Asthma Exposed to ETS at Home
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FIGURE 1 A, PEFR, but not FEV1 or FVC, is sensitive to ETS exposure in participants with asthma. ETS+: n = 131; ETS–: n = 279. Error bars = SEM. Shown are the P values for the comparison between ETS– and ETS+; NS indicates not significant. B, PEFR is more sensitive to ETS in the older age group.
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TABLE 3 Effect of Exposure to ETS at Home on Mean Percent Predicted FEV1 Between GST Genotypes in Young Children and Adolescents With Asthma
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TABLE 4 Effect of Exposure to ETS at Home on Mean Percent Predicted FVC Between GST Genotypes in Young Children and Adolescents With Asthma
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TABLE 5 Effect of Exposure to ETS at Home on Mean Percent Predicted PEFR Between GST Genotypes in Young Children and Adolescents With Asthma
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FIGURE 2 Dual GSTM1/GSTP1 genotype and peak expiratory flow in teenagers and young adults with asthma. GST– indicates GSTM1-null or GSTP1 Val105/Val105. Error bars = SEM.
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DISCUSSION
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Our study agrees with others that the overall frequency of GST variants in childhood asthma is similar to the general population2,11,12 and that environmental insults, such as tobacco smoke, are required to reveal the role of these genes and their influence on the susceptibility to asthma.12 The study demonstrates, in agreement with recent studies,3,13 that certain individuals who are null for GSTM1 or homozygous for the GSTP1Val105 allele are more susceptible to asthma associated with ETS exposure than those with more intact GST status. A caveat to this observation is that we have not examined the possibility that the GST status modulates the chances of coming from a smoking household per se. However, the previous studies that have examined the role of GST variants in children's lung growth and asthma susceptibility have not shown an association between these variants and either children or parental smoking,3,11,12 and, in a large study of >2000 adults with type 2 diabetes, we have shown that these GST variants are not associated with adult smoking status.1
The study also demonstrates for the first time that, in 13- to 21-year-olds with asthma, GSTM1/P1 status interacts with ETS exposure to reduce the percentage of mean predicted PEFR, whereas this effect is not observed in 3- to 12-year-olds. This result is in the context of lower PEFR in the 3- to 12-year-olds, which is associated with poorer overall disease control in this age group as judged by a significant increase in exacerbations seen in this group (data not shown).
Among GSTM1-null and GSTP1 Val105Val children, in utero ETS exposure has been associated with an increased prevalence of asthma in childhood2 and, furthermore, GSTM1-null status interacts with environmental ozone concentrations to cause lower forced expiratory flow in children and teenagers in Mexico City.14 Our results are, thus, consistent with these observations. Although we did not document details of in utero ETS exposure in our population, exposure to ETS is likely to present as a continuing insult that starts either from fetal life or from infancy in smoking families and extends through the life of the young person. The presence of a significant reduction in PEFR in GSTM1/P1-deficient asthmatic 13- to 21-year-olds and an absence of such an effect in 3- to 12-year-olds with asthma is consistent with a cumulative oxidant dose-dependent effect on GSTM1/P1-deficient small airways that becomes relevant for its effect on pulmonary function in the adolescent and young person in comparison with the younger child. We can exclude the possibility that active smoking in the older subgroup was responsible for this observation, because removal of the participants reporting active smoking (n = 3) from the analysis did not alter the association between PEFR measures and the GSTM1/P1 genotype (data not shown).
The reasons for an absence of any evidence of a difference in the lung function of the younger individuals in our study may be complex. The first, and most empirical, caveat to our study is that measurements of lung function are based on individuals who have been treated with various drugs that aim to normalize lung function. This would imply that the changes in PEFR detected in the 13- to 21-year-olds may be the consequence of more severe expiratory flow limitation, possibly resulting from small airway damage accumulating through prolonged exposure to tobacco smoke that is no longer irreversible by standard drug therapy. Low density areas of air trapping suggestive of irreversible pulmonary changes have been demonstrated on high-resolution computed tomography scans in a proportion of young people with asthma.15 A second, less obvious confounder for the lung function of the 3- to 12-year-olds in our study is the potential role of GSTs in the growth of lung function. Young children have growing lungs, and both GSTP1 and GSTM1, but not GSTT1, have been shown to modulate lung function growth.3 The role of GSTP1 in lung growth has been reinforced by findings that the lungs of gstp1-null mice are almost twice the size of the lungs from their wild-type litter mates.16,17 A recent study on asthmatic children and teenagers in the United Kingdom has observed an increase in FEV1 and FVC in GSTM1/GSTP1-deficient participants, but GSTT1 had no apparent role.11 In this study, the effects were only significant in the nonasthmatic siblings, which would agree with the existence of the confounding effects of disease and treatment on these measures in the children with asthma. We have observed similar trends in our data for FEV1 and FVC in 3- to 12-year-olds with asthma, although these relationships did not reach significance. Pollutants, such as ozone, have shown much stronger effects on forced expiratory flows and PEFR, in comparison with FEV1, in GSTM1-null children with asthma in Mexico City.14 Oxidants (ozone or through ETS exposure) thus seem to have their principal effects on expiratory flow rates and not on FEV1 and FVC in GST-null children with asthma. In addition, our study suggests that the negative effects of postnatal ETS exposure in GSTM1/P1-deficient asthmatics may not manifest until the teenage years.
More than 50% of our asthmatic children reported other features of atopic disease, namely, allergy or eczema. We also included this in our analysis; however, this did not seem to affect our observations (data not shown).
Although our findings are rather complex and may be viewed as exploratory, it is important to note that our subgroup observations were based on previous analyses in the literature, and the results confirm the findings obtained from 2 large populations of children and young people with and without asthma. In addition, the subgroup analysis is supported by statistically significant interaction terms when analyzing the entire population. Specifically, all of these studies support the role of GSTM1 in the pulmonary response to tobacco smoke exposure3,12 and suggest that GSTP1, which has a highly substantiated role in lung growth, also modulates sensitivity to such exposure. This combined GSTM1/GSTP1 genotype is also strongly implicated in susceptibility to lung cancer18 and sensitivity to DNA damage from tobacco smoke exposure.19 Together, these 2 genes define an at-risk subgroup that comprises 65% of the entire population that is sensitive to chronic ETS exposure and, as for long-term ozone exposure in Mexico City,13 may respond successfully to antioxidant, nutraceutical interventions.
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ACKNOWLEDGMENTS
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This study was funded by Scottish Enterprise Tayside, Perth and Kinross City Council, and the Gannochy Trust.
We thank the participants of this study and their carers and acknowledge the assistance of Vicky Alexander, Anna Crighton, and Dorothy Rodger (National Health Service Tayside).
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FOOTNOTES
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Accepted Feb 16, 2006.
Address correspondence to Somnath Mukhopadhyay, MD, PhD, FRCPH, Children's Asthma and Allergy Unit, Ninewells Hospital, Dundee DD1 9S4, United Kingdom. E-mail: s.mukhopadhyay{at}dundee.ac.uk
The authors have indicated they have no financial relationships relevant to this article to disclose.
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PEDIATRICS (ISSN 1098-4275). ©2006 by the American Academy of Pediatrics
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ABSTRACT
METHODS
