An interaction between Nrf2 polymorphisms and smoking status affects annual decline in FEV1: a longitudinal retrospective cohort study
© Masuko et al; licensee BioMed Central Ltd. 2011
Received: 20 February 2011
Accepted: 20 July 2011
Published: 20 July 2011
An Nrf2-dependent response is a central protective mechanism against oxidative stress. We propose that particular genetic variants of the Nrf2 gene may be associated with a rapid forced expiratory volume in one second (FEV1) decline induced by cigarette smoking.
We conducted a retrospective cohort study of 915 Japanese from a general population. Values of annual decline in FEV1 were computed for each individual using a linear mixed-effect model. Multiple clinical characteristics were assessed to identify associations with annual FEV1 decline. Tag single-nucleotide polymorphisms (SNPs) in the Nrf2 gene (rs2001350, rs6726395, rs1962142, rs2364722) and one functional SNP (rs6721961) in the Nrf2 promoter region were genotyped to assess interactions between the Nrf2 polymorphisms and smoking status on annual FEV1 decline.
Annual FEV1 decline was associated with smoking behavior and inversely correlated with FEV1/FVC and FEV1 % predicted. The mean annual FEV1 declines in individuals with rs6726395 G/G, G/A, or A/A were 26.2, 22.3, and 20.8 mL/year, respectively, and differences in these means were statistically significant (pcorr = 0.016). We also found a significant interaction between rs6726395 genotype and smoking status on the FEV1 decline (p for interaction = 0.011). The haplotype rs2001350T/rs6726395A/rs1962142A/rs2364722A/rs6721961T was associated with lower annual decline in FEV1 (p = 0.004).
This study indicated that an Nrf2-dependent response to exogenous stimuli may affect annual FEV1 decline in the general population. It appears that the genetic influence of Nrf2 is modified by smoking status, suggesting the presence of a gene-environment interaction in accelerated decline in FEV1.
Among pulmonary function test (PFT) measurements, forced expiratory volume in one second (FEV1) is the most reproducible . Therefore, it is suitable for analyzing changes in pulmonary function over time. Accelerated decline in FEV1 is considered as an important predictor for the development of inflammatory obstructive lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD) [2, 3]. A rapid decline in FEV1 may be affected by multiple factors, including environmental and genetic factors.
The most important environmental factor for FEV1 decline is cigarette smoking. In their landmark study, Fletcher et al.  demonstrated that smokers had a steeper decline in FEV1 than non-smokers. Subsequent studies have revealed that the rate of decline in FEV1 depends on pack-years smoked and that the accelerated decline in FEV1 in smokers slows to normal rates of decline upon smoking cessation [4–6]. Cigarette smoke contains high concentrations of oxidants, including reactive oxygen species and reactive nitrogen species . Oxidative stress due to cigarette smoking promotes direct injury to airway epithelium, expression of genes encoding proinflammatory mediators, and protease/antiprotease imbalance , all of which induce chronic inflammation in the lung of smokers that results in deterioration of lung function.
However, only 10-15% of smokers develop a severe impairment of lung function . In addition to environmental factors, genetic determinants play an important role in rapid decline in lung function. A pedigree-based study has shown that FEV1 levels have a heritability that is independent of cigarette smoking and disease status such as asthma . Furthermore, recent large-scale genome-wide association studies have identified several loci associated with FEV1 and the FEV1/forced vital capacity (FVC) ratio [10, 11].
It is possible that oxidant/antioxidant imbalance in the lungs of smokers results in an accelerated loss of lung function. Nrf2 is a major regulator of the antioxidant response . Nrf2 regulates the expression of several genes encoding antioxidant and detoxification proteins . In animal models, Nrf2 plays an important role in reducing inflammation associated with elastase-induced emphysema . In human studies, attenuation of Nrf2 due to the down-regulation of the Nrf2 mRNA has been detected in alveolar macrophages of COPD patients . Moreover, 3 single-nucleotide polymorphisms (SNPs) in the promoter region of the Nrf2 gene have an influence on the gene's transcriptional activity, and one of these SNPs is associated with the development of acute lung injury . Recently, one SNP (rs2364723) in the first intron of Nrf2 has been shown to be related to a lower FEV1 . All of these findings indicate that an Nrf2-dependent adaptive response is important in inhibiting the oxidant-induced lung inflammation that results in a rapid decline in lung function.
Therefore, we conducted a longitudinal retrospective cohort study of a general Japanese population in order to analyze associations between Nrf2 polymorphisms and annual decline in FEV1. We also assessed whether an interaction between the Nrf2 polymorphisms and smoking status affects FEV1 decline.
The Institutional Review Boards of the University of Tsukuba (IRB No. 136) and the Tsukuba Medical Center (IRB No. 2008-01-31) approved the study, and each subject provided written informed consent.
Pulmonary Function Test (PFT)
Spirometry was performed with an electronic spirometer (Autospiro SYSTEM7; Minato Medical Science Co., Ltd., Osaka, Japan) according to the standards recommended by the Japanese Respiratory Society (JRS) . The patients performed the maneuvers without any bronchodilators. The highest value for the sum of FVC and FEV1 was selected as the measurement for each PFT. FVC and FEV1 were expressed as a percentage of predicted values approved by the JRS . All available longitudinal data for each participant were collected retrospectively to estimate the annual decline in FEV1.
Single-Nucleotide Polymorphism (SNP) Selection and Genotyping
Using JPT (Japanese in Tokyo, Japan) genotype data (PhaseIII/Rel#2, Feb09, on NCBI B36 assembly, dbSNP b126) from the International HapMap project http://hapmap.org/, four tag SNPs (rs2001350, rs6726395, rs1962142, rs2364722) were identified in the 34.38 kb Nrf2 gene region (chromosome 2, position 177,803,285-177,837,663). We used the multi-marker predictor method implemented in the Tagger program . Tag set was generated using a threshold r2 of 0.8 and a minor allele frequency of > 0.1. Genomic DNA was extracted from samples of whole blood from each of the 915 participants by an automated DNA extraction system (QuickGene-610L, FUJIFILM, Tokyo, Japan). Genotyping of these 4 bi-allelic SNPs were attempted for each participant by the pre-designed TaqMan allele-specific polymerase chain reaction (PCR) assays according to the manufacturer's instruction (Applied Biosystems, Foster City, CA).
It has been reported that 3 SNPs (rs6721961, rs6706649, and rs35652124) located in the promoter region of the Nrf2 gene affect the transcriptional activity of Nrf2 . Genotyping for rs6721961 was carried out for each participant by the TaqMan technique using a pair of primers and a pair of oligonucleotide probes designed and synthesized by Applied Biosystems. The sequences of the primers were as follows: forward, 5'-CAGTGGGCCCTGCCTAG-3'; reverse, 5'-TCAGGGTGACTGCGAACAC-3'. The TaqMan fluorescence-labeled oligonucleotide probes were 5'-[VIC]-TGGACAGCGCCGGCAG-3' and 5'-[FAM]-TGTGGACAGCTCCGGCAG-3'. Because rs6706649 and rs35652124 are only 2 base pairs apart, the allele-specific probe technique was not appropriate for genotyping. Instead, for these 2 SNPs, direct DNA sequencing analysis was performed for 50 subjects (25 major allele homozygotes and 25 minor allele homozygotes for rs6726395 SNP). PCR amplification was carried out with 50 ng genomic DNA and a pair of primers flanking the 2 SNPs by a GeneAmp PCR System (Applied Biosystems). The primer sequences were as follows: forward, 5'-AGAGGTTCTCTTGGGGTTCC-3'; reverse, 5'-AGAACCTTGCCCTGCTTTTA-3'. The amplified 343-bp PCR DNA products were sequenced using the same primers and the dideoxynucleotide chain termination method available as a fluorescent sequencing kit (DNA Sequencing Kit; Applied Biosystems) and an automated sequencer (ABI PRISM 3130; Applied Biosystems) according to the manufacturers' instruction.
Data are expressed as mean ± SD, unless otherwise stated. Statistical analysis was performed using SYSTAT software, version 13 (Systat Software, Inc., Chicago, IL). Statistical tests with a p value < 0.05 were considered significant.
Values of annual FEV1 decline were computed for each individual across the repeated measurements using a linear mixed-effect model. We used a random intercept to take into account the heterogeneity across subjects and the correlation induced by having repeated observations on the same subjects.
We performed univariate analysis to evaluate association of annual decline in FEV1 with clinical characteristics. For categorical variables such as gender and smoking status, Student's t tests and one-way analyses of variance with Bonferroni post hoc correction were used for comparisons of 2 and 3 group means, respectively. For continuous variables such as age, body mass index (BMI), PFT measurements, and total serum IgE levels, the correlation with annual decline in FEV1 was assessed by Pearson correlation coefficient analysis.
All polymorphisms were tested for Hardy-Weinberg equilibrium using Haploview 4.2 software http://www.broadinstitute.org/haploview. Estimates of pairwise linkage disequilibrium (LD) between the loci were calculated using r2 . The associations of genotypes with annual decline in FEV1 were analyzed by multivariate linear regressions adjusted for potential confounding factors such as sex, age, BMI, FEV1/FVC ratio, total serum IgE levels, smoking status (never, ex, or current), smoking index (0, 0-200 or > 200), and affection of bronchial asthma. Correction for multiple comparisons was done by the Bonferroni's method. The interaction effect of genotypes and smoking status on the annual decline in FEV1 was analyzed using general linear models adjusted for the same confounding factors except for smoking behavior.
Association of the rs6726395 genotypes with the mRNA expression levels of Nrf2 was analyzed using GENEVAR database http://www.sanger.ac.uk/humgen/genevar/, which shows mRNA expression profiles of 3 cell types (fibroblast, lymphoblastoid cell line and T-cell) derived from umbilical cords of 75 Geneva GenCord individuals .
For analyses of association between haplotypes and annual FEV1 decline, we used the Haplo. score program http://mayoresearch.mayo.edu/mayo/research/biostat/schaid.cfm, which adjusts for the same covariates and calculates simulation p values for each haplotype .
Characteristics of the participantsa
Participants (N = 915)
52.1 ± 8.3
Male sex - N (%)
Total duration of follow-up (years)
11.1 ± 4.6
Number of visits
9.1 ± 3.7
23.1 ± 3.0
3.12 ± 0.78
FVC % predicted (%)
100.5 ± 13.6
2.56 ± 0.64
82.2 ± 5.6
FEV1 decline (mL/year)
23.8 ± 26.3
Serum IgE (Log IU/mL)
1.78 ± 0.59
Smoking habits - N (%)
Smoking indexb - N (%)
Asthma - N (%)
Univariate analysis comparing annual decline in FEV1 and clinical variables
Annual decline in FEV1
Mean ± SD (mL/yr)
25.0 ± 28.2
22.8 ± 24.5
21.7 ± 24.5
26.7 ± 27.6
28.5 ± .30.5*
21.7 ± 24.5
28.2 ± 29.6
27.0 ± 28.3*
FVC % predicted (%)
FEV1 % predicted (%)
Serum IgE (Log IU/mL)
Multivariate linear regressionsa for association between genotypes at the Nrf2 SNPs and decline in FEV1
Annual decline in FEV1
Mean ± SD (mL/year)
(corrected p value)
24.7 ± 7.5
24.2 ± 7.4
23.8 ± 7.9
26.2 ± 8.3
22.3 ± 7.4
20.8 ± 8.8
28.1 ± 10.4
23.6 ± 6.8
24.0 ± 8.0
24.9 ± 7.9
24.0 ± 7.4
23.4 ± 8.1
23.6 ± 7.8
24.5 ± 7.7
24.4 ± 7.3
Previous results indicate that 3 SNPs (rs6721961, rs6706649 and rs35652124) in the promoter region of Nrf2 are functionally relevant . Although it has been reported that the minor allele of rs6721961 SNP diminishes promoter activity of the Nrf2 gene, there was no significant association between this SNP and annual FEV1 decline in the present study (Table 3). We estimated the extent of linkage disequilibrium between rs6726395 and the other 2 functional SNPs (rs6706649 and rs35652124) by sequencing 50 subjects. The SNP rs6726395 was not in tight LD with rs6706649 (r2 = 0.02) or with rs35652124 (r2 = 0.67), suggesting that genetic effects of these 2 functional SNPs do not underlie the association of rs6726395 with FEV1 decline. The SNP rs35652124 was in tight LD (r2 = 0.92) with rs2364722, which did not have a significant association with annual decline in FEV1 in the present study (Table 3).
Estimated haplotype frequencies and haplotype association with annual decline in FEV1
Simulation p value
Global simulation p value
Cigarette smoke (CS) contains a high concentration of oxidants and leads to oxidative stress . In smokers, increased oxidative stress in the airways is a predominant cause of accelerated decline in lung function [26, 27]. Nrf2 plays a central role in protecting the lung against CS-induced oxidative stress by up-regulating multiple genes encoding antioxidant and detoxification proteins, e.g., heme oxygenase-1, NADPH, and glutathione S-transferase [12, 13]. CS-induced reactive oxygen species production via NADPH oxidase activation is involved in the positive regulation of the Nrf2/ARE pathway. NADPH oxidase, as a critical regulator of innate immunity, also limits lung inflammation by attenuating NF-κB and by activating Nrf2[28, 29]. On the other hand, CS activates the NF-κB pathway, which participates in the negative regulation of Nrf2/ARE signaling[30, 31]. In addition, protein carbonylation induced by CS is involved in the suppression of the Nrf2/ARE pathway. Because several studies have demonstrated that an Nrf2-dependent adaptive response is important in preventing CS-induced lung inflammation and injury[15, 33, 34], we reasoned that Nrf2 polymorphisms have a genetic impact on the CS-induced deterioration of lung function.
In the present study, we showed that a variant of the Nrf2 gene was associated with accelerated decline in FEV1 in a general population sample including non-smokers, moderate smokers, and heavy smokers. A stronger effect of the rs6726395 SNP on annual FEV1 decline was observed in smokers than in never-smokers, indicating a gene-smoking interaction in FEV1 decline. Such an interaction is reasonable because Nrf2 activation protects tissues against oxidative stress. The minor allele of rs6726395 in the homozygous state (A/A) was associated with a smaller FEV1 decline; therefore, this allele was thought to be protective against FEV1 decline.
The mechanisms mediating the relationship between rs6726395 and FEV1 decline were not determined in this study. Although the rs6726395 SNP is located in the first intron of the Nrf2 gene, rs6726395 variants did not correlate with different Nrf2 mRNA levels according to the GENEVAR database. However, given that GENEVAR utilizes only three cell types (fibroblast, lymphoblastoid cell line and T-cell), the possibility remains that rs6726395 has some genetic influence on Nrf2transcriptional activity in alveolar macrophages because Nrf2 appears to exert its protective effects through the transcriptional activation of antiprotease and antioxidant genes in alveolar macrophagesand its mRNA expression is decreased in macrophages of COPD patients. As the rs6726395 SNP was not in significant LD with any of the 3 functional SNPs known to reside in the promoter region, rs6726395 may be in LD with other causal SNPs in or nearby the Nrf2 gene; the allele responsible for the protective effects observed in this study could be on the extended rs2001350T/rs6726395A/rs1962142A/rs2364722A/rs6721961T haplotype. We have not comprehensively assessed the genetic variation in Nrf2, and the functional impact of the Nrf2 SNPs carried on different haplotypes is still unknown. The identification of functional variants in Nrf2 loci will require fine mapping efforts using large populations. However, the lack of suitable Japanese cohorts with the measurements of annual decline in FEV1 and genomic DNA samples available for genotyping prevented us from performing a replication of this study on the association of rs6726395 with FEV1 decline.
In the present study, the annual declines in FEV1 were estimated by longitudinal retrospective measurements. The natural course of FEV1 over time is divided into three phases; a lung growth phase occurs during childhood and adolescence, this grow phase is followed by a plateau phase, and a decline phase begins at about 25 years of age . The level of FEV1 at a given time in adulthood is affected by any deterioration that occurred in any of these 3 phases. Because we planned to analyze the effects of oxidative stress caused by cigarette smoking on lung function, FEV1 decline over time calculated in a longitudinal study was more valuable than absolute values of pulmonary function measurements in a cross-sectional study. As the ages of all the participants in this study were over 25 years at the first visit, all the subjects were thought to be in the decline phase of lung function.
Siedlinski et al.  have reported that the heterozygote genotype of rs2364723, which is in the first intron of Nrf2, is associated with a lower level of FEV1 in Caucasian smokers. However, they showed no relationship between rs2364723 and annual decline in FEV1. Because rs2364723 is in complete LD with rs2364722 (r2 = 1.00) in the JPT population of the HapMap group, the finding from the Siedlinski et al. investigation may be compatible with the result from the present study. Our results are also consistent with findings from a previous Japanese case-control association study that showed no relationship between the 3 previously identified functional SNPs in the promoter region (rs6721961, rs6706649, and rs35652124) and susceptibility to COPD . Recently, two large-scale genome-wide association studies have identified several loci associated with FEV1 and FEV1/FVC ratio [10, 11]. Reports from the studies included lists of the top 2,000 SNPs related to the pulmonary function measurements. SNPs in or nearby the Nrf2 gene including the SNPs in the current study were not among these top 2,000 SNPs.
In order to ensure a reliable estimate of FEV1 decline, we selected the subjects who provided at least 4 valid PFT measurements over a period of at least 4 years. Variation in the follow-up periods and the numbers of visits could contribute to bias in estimations of annual FEV1 decline; therefore, we used a linear mixed-effects model to control for correlations among repeated measures from each subject. Moreover, inhaled corticosteroids, leukotriene receptor antagonists, and bronchodilators (e.g., β-adrenoceptor stimulants and anticholinergic agents) can improve FEV1 measurements in asthmatic and/or COPD subjects; therefore, we excluded those subjects with a history of asthma and/or COPD treated with these medications during the retrospective study period. Nevertheless, because this study was retrospective, attrition could affect the estimation of FEV1 decline. Subjects with accelerated decline in FEV1 are more likely to drop out from the annual health checkup. However, as attrition is likely to result in an underestimate of annual FEV1 decline, it would bias the study against finding an effect.
We demonstrated an association between a SNP (rs6726395) in the first intron of the Nrf2 gene and annual decline in FEV1. In smokers, individuals carrying the major allele of this SNP showed greater decline in FEV1 than those homozygous for the minor allele; however, this effect was not seen in never-smokers. Although the direct functional effect of rs6726395 on the Nrf2 gene is unknown, this risk allele may be useful as a clinical marker for identifying individuals particularly susceptible to loss of lung function due to cigarette smoking. Our study suggests that pharmacological activation of Nrf2 by chemopreventive and phytochemical agents may be the strategy capable of exerting protective effects against various stress conditions including increased annual decline in FEV1associated with CS.
This study was partly supported by a Grant-in-Aid for Scientific Research (B), No. 21390254, from the Japan Society for the Promotion of Science.
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