Association of Genetic Polymorphism of GSTT1, GSTM1 and GSTM3 in COPD Patients in a North Indian Population

Abstract

Environmental exposures and genetic susceptibility can contribute to lung function decline in chronic obstructive pulmonary disease (COPD). Cigarette smoking is the main etiological factor for decline in lung function in COPD. However, only 10–20% chronic smokers develop symptomatic COPD. Genetic susceptibility to COPD might depend upon the variation of enzyme activities that detoxify cigarette smoke components. We performed a case control study to assess the association of Glutathione- S-transferase T1(GSTT1),Glutathione- S-transferase M1 (GSTM1), and Glutathione-S-transferase M3(GSTM3) common polymorphisms with the susceptibility to COPD patient in a north India population. In the present study, the genotypes of 412 subjects, (204 COPD patients and 208 healthy controls) were analyzed. Statistical analysis revealed that the frequency of homozygous GSTM1 null genotype was found to be significant higher in COPD patients as compared with healthy controls (OR, 2.58; 95% CI, 1.73–3.84; P = 0.001), but there were no significant differences in the distribution of homozygous null GSTT1 and 3-bp deletion polymorphism (rs1799735) in intron 6 variant allele in GSTM3 between COPD patients and healthy controls. Our study results suggest that GSTM1 null polymorphism is associated with genetic susceptibility to COPD. Moreover, we also found association between this polymorphism with pulmonary function test in smokers as well as nonsmokers.

Keywords::

• COPD
• Polymorphisms
• Xenobiotic metabolizing enzymes
• Genetic susceptibility

INTRODUCTION

Chronic obstructive pulmonary disease (COPD) represents a major public health care problem worldwide due to its increasing prevalence, morbidity, and mortality (1). Cigarette smoking is the most important risk factor for COPD, 10–20% of all cigarette smokers develop COPD (2, 3). In addition to smoking, other environmental and genetic factors and gene environment interactions influence the development of COPD (4), suggesting that individual susceptibility or genetic factors may play a role (5).

The enzymes that detoxify constituents of tobacco smoke or its metabolites (3) are good candidates for the study of inter-individual susceptibility to COPD. These enzymes include microsomal epioxide hydolase (mEPHX), glutathione-S-transferases (GSTs), and cytochrome P4501A1 (CYP1A1) (6). Glutathione-S-transferases (GSTs) play inportant role in the detoxification of carcinogenic compounds contained in cigarette smoke and also in the antioxidant protection (7, 8). The null genotypes of GSTM1 and GSTT1 genes affect the GSTmu and GSTtheta enzyme activities. The GSTmu has been recognized to detoxify smoke-derived carcinogens, e.g., polycyclic aromatic hydrocarbon and aromatic amines (9). The homozygous GSTM1-null genotype has also been reported to show some association with the susceptibility of lung cancer (10), emphysema (11), and COPD (12, 13).

GSTtheta conjugates glutathione and various potential carcinogens including halomethanes, (14) which are present in cigarette smoke, and its null type mutant allele has been suggested as a risk factors for various diseases (6,15). Hu et al. found GSTT1 null genotype to be significantly associated with COPD in the Chinese population (16, 17, 18), but other studies showed no association of null GSTT1 in COPD patients (13 19, 20, 21).

GSTM3 plays a role in the metabolism of harmful agents, like polyaromatic hydrocarbons benzo(α)pyrene, and has overlapping substrate specificity with GSTM1 (13). GSTM3 gene, 3-bp deletion polymorphism (rs1799735) in intron 6 has been described as GSTM3A wild-type and GSTM3B variant alleles. The GSTM3B variant allele contains a recognition motif for the YY1 transcription factor, which has been postulated to regulate gene expression (22). The GSTM3 polymorphism could, therefore, confer different efficiencies in the metabolism of tobacco smoke. GSTM3AA genotype has been reported to be associated with increased risk of larynx cancer while GSTM3AB genotype confers high risk for cervix cancer (23) and GSTM3 AB+BB genotype were risk for gastric and esophageal cancers (24, 25).

When multiple genes operate in the pathogenesis of disease, influence of each gene might be relatively weak (26). Multiple genetic polymorphisms should be investigated to clarify whether the genetic events have additive effects or can predict the risk of developing COPD. Some previous studies have examined the role of GSTT1 and GSTM1 polymorphisms in COPD but no study has been carried out for GSTM3 polymorphism. Here, we have aimed to analyze the association GSTM1, GSTM1, and GSTM3 gene polymorphism with COPD patients in a cohort from a North Indian population, which is under-studied in terms of genetic association studies in COPD.

MATERIAL AND METHODS

Patients of COPD were diagnosis according to Global Initiative for chronic obstructive lung disease (GOLD) (1, 2) were recruited from the outpatient Department of Pulmonary Medicine, Chhatrapati Shahuji Maharaj Medical University Lucknow, CSMMU (Erstwhile King George's Medical College). A total of 412 subjects (204 COPD patients and 208 healthy controls) were enrolled after evaluation of lung function by spirometry.

The COPD group consisted of 179 males and 25 females (mean age 57.83 ± 10.58 years) and healthy control group had 166 males and 42 females (mean age 56.35 ± 8.12 years). Exclusion criteria were respiratory disorders other than COPD, such as interstitial lung disease, any type of malignancy, previous history of asthma, tuberculosis. Healthy age-matched subjects without pervious medical history of respiratory disorders and without any present respiratory symptoms (Persistent cough, sputum production, or dyspnoea), seen by their practitioner for regular checkup, were recruited consecutively as controls. The participants were residence of geographic area of north India. The study was approved by ethical committee of CSMMU (Approval Code No-XXVIII ECM-B/P3), and the subjects were enrolled in accordance with the ethical standards of the Helsinki declaration of 1975. All subjects gave their written informed consent.

Spirometry

In all the subjects, pulmonary function tests were performed using Spiro 230 system (Morgan Scientific Inc, USA) 20 minutes before and after 200 μg dose of inhaled salbutamol (Foracort, CIPLA). Short-acting inhaled drugs were not used 6 hours before the testing, and long acting bronchodilator therapy was stopped for 12 hours before the test. All pulmonary function tests were performed according to the GOLD (2) with the patients in the sitting position. FEV₁ (forced expiratory volume in 1 second) adjusted for age, height, and sex. the inclusion criteria predicted post bronchodilator FEV₁ <80%, FEV₁/ forced vital capacity (FVC) <70% and improvement in FEV₁ <12% or 200 ml after inhalation of 200 μg salbutamol of pre-bronchodilator (2).

Blood collection and DNA extraction

EDTA-buffered whole blood (5 ml) was drawn for subsequent DNA extraction by standard salting-out method (27).

Genotype analysis

Null alleles ofGSTM1 and GSTT1 were determined by using multiplex polymerase chain reaction (PCR) with the CYP1A1 gene as an internal positive control (28). Briefly, a 215-bp region between exons 4 and 5 of the GSTM1 gene and 480-bp products for were amplified along with a 312-bp size product of CYP1A1. The PCR products were electrophoresed on a 2% agarose gel. The absence of 480 and 215 bp bands indicated homozygous null genotypes of GSTM1 and GSTT1, respectively.

A simple PCR strategy was used to identify A and B alleles of the GSTM3 gene. Primers for intron 6 of GSTM3 were used to amplify a 79/76 bp product and electrophoretic separation on 20% polyacrylamide gel (25). All reactions were carried out in an MJ Research 200 thermal cycler. PCR products and molecular weight markers were visualized after staining with ethidium bromide (Etbr).

DNA sequencing

PCR amplicons of GSTM3 were purified and sequenced using automated DNA sequencer. The sequence analysis was performed using DNA STAR and Chromas 231 (available online) software. Figure 1.

Figure 1.  GSTM3 allele A 79bp and allele B 76bp (3bp deletion in B allele).

Statistical analysis

Descriptive statistics of patients and controls were presented as mean and standard deviations for continuous measures whereas frequencies and percentages were used for categorical measures. Effective sample sizes were calculated by the Quanto software version 1.2(Gauderman WJ, Morrison JM. QUANTO 1.1 a computer program for power and sample size calculations for genetic-epidemiology studies <http://hydra.usc.edu/gxe> (2006). Independent sample t-test was performed to compare the mean values of continuous data and χ2-test use for to compare the genotype frequencies between patient and control. The clinical parameters were expressed as Mean ± SD. Odds ratio (OR), 95% confidence interval (CI) and p value of genotype data were adjusted for confounding factors such as age, sex and pack-years of smoking. A p value of ≤0.05 was considered statistically significant.

RESULTS

Patients and Controls

Twenty-five of the 179 COPD patients and 42 of the 166 healthy controls were female, while the rest were male (Table 1). No significant differences were observed in age (p = 0.111). Numbers of smokers were higher in patient's comparison to healthy controls. Significant difference was found in pack-year smoked (p = 0.001), in control was 13.51 ± 6.97 pack-years. But patients with COPD had a 23.67 ± 17.89 pack-year's history of smoking. In COPD patient significant lower lung function was observed as compared with the healthy control, their pulmonary function tests showed predicted mean post-bronchodilator FEV₁ of 40.34 ± 12.96%, and predicted (FVC) of 53.31 ± 12.90%, with mean FEV₁/FVC ratio 54.85 ± 14.22%, while healthy control showed FEV₁ of 80.82 ± 23.36%, FVC 84.75 ± 19.80%, with mean FEV₁/FVC ratio 94.54 ± 14.04%.

Table 1.  Demographic characteristic of COPD cases and controls according to GOLD criteria^*

	Control n = 208	Cases n = 204	p-value
Sex
Male (n,%)	166 (79.8%)	179 (87.7%)	0.058
Female (n,%)	42 (20.2%)	25 (12.3%)
Age (Mean ± SD)	56.35 ± 8.12	57.83 ± 10.58	0.111
Smoking History
Smoker(n,%)	50 (24.0%)	148(72.5%)
NonSmoker (n,%)	158 (76.0%)	56 (27.5%)
Pack-year (Mean ± SD)	13.51 ± 6.97	23.67 ± 17.89	0.001
Pulmonary function test
FEV1% (Predicted) post ± SD	80.82 ± 23.36	40.34 ± 12.96	0.001
FVC% (Post) ± SD	84.75 ± 19.80	53.31 ± 12.90	0.034
FEV1/FVC% (Pre) ± SD	94.54 ± 14.04	54.85 ± 14.22	0.001

^*Data are presented as n (%) or Mean ± Standard deviation, ^†person who reported smoking more than 1 cigarette per day for at least 12 month were defined as cigarette smokers. COPD = Chronic obstructive pulmonary disease; Gold = Global Initiative for Chronic Obstructive lung disease; FEV₁ = forced expiratory volume in 1 second; FVC = forced vital capacity; Non-smoker (farmer, factory work, person who reported regularly chewing betal nut <2 times per week for at least 6 months were, FEV₁/FV; C <70%).

Genotypes of GSTM1 and GSTT1

The distribution of genotypes and allele frequencies of GSTM1 and GSTT1 in patients and controls and the respective ORs are presented in Table 2. The frequency of GSTM1 null genotypes was higher (60.8 vs. 37.5%) and imposed significant 2-fold risk for COPD patients (OR, 2.58; 95% CI, 1.73–3.84; P = 0.001). It was observed that when FEV₁/FVC less than 70% was significantly associated with GSTM1 null genotype, data was not showed. However, the frequency of GSTT1 null genotypes were not found to be significantly different between COPD patients and healthy control (17.6 vs 24.5%,).

Table 2.  Frequency distribution of GSTM1, GSTT1 and GSTM3 between COPD cases and control

	Controls		Cases
	N = 208	Percentage	N = 204	Percentage	OR (95%CI) P-value
GSTT1 Present	157	75.5%	168	82.4%	1 Reference
GSTT1 Null	51	24.5%	36	17.6%	0.66 (0.41–1.06) 0.092
GSTM1 Present	130	62.5%	80	39.2%	1 Reference
GSTM1 Null	78	37.5%	124	60.8%	2.58 (1.73–3.84) 0.001
GSTM3 intron 6 3-bp deletion (rs1799735)
AA	145	69.7%	132	64.7%	1 Reference
AB	60	28.8%	70	34.3%	1.75 (0.28–10.82) 0.547
BB	3	1.4%	2	1.0%	1.36 (0.23–8.30) 0.735
A	350	84.1%	334	81.9%	1 Reference
B	66	15.9%	74	18.1%	0.85 (0.59–1.22) 0.386

^*adjusted for age and sex; OR = adjusted odd ratio; CI = confidence intervals.

Genotype of GSTM3

In controls, observed genotype frequency of GSTM3 polymorphism did not deviate from Hardy-Weinberg equilibrium The frequency of AB genotype of GSTM3 was higher in COPD patient in comparison to healthy control (34.3% vs. 28.8%), but difference was non-significant (OR, 1.75; 95% CI, 0.28–10.32; P = 0.547) (Table 2).

Gene-environment interaction

Table 3 shows the association between the different genotypes with COPD in smokers and nonsmokers. The GSTM1 null polymorphism was associated with an increased risk of developing COPD in both the smokers (OR, 4.05; 95% CI, 1.99–8.27; P = 0.001), as well as non-smokers (OR, 2.78; 95% CI, 1.47–5.27; P = 0.002), while no differences were observed for GSTT1 and GSTM3 polymorphisms.

Table 3.  Association of GSTT1, GSTM1 and GSTM3 polymorphism with COPD cases and control on basis of cigarette smoking

		Controls = 208	Cases = 204	P-value	OR (95%CI)
Smoker	GSTT1Present	35 (70.0%)	123(83.1%)	0.065	1Reference
	GSTT1 Null	15 (30.0%)	25 (16.9%)		0.47 (0.23–0.99)
Non-Smoker	GSTT1 Present	122 (77.2%)	45 (80.4%)	0.789	1
	GSTT1 Null	36 (22.8%)	11 (19.6%)		0.83 (0.38–1.76)
Smoker	GSTM1 Present	37 (74.0%)	61 (41.2%)	0.001	1
	GSTM1 Null	13 (26.0%)	87 (58.8%)		4.05 (1.99–8.27)
Non-Smoker	GSTM1 Present	93 (58.9%)	19 (33.9%)	0.002	1
	GSTM1 Null	65 (41.1%)	37 (66.1%)		2.78 (1.47–5.27)
Smoker	GSTM3 AA	33 (66.0%)	92 (62.2%)	0.735	1
	GSTM3 AB+BB	17 (34.0%)	56 (37.8%)		1.18 (0.60–2.32)
Non-Smoker	GSTM3 AA	112 (70.9%)	40 (71.4%)	1.00	1
	GSTM3 AB+BB	46 (29.1%)	14 (28.6%)		0.97 (0.49–1.91)

^*Adjusted for age and sex; OR = odds ratio; CI = confidence intervals.

DISCUSSION

There is likely to be a complex interplay between environmental and genetic factors for the development of COPD. It has been suggested that individual susceptibility due to genetic predisposition is likely to be an important determinant of this disease. Genetic polymorphisms in xenobiotic enzyme genes may play a role in susceptibility to oxidant related lung disease, and several studies have assessed the association between COPD and candidate genes of enzymes involved in the metabolism of mutagens and carcinogens originating in cigarette smoke components that are believed to cause some of the lung damage characteristic of COPD (29).

Glutathione S-transferases (GSTs) are a large family of enzymes participating in detoxification of endogenous and exogenous toxic substrates including tobacco derived toxins; they also exhibit peroxidase activity and thus might play an important role in oxidative stress (30).

Our results show clear association of null genotype of GSTM1 with COPD while GSTT1 null or GSTM3 intron 6 polymorphism do not play role in the disease susceptibility, In case of COPD patients, the frequency of GSTM1 null genotype was higher in cases compared to controls (OR, 2.58; 95% CI, 1.73–3.84; P = 0.001). The results in GSTM1 are agreement with previous studies on COPD (15, 31). The null allele of GSTM1 may result in defective detoxification of the polycyclic aromatic hydrocarbons of cigarette smoke and may promotes cellular and tissue damage of the lung due to an excess of oxidants and free radicals (32).

The frequency of GSTM1-null genotype in the control group was closer to that found in Western countries as well as other Indian reports (37.5 versus 46.9–53%),but it was lower than of Koreans population (65%) (4, 11, 25, 33). The frequency of GSTT1-null genotype shows distinct difference in various ethnic groups, it is 10–18%, in Caucasian population and 22–29% in African Americans (34). In the present study, frequency of controls group (24.5%) was similar to other north India populations but lower than various Asian populations (19), However, no association of null GSTT1 with COPD (12, 19, 21) is in conformity with several previous studies (12, 13, 19, 21).

GSTM3 3bp deletion intron 6 has two alleles: GSTM3A and GSTM3B, The 3bp deletion generates a site for the binding of transcription factor YY1 which can both repress and activate transcription. However, molecular mechanisms controlling its behavior are complex and still not understood (6), The frequency of GSTM3 B mutant allele has been rated as 15% to 24% (35) in Caucasians and 68% in African Americans (35). In the present study, frequency of GSTM3 B allele was 15.9% in controls, which is closer to Caucasians but different from other Indian studies (1, 25, 36). To the best of our knowledge, this is the first study to examine association of GSTM3B mutant allele in COPD but genotype and allelic frequencies of GSTM3 polymorphic alleles were similar in COPD and control groups.

In our study we analyzed the association of genotypes of the GSTT1, GSTM1, and GSTM3 genes with the smoking history and pulmonary function test (post-bronchodilator FEV₁/FVC%). We observed significant association of GSTM1 null in both smokers and non-smokers. Although cigarette smoking is major risk factor for COPD but, there is a substantial body of epidemiological evidence linking occupational exposures to dusts, gases/vapors, and fumes to chronic airflow destruction, with a substantial population attributable risk (15–20%) in non-smokers (37). In India 5% male and 2.7% females subjects are affected by COPD (38), and tobacco smoking is common in most male patients. but non-smoker patients (especially women) are significantly exposed of solid fuels or occupational exposures to dust and fumes (39). The smoke from combustion of solid fuels such as dried dung, wood and crop residue used for cooking and heating, especially in villages, semi-urban and slum areas, is an important cause of pollution of the indoor air, and it is responsible for large number of COPD patients in the rural inhalation in general and women in particular (39).

Pillai et al. (26) conducted a genome wide association study (GWAS) for gene environment interaction in COPD in a cohort of Norwegian samples and observed strong association with SNPs belonging to a-nicotinic acetylcholine receptor (CHRNA 3/5) locus. The absence of genes belonging to GST family in the GWAS may be related to population specific variations in candidate gene approaches.

The limitation of this study is small sample sizes and the fact that only a few genes involved in the detoxification of smoke products were studied. In conclusion, we found association of GSTM1 null polymorphism with COPD in a north India population. Moreover, we also found association between this polymorphism with pulmonary function test in smokers as well as nonsmokers with COPD. However, COPD is a multifactorial disease and role of other genes and their interactions should also be considered before their clinical applications.

ACKNOWLEDGMENT

The study was supported by a research grant (UPCST/SERPD-D-3404).from Council of Science & Technology, U.P., Lucknow.

Conflict of interest

None of the authors have conflict of interest to declare in relation to this work. The authors alone are responsible for the content and writing of the paper.