Glutathione S-transferase genotypes modify lung function decline in the general population: SAPALDIA cohort study

The SAPALDIA cohort study has been described in details elsewhere [21, 22]. In brief, participants predominantly of European-Caucasian ethnicity and Swiss nationality, were randomly selected from eight regional population registries [21, 22]. Health examinations at baseline (1991) and follow-up (2002) included an interview about respiratory health, occupational and lifestyle exposures as well as spirometry, a methacholine bronchial challenge test and end-expiratory carbon monoxide measurement. Participants gave informed consent at both surveys separately for health examination, interview and blood analysis. The SAPALDIA cohort study complies with the Helsinki Declaration and has received ethical approval by the central ethics committee of the Swiss Academy of Medical Sciences and the Cantonal Ethics Committees for each of the eight examination areas.

Participation rate in SAPALDIA at baseline was 59.3%. Of 9651 participants examined at baseline, 8047 subjects (86%) agreed to participate fully or partially at follow-up. For the present investigation no selection of SAPALDIA participants was made, but we included all subjects with complete information on outcome and covariate data. 5973 subjects (62%) completed the entire follow-up protocol including spirometry and blood sampling. For 275 participants no DNA was available for genetic testing due to refusal or insufficient blood sample volume. Valid spirometry data on FEV1, FVC and FEF₂₅-₇₅ were not available from both surveys for 215, 310 and 373 participants, respectively. Genotyping for one or more genetic polymorphisms failed in 13 participants. Missing information on one or more covariates included in the regression models further diminished the sample size (n = 784). The final sample size was 4686, 4591 and 4528 subjects for the investigation of annual change in FEV1, FVC and FEF₂₅-₇₅, respectively. Comparison of the baseline characteristics of SAPALDIA cohort participants included in and excluded from this analysis revealed that excluded SAPALDIA participants were on average older, more likely to have been smokers at baseline examination, and had reported a slightly higher number of pack-years at baseline (Table 1). Accordingly lung function was slightly lower and the proportion of subjects with an FEV1/FVC ratio below 70% was slightly higher in non-participants excluded from this current investigation.

INSERT [Table 1]

The spirometry measurement procedures at both time points have been described elsewhere in detail [21, 22]. Briefly, identical spirometer devices (Sensormedics model 2200, Yorba Linda, USA) and protocols were used at baseline and follow-up and their comparability was assessed prior to the follow-up study [23]. Three to maximal eight forced expiratory lung function manoeuvres were performed by each participant and a minimum of two acceptable forced expiratory flows, forced vital capacity (FVC), forced expiratory volume in the first second (FEV₁) and forced expiratory flows during the middle half of the FVC (FEF_25–75) complying with American Thoracic Society criteria [24] were obtained. Expiratory flow measures with the highest sum of FVC, FEV₁ and FEF_25–75 were taken from the same flow-volume curves.

Bronchial hyperresponsiveness (BHR) to methacholine chloride (Provocholine^®, Roche, Nutley, New Jersey, USA) was defined as presence of a 20% or greater drop in FEV1 compared to the highest FEV1-value measured during the test. Increasing concentrations of methacholine (0.39, 1.56, 6.25, and 25.0 mg/ml solutions in a phosphate buffer without phenol) were administered through an aerosol dosimeter (Mefar MB3, Bovezzo, Italy) up to a cumulative dose of 2 mg (8.37 ug/mol).

DNA was extracted from EDTA blood using the PUREGENE™ DNA purification kit (GENTRA Systems, Minneapolis, USA)[21]. In all subjects GSTM1 and GSTT1 gene deletions and a single nucleotide polymorphism (SNP) in GSTP1 leading to the amino acid substitution Ile105Val were genotyped on the ABI Prism 7000 sequence detection system (Applied Biosystems, Rotkreuz, Switzerland) using 5'nuclease real time PCR (TaqMan^®) assay and fluorescently labeled allele-specific probes. Following primers and probes were used for GSTM1: forward 5'-GGACATTTTGGAGAACCAGACC-3' and reverse 5'-CTGGATTGTAGCAGATCATGCC-3' primers and GSTM1-specific probe 5'-VIC-TGGACAACCATATGCAG-MGB-3'; for GSTT1: forward 5'-GTCATTCTGAAGGCCAAGGACTT-3' and reverse 5'-GGCATCAGCTTCTGCTTTATGGT-3' primers and GSTT1-specific probe 5'-FAM-CACCTGCAGACCCC-MGB-3'; for GSTP1 Ile105Val: forward 5'-CCTGGTGGACATGGTGAATGAC-3' and reverse 5'-CAGATGCTCACATAGTTGGTGTAGA-3' primers and Ile105 -specific probe 5'-VIC-CTGCAAATACATCTCC-MGB-3'and Val 105 -specific probe 5'-FAM- CTGCAAATACGTCTCC-MGB-3'. GSTM1/GSTT1 assays were repeated for all DNA samples carrying double homozygous GSTM1 and GSTT1 deletions using internal positive GSTP1 controls. All double homozygous deletion carriers could be confirmed. With this approach, hemizygous GSTM1 or GSTT1 carriers were not distinguishable from homozygous carriers. In addition a 5% random sample of all DNA samples was regenotyped with highest reproducibility (>99.5%). Hardy-Weinberg equilibrium (HWE) was tested for GSTP1 Ile105Val using Arlequin (Version 2.000) software [25].

The dependent variable, annual change in lung function, was calculated by dividing the difference between follow-up and baseline lung function by the number of follow-up years. Multiple linear regression analysis was used to estimate in a fixed effect model of the association of GST genotypes with annual change in lung function. Covariates included in the models were baseline lung function, age, sex, height, weight change during follow-up, study center, level of education, exposure to gas and dust at work at baseline, smoking status at baseline and at follow-up, pack-years smoked at baseline and during follow-up. Cumulative cigarette smoking exposure was summarized in two separate variables: "pack-years smoked up to baseline" and "pack-years smoked during follow-up". The following categories of smoking status were derived for the current study: "Never smokers" reported to be non-smokers at both surveys (n = 2258). "Ever smokers" had to have smoked more than 20 packs of cigarettes or more than 360 g of tobacco (n = 2428) in their lifetime by the end of the follow-up period. Ever smokers were further divided into: "persistent smokers" reported current smoking at both surveys (n = 1026), and "others" were all remaining subjects, comprising participants reporting at both surveys former smoking (n = 944), quitting smoking during follow-up (n = 387), starting smoking during follow-up (n = 38), non-smoking at baseline and former smoking at follow-up (n = 29) and former smoking at baseline and current smoking at follow-up (n = 4). 48 participants provided inconsistent smoking information. Exclusion of these subjects in a sensitivity analysis did not change the strength or the direction of the association observed. Effect modification of genotype/lung function associations by gender, smoking status, and smoking intensity (pack-years up to baseline and during follow-up in ever smokers), as well as BHR and age, was assessed by including according multiplicative interaction terms in the regression models. Trend tests for the combination of GSTT1 and GSTM1 genotypes were conducted by using a genotype combination variable coded as "presence of zero, one and two gene deletion polymorphisms" as ordinal variable in the model. Two-sided p-values of <0.05 and <0.10 were considered as statistically significant for main effects and interactions [26], respectively. Correction for multiple testing was done using the conservative Bonferroni correction. The associations were corrected for the number of statistical tests performed (main effects and interactions with gender and smoking intensity) (thirty comparisons per lung function parameter investigated, consisting of fifteen tests in men and fifteen tests in women: all; never smokers; persistent smokers). The Bonferroni corrected significance level for the a priori hypotheses regarding association between GST genotypes and lung function change in men and women including the a priori assessment of interaction with gender and smoking and was P > 0.0017. Sensitivity analyses regarding age and BHR were not corrected for multiple testing. All analyses were conducted using STATA SE version 8.0 (Stata Corporation, TX, USA).

No independent association of GSTM1 or GSTP1 genotype with any of the lung function parameters was observed, irrespective of gender. GSTT1 gene deletion alone or in combination with GSTM1 deletion was associated with accelerated lung function decline in men, but not women. Men homozygous for the GSTT1 gene deletion exhibited an excess annual change in FEV1 of -5.3 ml/yr (P = 0.001). The GSTT1 effects on FVC and FEF_25–75 were comparable in size and direction, but did not reach statistical significance. Men carrying the double homozygous gene deletions of GSTT1 and GSTM1 had on average a -8.3 ml/yr greater annual decline in FEV1 than men with at least one copy of both, the GSTT1 and the GSTM1 gene (P for trend <0.001); the according excess change was -6.5 ml/yr (P = 0.045) for FVC and -7.8 ml/yr (P = 0.094) for FEF_25–75. The interactions between gender and GSTT1 deletion alone or in combination with GSTM1 deletion were statistically significant for FEV1, FEF_25–75 and FVC (for GSTT1/GSTM1 combination only).

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The majority of the reported association results did not withhold the conservative Bonferroni correction; however the GSTT1 genotype alone or in combination with GSTM1 genotype showed a significant association with annual change in FEV1 even after Bonferroni correction. The effect of double homozygous GSTT1 and GSTM1 deletion on lung function decline is graphically presented as predicted mean annual FEV1 decline in different genotype/gender strata.

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An important determinant of premature lung function decline is active smoking. In our study population, persistent male smokers exhibited on average an -6.6 ml/yr greater annual FEV1 change than never smoking men; the average change in FEV1 was -42.8 ml/yr (± 35.6) in male persistent smokers and -36.2 ml/yr (± 33.2) in male never smokers. In persistent smokers each pack-year smoked during follow-up was associated with an excess average annual FEV1 change of -0.8 ml/yr. We assessed the impact of genetic GST deficiency on lung function decline separately for never smokers and persistent smokers; associations observed in ever-smokers were similar to those reported here for persistent smokers (data not shown). Irrespective of gender or smoking status no independent effects of GSTM1 or GSTP1 Ile105Val genotype on accelerated decline of FEV1, FVC or FEF_25–75 were observed. Male persistent smokers with GSTT1 null genotype exhibited on average an excess annual decline in FEV1 of -8.0 ml/yr (P = 0.013) when compared to persistent smokers with GSTT1 non-null genotype. The according GSTT1 effect in male never smokers was -5.6 ml/yr (P = 0.025). The difference in GSTT1 effect between persistent and never smokers was not statistically significant (P for interaction>0.10). The GSTT1 effect in persistent smokers was modified by packyears smoked to baseline (P for interaction<0.001) and during follow-up (P for interaction = 0.029). Similar trends for the GSTT1 effect on FEV1 decline in smoking strata, though lacking statistical significance, were observed for FVC and less clearly for FEF_25–75. The GSTT1 genotype alone or in combination with GSTM1 genotype was not associated with excess lung function change in women, irrespective of smoking status. There was a suggestion that heterozygosity for the GSTP1 Ile105Val SNP was associated with slower decline in FVC in persistent smokers (P = 0.030), but no according heterozygous effects on FEV1 or FEF_25–75 were observed.

INSERT [Table 4]

The GST genotypes have previously been associated with asthma and BHR [28]. Restriction of the analysis to subjects without a report of asthma (data not shown) and without the presence of BHR at either baseline or follow-up (Table 5) revealed comparable associations in size between GST genotypes and lung function change as reported for the whole study population (Table 3), irrespective of gender and lung function parameter. Thus the observed GST/lung function decline associations are not merely due to an effect of GST on asthma or BHR.

BHR was previously shown to be predictive of COPD [29]. Results of the investigation of the GST effects on decline in lung function among BHR positive subjects (Table 5) suggested that the respective impact of GSTT1 and GSTM1 gene deletion might be modified by BHR. The interaction between GST genotypes and BHR did not reach statistical significance, though. In male BHR positive subjects, GSTM1 rather then GSTT1 deficiency was associated with accelerated decline in FEV1 (-8.2 ml/yr, P = 0.017) and FEF_25–75 (-12.4 ml/yr, P = 0.051). Again, the lung function decline was strongest for the combined GSTM1/GSTT1 genotypes, consistent with a gene dose-response. For both, FEV1 and FEF_25–75 effect estimates for GSTM1T1 both null were stronger than those observed among male BHR negative subjects. No association of GST genotype with FVC was observed in male BHR positive subjects. In BHR positive women again no statistically significant GST genotype/lung function associations were observed.

INSERT [Table 5]

Both, lung function growth and decline are age-dependent processes. The SAPALDIA cohort also includes young adults (age at baseline 18 to 60 years). To confirm that the observed associations between GST genotype and lung function change are due to an impact of these genotypes on age-related decline, we restricted analysis to subjects older than 30 years, an age at which lung growth has ceased and age-related lung function decline started [30] (data not presented). In men we observed associations of GSTT1 alone or in combination with GSTM1 with change in FEV1 and FVC that were similar in trend to the ones observed in the entire study sample (for GSTT1 and FEV1: -5.8 ml/yr (P = 0.001); for GSTM1 and GSTT1 both null and FEV1: -7.4 ml/yr (P = 0.009)). The association with change in FVC was more pronounced (for GSTT1: -4.4 ml/yr (P = 0.08); for GSTM1 and GSTT1 both null: -9.5 ml/yr (P = 0.005)). In contrast, the non-significant association observed for GSTT1 genotype and change in FEF_25–75 was no longer present in men aged 30 years or older (for GSTT1: -0.4 ml/yr (P = 0.89); for GSTM1 and GSTT1 both null: -1.9 ml/yr (P = 0.72)). Instead, the association between GSTM1 null genotype and excess annual change in FEF_25–75 became statistically significant (for GSTM1: -7.6 ml/yr (P = 0.024)). In women over age 30 at baseline, we did not observe any GST genotype/lung function decline association.