American Journal of Respiratory and Critical Care Medicine

The effects of glutathione-S-transferase (GST) M1, GSTT1, and GSTP1 genotypes on lung function growth were investigated in 1,940 children enrolled in the Children's Health Study as fourth graders (aged 8–11 years) in two cohorts during 1993 and 1996 and were followed annually over a 4-year period. Genotypes for GSTM1 and GSTT1 and GSTP1 codon 105 variants (ile105 and val105) were determined using DNA from buccal cell specimens. We used two-level regression models to estimate the effects of GSTM1, GSTT1, and GSTP1 genotypes on the adjusted annual average lung function growth. GSTM1 null was associated with deficits in annual growth rates for FVC (−0.21%; 95% confidence interval [CI], −0.40, −0.03) and FEV1 (−0.27%; 95% CI, −0.50, −0.04). Children who were homozygous for the GSTP1 val105 allele had slower lung function growth (FVC −0.35%; 95% CI, −0.62, −0.07; and FEV1 −0.34%; 95% CI, −0.68, 0.00) than children with one or more ile105 alleles. Children with asthma who were homozygous for the GSTP1 val105 allele had substantially larger deficits in FVC, FEV1, and maximal mid-expiratory flow than children without asthma. The deficits in FVC and FEV1 growth associated with both GSTM1 null and the GSTP1 val105 allele were largest and were statistically significant in non-Hispanic white children. We conclude that GSTM1 and GSTP1 genotypes are associated with lung function growth in school children.

Normal lung growth and development during childhood are essential to reach maximum attainable adult lung function. Reduced growth in lung function leads to a lower attained lung volume and air flow, which increase the adulthood risks for acute symptoms from exacerbations of asthma or respiratory infections, adverse effects from exposure to respiratory toxins, and risks for chronic diseases such as chronic obstructive pulmonary disease (COPD) (15). Associations of low FEV1 with increased risk of cardiovascular disease and overall mortality emphasize the importance of attaining maximum lung function (6, 7).

A broad spectrum of determinants for childhood lung function growth has been identified (1, 2, 811). Experiences during the in utero period affect lung function growth, as indicated by associations of adult lung function level with exposure to maternal smoking, gestational age, and birth weight (8, 1215). Early childhood respiratory infections, asthma, and airway hyper-responsiveness as well as childhood exposures to air pollution and tobacco smoke are also associated with reduced lung function growth, lower adult lung function, and increased risk for COPD. Familial aggregation of FEV1 and associations of COPD with candidate genes such as glutathione-S-transferase (GST) P1, epoxide hydrolase, and α1-antitrypsin gene indicate that a genetic variation is likely to affect childhood lung growth; however, genetic determinants of childhood lung function growth have not been extensively studied (1622). Understanding the contribution of common genetic variants to the complex phenotype of lung function growth may clarify the pathophysiologic pathways that are important for normal lung development and identify susceptible groups for interventions.

We adopted a candidate gene approach to investigate loci with common functional allelic variants that may affect lung function growth (23). We identified candidate genes based on evidence that antioxidant defenses are important determinants of lung function. The growing lung is exposed to a substantial burden of endogenously produced and inhaled oxidants and pro-oxidants, including O2 and a wide variety of toxic aerosols (2426). If antioxidant defenses are inadequate, substantial oxidative stress can occur that may interfere with normal lung growth and may contribute to increased incidence, prevalence, and severity of respiratory diseases such as COPD, asthma, and viral infections. Because oxidative stress is involved in the pathogenesis of conditions that affect adult lung function such as asthma and COPD, we hypothesized that polymorphic variants of genes functioning in antioxidant pathways that modulate oxidative stress are determinants of lung function growth. We focused this study on GSTM1, GSTT1, and GSTP1 genotypes because these genes are expressed in the lung, are involved in antioxidant defense pathways, and have common functional variant alleles (23, 2729).

GSTs (E.C. 2.5.1.18) are a superfamily of enzymes consisting of α, μ, π, and θ families in humans (28, 30). Members of the GST families have sequence similarity and shared catalytic properties for reaction of glutathione (GSH) with reactive substrates. GSTs are well known as phase II xenobiotic detoxifying enzymes. One of more recently recognized roles of GSTs is in oxidative defenses, where members of this family function as peroxidases to detoxify products of oxidative attack (27). Because antioxidants play a role in the pathobiology of a variety of diseases and variants in the GST superfamily are common, members of this superfamily may be determinants of respiratory health.

Several common variants of GSTs are well characterized (28). GSTM1, a member of the μ family located on chromosome 1p13.3, has two alleles: a wild-type allele GSTM1*1 and a variant allele that results in no protein expression GSTM1*0. Approximately 50% of Europid populations are homozygous for the GSTM1*0, and individuals with this null genotype express no GSTM1. GSTT1, a member of the θ family located on chromosome 22q11.2, has a similar null genotype; however, the frequency of the null genotype is approximately 25% in Europid, but it reaches 40% in some Asian populations. GSTP1 variants have been associated with a reduced risk for asthma and related phenotypes. GSTP1 has four alleles formed from two linked single nucleotide polymorphisms in codons 105 (exon 5) and 114 (exon 6) (wild-type P1*A [ile105, ala114], P1*B [val105, ala114], P1*C [val105, val114], and P1*D [ile105, val114]) (31). The ile105 wild-type allele has higher catalytic rates than the val105 variant for 1-chloro-2,4-dinitrobenzene but a lower efficiency for polycyclic aromatic hydrocarbon diol epoxides. Because the val105 and val114 are in highly significant linkage disequilibrium and the val114 does not appear to affect substantially the function of the 105 variants, we limited our genotyping the single base G to A change in codon 105 in this study.

The Children's Health Study, a cohort study of children's respiratory health, offered an opportunity to investigate the effects of GSTM1, GSTT1, and GSTP1 genotypes on lung function growth in school children. We examined longitudinal lung function data collected over 4 years to determine whether genotypes were associated with annual average FEV1, FVC, and maximal mid-expiratory flow (MMEF) growth among 1,940 school children who resided in 12 southern California communities.

Study Subjects

The elements of the Children's Health Study have been described previously (32). Two cohorts of fourth-grade children were recruited from public school classrooms in 12 southern California communities that were selected based on historical measurements of air quality, demographic similarities, and a cooperative school district. The first cohort of 1,806 fourth-grade children was enrolled in 1993, and the second cohort of 2,081 children was enrolled in 1996. The parents or guardians of each participating student provided written informed consent and completed a written questionnaire, which provided demographic information and characterized history of respiratory illness and its associated risk factors, exposure information, and household characteristics. We collected environmental tobacco smoke data using parent-completed questionnaire items about household smokers, as previously described (33). Personal smoking habits of participants were assessed in a private interview at lung function testing sessions.

Students in each community had lung function tests during visits to each school, as previously described (34). Maximum forced expiratory flow–volume maneuvers were recorded using rolling-seal spirometers (Spiroflow; P.K. Morgan Ltd., Gillingham, UK). Each subject was asked to perform three satisfactory maneuvers based on the American Thoracic Society's recommendations modified for children (35). At the time of testing, students had height and weight measured and had a private interview about personal smoking habits and recent respiratory illness history and whether they had performed vigorous exercise within 30 minutes of the test. Genotype information was available from 1,940 of the 3,135 children (62%) enrolled in the two fourth-grade cohorts with complete covariate data from the questionnaire and at least two lung function tests.

Laboratory Methods

Buccal cells were collected from participants as a source of genomic DNA for genotyping assays. Details of buccal cell processing and genotyping assays are provided in an online data supplement. Briefly, DNA was extracted using a PUREGENE DNA isolation kit (cat #D-5000; GENTRA, Minneapolis, MN). Genotypes for GSTM1 were determined using two methods. The first 380 samples were analyzed by enzymatic amplification of the polymorphic GSTM1 locus. Polymerase chain reaction products were visualized by electrophoresis on 2.5% agarose gel. The remaining GSTM1 samples and all of the GSTT1 and GSTP1 genotypes were determined using real-time polymerase chain reaction using a TaqMan 7,700 (Applied Biosystems, Foster City, CA). The presence or absence of a fluorescent amplification signal was used as an indication of whether the GSTM1 and GSTT1 alleles were present or absent in a particular genomic DNA sample. Samples showing no signal or late cycle number for start of amplification were repeated and further analyzed with primers and probes for the actin gene to verify the presence of amplifiable DNA. Analysis of the single nucleotide polymorphism at codon 105 in the GSTP1 gene was performed using allele-specific probes.

Statistical Analysis

We used a two-level regression modeling approach to investigate the relationship between genotypes and lung function growth. The first-level model was a mixed effects linear regression of log-transformed lung function measures on age to estimate the 4-year average growth slope for each subject. This model included adjustment for the following time-varying covariates selected on the basis of lung growth biology reported in the literature, published reports, and previous analyses of the Children's Health Study lung function data (11, 32): height, body mass index (linear and quadratic terms), yearly report of physician-diagnosed asthma (active asthma), personal smoking, respiratory symptoms on the day of the test, exercise in the 30 minutes before the test, interaction of each of these factors with sex to allow for possible male/female differences, and spirometer testing variables (barometric pressure, temperature, technician, and spirometer). The second level of the model was an inverse variance-weighted regression of the subject-specific adjusted growth slopes (from the first model) on genotype. This model included adjustment for community of residence, cohort membership, sex, cohort-specific effects of ethnicity, environmental tobacco smoke exposure, and asthma status at study entry. The quantity of interest was the regression coefficient for genotype, which we report as the percentage of difference in annual lung function growth for subjects with one genotype relative to those with another genotype. For example, for GSTM1, we report the percentage difference in lung function growth for those with the GSTM1 null genotype compared with those with GSTM1 present genotypes. An analogous comparison is made for GSTT1, whereas for GSTP1, we present effect estimates under codominant, dominant, recessive, and additive genetic models for the GSTP1 val105 variant allele. In the additive model, genotype was coded as zero, one, or two for the number of val alleles.

We considered a number of a priori potential modifiers of the genetic effects on lung function growth, including ethnicity, sex, environmental tobacco smoke exposure, family income, parental education, personal smoking, family history of asthma or atopy, asthma, and wheezing status. These variables were identified as potential modifiers of the associations of the genotypes with lung function based on consideration of factors that may contribute to genetic and environmental interactions. We tested the statistical significance of effect modifiers by comparing models with and without the appropriate interaction terms and report stratified results for variables that show statistical evidence for effect modification of the genetic associations (p < 0.10). All analyses were conducted using the MIXED procedure in the SAS software and the MLn program (36, 37), and all reported p values are based on a two-sided alternative hypothesis.

The group of 1,940 eligible Children's Health Study participants with genotype data consisted of an approximately equal number of boys and girls (Table 1)

TABLE 1. Characteristics of the eligible* chs fourth-grade children with and without genotype



Genotyped

Not Genotyped
Variables
Number
 (n = 1,940)
Percentage
Number
 (n = 1,195)
Percentage
Sex
Girls98950.957648.2
Boys95149.061951.8
Ethnicity
White1,17360.560750.1
Hispanic52627.140934.2
Black824.2746.2
Asian965.0645.4
Other633.3413.4
Asthma
Ever diagnosed29215.015212.7
Never diagnosed1,64885.01,04387.3
Cohort
183843.261951.8
21,10256.857648.2
ETS
Present31216.427723.8
Absent1,59583.688976.2
Personal smoking
Smokers1176.013411.2
Nonsmokers1,82394.01,06188.8
Mean§ (SD)
Height, m1.4 (0.1)1.4 (0.1)
Weight, kg35.9 (8.8)36.9 (9.5)
BMI, kg/m218.2 (3.4)18.6 (3.6)
Age, years
10.1 (0.5)
10.1 (0.6)

*At least two-lung function tests available during the 4 years of follow-up for 2 fourth-grade cohorts recruited in 1993 and 1996; distributions at study entry.

Physician-diagnosed asthma at study entry.

Child smoker at the end of 4 years of follow-up.

§Mean and SD at study entry.

Definition of abbreviations: BMI = body mass index; CHS = Children's Health Study; ETS = environmental tobacco smoke.

. The majority of participants were non-Hispanic white (60.5%) and Hispanic (27.1%). Physician-diagnosed asthma was reported for 15.1% of children. Children ranged in age from 8 to 11 years at study entry, with a mean age of 10.1 years. An average of 4.4 lung function tests was recorded on each participant. Over the 4-year period of study, participants' FEV1 and FVC both grew at an average rate of 11.9% per year. Children in the Children's Health Study with and without genotype data did not differ substantially by age, sex, height, and weight. Compared with children with genotyping data, children without genotyping data participated more frequently in the earlier cohort (52% versus 43%), had more environmental and personal tobacco smoke exposure (24% versus 16%), had slightly less asthma (13% versus 15%), and were less likely to be non-Hispanic white (51% versus 60%). The differences in these characteristics were related to differences in the socioeconomic status of the groups, as indicated by differences in parental education and family income. In the group with genotype date, 26% of parents completed college and 27% had a high school education or less compared with 16% and 40%, respectively, among the groups without genotyping. The proportion of families with annual income that was less that $22,500 was higher in the group without genotyping (39%) than in the group with genotyping (26%). The loss to follow-up for buccal cell sample collection was largely caused by families moving out of the study area for economic reasons, as ascertained by a questionnaire that asked about the reason for moving. The families with lower education and income were more likely to move. This loss to follow-up is unlikely to introduce bias into the study because moving for economic reasons is unlikely to be related to genotype.

GSTM1 and GSTT1 null genotypes were present in 43.4% and 22.8% of children, respectively. Fewer children were homozygous for the GSTP1 val105 variant allele (13.3%). The frequency of genotypes and alleles for GSTP1, GSTM1, and GSTT1 varied among the ethnic populations in our study (Table 2)

TABLE 2. Allele and genotype frequencies for GSTP1, GSTM1, and GSTT1 among participants in the children's health study



Ethnicity
NHW
Hispanics
Blacks
Asians
Others
Genotypes
n
%
n
%
n
%
n
%
n
%
GSTP1
Ile/Ile51244.614828.62531.64749.02336.5
Ile/Val51544.927252.64050.64243.82641.3
Val/Val12110.59718.81417.777.31422.2
Observed allele frequency
Ile0.670.550.570.680.57
Val0.330.450.430.320.43
GSTM1
Null53145.321540.92125.64647.93047.6
GSTT1
Null
256
21.8
112
21.3
16
19.5
44
45.8
14
22.2

Definition of abbreviations: GST = glutathione-S-transferase; NHW = non-Hispanic whites.

GSTP1 alleles are consistent with Hardy–Weinberg Equilibrium in each group.

. African Americans had the lowest frequency of the GSTM1 null genotype. A higher proportion of Asians had the GSTT1 null genotype than in the other ethnic groups. The allele frequency for the val105 variant was highest in Hispanics (0.45) and lowest in Asians (0.32). The alleles of GSTP1 were in Hardy-Weinberg equilibrium within each ethnic group. The frequencies in our study population are within the ranges reported in other study populations (38, 39).

We found that GSTM1 and GSTP1 genotypes were associated with statistically significant deficits in annual lung function growth (Table 3)

TABLE 3. Effects of GSTM1, GSTT1, and GSTP1 genotypes on annual lung function growth among chs participants




Percent Deficit in Growth Rate*
Gene
Genetic Model
FVC
 
 Percentage Deficit (95% CI)
FEV1
 
 Percentage Deficit (95% CI)
MMEF
 
 Percentage Deficit (95% CI)
GSTM1Present
Null−0.21 (−0.40, −0.03)−0.27 (−0.50, −0.04)−0.30 (−0.78, 0.19)
GSTT1Present
Null−0.13 (−0.36, 0.10)−0.09 (−0.37, 0.20)0.27 (−0.32, 0.86)
GSTP1Recessive
ile/ile or ile/val
val/val−0.35 (−0.62, −0.07)−0.34 (−0.68, 0.00)−0.03 (−0.74, 0.69)
Dominant
ile/ile
val/val or ile/val0.04 (−0.16, 0.24)0.05 (−0.20, 0.29)0.09 (−0.41, 0.59)
Codominant
ile/ile
ile/val0.13 (−0.08, 0.33)0.13 (−0.12, 0.39)0.10 (−0.42, 0.63)
val/val−0.27 (−0.58, 0.03)−0.27 (−0.64, 0.11)0.03 (−0.74, 0.81)

Additive
−0.07 (−0.21, 0.07)
−0.06 (−0.24, 0.11)
0.04 (−0.32, 0.40)

*Growth rates were adjusted for the covariates listed in METHODS.

p < 0.05.

p < 0.052.

Definition of abbreviations: CHS = Children's Health Study; CI = confidence interval; GST = glutathione-S-transferase; MMEF = maximal mid-expiratory flow rate.

. The GSTM1 null allele was associated with a 0.21% annual deficit in FVC growth and a 0.27% annual deficit in FEV1 growth. For GSTP1, the recessive model for the val105 allele indicates a 0.35% deficit per year in FVC growth and a 0.34% reduction in growth for FEV1. The codominant model was consistent with decreased growth in FVC and FEV1 for the homozygous val105 genotype. These findings are consistent with a decrease in lung volume growth.

Children with asthma had larger deficits in lung function growth than children without asthma (Table 4)

TABLE 4. Effects of GSTM1, GSTT1, and GSTP1 genotypes on annual lung function growth among children with and without physician-diagnosed asthma at study entry





Percent Deficit in Growth Rate*
Parameter
Gene
Genetic Model
Control Subjects
 
 (n = 1,620)
 Percentage Deficit (95% CI)
Patients with Asthma
 
 (n = 287)
 Percentage Deficit (95% CI)
p Value for
 Interaction
FVCGSTM1Present
Null −0.19 (−0.40, 0.01) −0.52 (−1.02, −0.01)0.70
GSTT1Present
Null −0.12 (−0.37, 0.12) −0.15 (−0.83, 0.53)0.70
GSTP1Recessive
ile/ile or ile/val
val/val −0.27 (−0.57, 0.04) −0.88 (−1.60, −0.15)0.20
FEV1GSTM1Present
Null −0.27 (−0.52, −0.02) −0.49 (−1.12, 0.14)0.98
GSTT1Present
Null −0.07 (−0.37, 0.24) −0.14 (−0.97, 0.70)0.51
GSTP1Recessive
ile/ile or ile/val
val/val −0.18 (−0.55, 0.20) −1.21 (−2.10, −0.32)§0.04
MMEFGSTM1Present
Null −0.34 (−0.84, 0.17) −0.16 (−1.60, 1.30)0.61
GSTT1Present
Null 0.33 (−0.29, 0.95) −0.01 (−1.91, 1.94)0.45
GSTP1Recessive
ile/ile or ile/val


val/val
 0.31 (−0.44, 1.08)
 −1.38 (−3.43, 0.72)
0.06

*Growth rates were adjusted for the covariates listed in METHODS.

Two-way p value for interaction between asthma at baseline and gene.

p < 0.05.

§p < 0.01.

Definition of abbreviations: CI = confidence interval; GST = glutathione-S-transferase; MMEF = maximal mid-expiratory flow rate.

. The annual deficits in FEV1 growth associated with the homozygous val105 genotype were statistically significantly larger among children with asthma compared with those without asthma. We found no statistically significant effects of genotypes at the three loci on MMEF in children with or without asthma; however, children with asthma and the homozygous val105 genotype had a deficit of 1.4% compared with children with asthma and the ile105 genotypes that was larger than for children without asthma (interaction p value of 0.06).

The annual deficits in lung function growth associated with GSTM1 and GSTP1 genotypes were larger and statistically significant when the analyses were restricted to non-Hispanic white children but not when restricted to Hispanic children (Table 5)

TABLE 5. Effects of GSTM1, GSTT1, and GSTP1 genotypes on annual lung function growth for hispanic and nonhispanic white children





Percent Deficit in Growth Rate*
Parameter
Gene
Genetic Model
Hispanics
 
 (n = 526)
 Percentage Deficit (95% CI)
Non-Hispanic Whites
 
 (n = 1,173)
 Percentage Deficit (95% CI)
p Value for
 Interaction
FVCGSTM1Present
Null −0.06 (−0.45, 0.33) −0.28 (−0.51, −0.05)0.32
GSTT1Present
Null −0.09 (−0.56, 0.38) −0.14 (−0.42, 0.15)0.81
GSTP1Recessive
ile/ile or ile/val
val/val −0.23 (−0.72, 0.26) −0.46 (−0.83, −0.09)0.35
FEV1GSTM1Present
Null −0.10 (−0.60, 0.40) −0.39 (−0.67, −0.11)§0.28
GSTT1Present
Null 0.02 (−0.58, 0.63) −0.12 (−0.47, 0.23)0.67
GSTP1Recessive
ile/ile or ile/val


val/val
 0.01 (−0.62, 0.64)
 −0.56 (−1.01, −0.11)
0.09

*Growth rates were adjusted for the covariates listed in METHODS.

Two-way p value for interaction between race and gene.

p < 0.05.

§p < 0.01.

Definition of abbreviations: CI = confidence interval; GST = glutathione-S-transferase.

. Among non-Hispanic whites, deficits in FEV1 growth were 0.39% for the GSTM1 null genotype and 0.56% for the GSTP1 val105/val105 genotype. Deficits in FVC among non-Hispanic whites were also larger and statistically significant for the GSTM1 null genotype and the GSTP1 val105/val105 genotype. Among Hispanic children, the deficits in FVC and FEV1 associated with GSTM1 null genotype and GSTP1 val105/val105 genotypes were smaller and were not statistically significantly different from zero. We found little evidence for modification of the genotype associations sex, family income, environmental tobacco smoke exposure, family income, parental education, personal smoking, family history of asthma, or atopy.

Consideration of the joint effects of GSTM1 and GSTP1 indicates that the genes may be operating through independent biologic pathways. In models among non-Hispanic white children, deficits in FVC and FEV1 growth associated with GSTM1 null genotype and the GSTP1 val105/val105 genotype were approximately additive (Table 6)

TABLE 6. Associations of GSTP1 and GSTM1 genotypes with lung function growth rates in nonhispanic whites



Percent Deficit in Growth Rate*
 
 GSTM1

FVC
FEV1
GSTP1PresentNullPresentNull
ile/ile or ile/val−0.24 (−0.48, 0.01)−0.30 (−0.60, −0.01)§
val/val
−0.37 (−0.89, 0.15)
−0.76 (−1.27, −0.24)
−0.44 (−1.07, 0.19)
−0.95 (−1.57, −0.33)

*Growth rates were adjusted for the covariates listed in METHODS.

Two-way interaction between GSTP1 and GSTM1, p = 0.68.

Two-way interaction between GSTP1 and GSTM1, p = 0.65.

§p < 0.05.

p < 0.01.

Definition of abbreviations: GST = glutathione-S-transferase.

.

We found that children who had the GSTM1 null genotype and those who were homozygous for the GSTP1 val105 variant allele had lower lung function growth than children with the more common alleles at the two loci. The deficits in FVC and FEV1 growth were largest and statistically significant for children with asthma and non-Hispanic white children. Although the magnitude of the cumulative effects on lung function over the period of study was relatively small, approximately 1%, a larger absolute deficit in maximum attained lung function may occur, and individuals with the variant genotypes may be susceptible to respiratory symptoms and conditions associated with low adult lung function, such as COPD. Furthermore, children with asthma had larger cumulative deficits, approximately 5%, which may compound deficits in lung function associated with asthma. We are not aware of other studies that have examined the associations between genetic variants and lung function growth in children.

We observed effects of GSTM1 and GSTP1 genotypes on FVC and FEV1 growth, but not MMEF. The similar magnitude of effects on FVC and FEV1 growth suggests that GSTM1 and GSTP1 genotypes primarily affect growth in lung volume with little effect on measures of air flow. The explanation for the predominant effects on growth of lung volume is unclear. In adults, GSTM1 and GSTP1 variant alleles have been associated with COPD and asthma, suggesting a role for these loci in the pathogenesis of airflow limitation at older ages (4043). It may be that the rapidly increasing lung volumes of children are more susceptible to reduced growth rates than are small airway flows such as MMEF. In addition, the measurement of MMEF has larger uncertainties than measurements of FVC and FEV1, and the larger measurement error may result in artifactually reduced estimates for MMEF. No formal efforts were made to adjust tests of significance for multiple comparisons in these analyses. The level of significance and the consistency of the results for lung function measurements reduce the likelihood that the results were caused by chance alone.

We found some indication of ethnic differences in the effects of GSTM1 and GSTP1 genotypes on FVC and FEV1 growth. Lung function growth is a complex trait that probably involves multiple genes and pathways. The effects of variation at one locus may depend on the genetic background of each ethnic group. Non-Hispanic white children may have a genetic background that allows a larger effect of GSTM1 and GSTP1 genotypes than Hispanic children. In addition, the associations may have been confounded by ethnic admixture. Lung function varies by ethnicity and genes such as GSTP1 have different frequencies in our Hispanics and non-Hispanic white populations. Hispanics are likely to show stronger population stratification caused by recent admixture that could result in biased estimates from uncontrolled confounding. We did not collect information to allow control for confounding from admixture within the ethnic groups. Because ethnic differences in lung function growth are small, we believe that the potential magnitude of a bias from confounding of the lung function growth results by admixture is small. The genes are on different chromosomes and thus are not in linkage disequilibrium. However, each locus could be in linkage disequilibrium with an unknown causal gene(s). The potential for linkage disequilibrium is a fundamental limitation of the candidate gene association approach and depends on the linkage disequilibrium surrounding each locus in the study populations. Different patterns of linkage disequilibrium could explain the ethnic differences. The sample of the study population with genotyping had a greater proportion of non-Hispanic white children than the group without genotyping data (60% versus 50%). We adjusted for ethnicity and found no other substantial differences in characteristics of those with genotypes compared with those without genotypes, indicating that any bias from selection by availability of DNA is likely to be small. We had an insufficient number of children from African American, Asian, and other ethnic groups to allow assessment of the genetic effects within these groups.

We identified three GSTs with common functional polymorphisms as candidate genes based on a mechanistic hypothesis about pathways involved in lung function growth (23, 2730, 44). A broad spectrum of determinants for reduced lung function in children and lung function decline and COPD in adults has been identified; however, a unifying mechanistic framework for the inter-related determinants has yet to be established (22, 25, 43, 45). An emerging body of evidence indicates that COPD, asthma, and respiratory infections increase pulmonary and systemic levels of oxidative stress (22, 25, 4648). We hypothesized that excess oxidative stress provides a mechanistic framework that unifies inter-relationships between childhood lung function growth, asthma and respiratory infections, environmental exposures such as air pollution and tobacco smoke, and factors such as diet and genetics (23). Excess oxidative stress is the proximal event leading to inflammation, cell death, and subsequent airway remodeling among individuals with inadequate defenses. Acute events may lead to long-term effects in the setting of chronic excess oxidative stress. The measurement of chronic levels of oxidative stress is not currently feasible for large epidemiologic studies. Genetic variation may provide a useful method to classify chronic levels of oxidative stress. GSTM1 and GSTP1 are important antioxidant enzymes in the lung that function as antioxidants in xenobiotic, peroxide, and hydroperoxides metabolism pathways to reduce oxidative stress (29, 30). Our findings support the proposed mechanistic framework and the usefulness of studies focusing on genes involved in oxidative stress pathways. The relatively modest effects of GSTM1 and GSTP1 variants on lung function growth suggest that other genes may influence lung function growth (23). Investigations of the effects of additional loci on lung function growth are warranted.

The larger effects of GSTP1 genotype on lung function growth among children with asthma are consistent with the proposed oxidative stress mechanism. A number of reports indicate that children with asthma have lower lung function growth than children without asthma (49). The reasons for the slower growth have yet to be clarified, but treatment with inhaled steroids does not appear to contribute to or reduce the deficits (50). An alternative explanation is that the excess oxidative stress in the airways of children with asthma may interfere with normal lung growth and development. Children with the val105 genotype may be less able to defend their airways from the adverse effects of excess oxidative stress associated with asthma.

We conclude that school children with a GSTM1 null allele or a GSTP1 val105/val105 genotype show decreased growth in FVC and FEV1. We consider lung function to be a quantitative trait that is determined by multiple genes and exposures. Each gene is likely to contribute modestly to variation in lung function growth. In this study, we show that the GSTM1 and GSTP1 may contribute to variation in growth and, based on lung physiology and the functional biology of the genes, implicate oxidative stress as a potentially important pathway for further investigation. Although the effects of GSTM1 and GSTP1 val105 allele are modest in magnitude for individuals, this genetic variation may have public health importance, especially for children with asthma. The variants are common, and small changes in the population distribution of lung function mean level can substantially increase the number of individuals with symptomatically low lung function. Children with these genotypes, especially those with asthma, may have lower attained lung function at maturity and be more susceptible to a spectrum of adverse respiratory outcomes associated with chronic excess oxidative stress. Further follow-up of the Children's Health Study cohort will be necessary to determine whether the reduction in lung function growth results in lower attained lung function. Investigation of the relationships between genes involved in oxidative stress pathways and lung function growth is warranted.

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Correspondence and requests for reprints should be addressed to Frank Gilliland, Department of Preventive Medicine, University of Southern California Keck School of Medicine, 1540 Alcazar Street, CHP 236, Los Angeles, CA 90033. E-mail:

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