Research Article
Naushad Ahmad
Khan1,2, Naresh
Kumar3, Syed Akhtar
Husain4, Mradul Kumar
Daga5
1,3,5
Department of Medicine,
Maulana Azad Medical
College, New Delhi-110002.
2,4
Department of
Biosciences, Jamia Millia
Islamia University, New Delhi
-110025
Correspondence to: Dr.
Naushad Ahmad Khan,
Maulana Azad Medical
College, New Delhi-110002.
E-mail Id:
naushadkhan82@gmail.com
How to cite this article:
Khan NA, Kumar N, Husain
SA et al. Association of
Xenobiotic Metabolizing
Gene Polymorphisms and
Chronic Obstructive
Pulmonary Disease in Indian
Population. J Adv Res Med
2016; 3(1): 5-14.
ISSN: 2349-7181
© ADR Journals 2016. All Rights Reserved.
Association of Xenobiotic Metabolizing
Gene Polymorphisms and Chronic
Obstructive Pulmonary Disease in Indian
Population
Abstract
Background and Aims: Genetic susceptibility to the development of chronic
obstructive pulmonary disease might depend on variation in the activities of enzymes
that detoxify cigarette smoke products. We studied the relationship of GSTP1,
GSTM1, and GSTT1 gene polymorphisms with COPD risk in a case-control study of
Indian patients and controls.
Material and Methods: A total of 186 patients with COPD and 160 healthy controls
were included in the study. The frequencies of GSTP1, GSTM1 alleles were
determined by using conventional multiplex PCR and GSTP1 by polymerase chain
reaction and restriction fragment length polymorphisms technique.
Results: A significant case-control difference was observed for the presence of null
GSTM1, (61.8% vs 55.0%, P=0.04). No difference was observed in the frequency of
GSTT1 Null genotype and COPD susceptibility (54.8% vs 50.6% OR: 1.26; CI: 0.87-1.84;
P value=0.82). For GSTP1 polymorphism, we found that subjects homozygous
variants Val/Val were at increased risk of developing COPD (OR: 2.58; CI: 1.2-4.8) as
compared to heterozygote variants Ile/Val (OR: 1.28 CI: 0.7-2.14). Also, the mutant
allele frequency (Val) was significantly higher in patients as compared to controls and
the difference was found to be statistically significant. (OR: 1.8 CI: 1.4-4.2; P
Value=0.001).
Conclusion: We propose that subjects with GSTM1 null allele and GST P1 homozygous
isoleucine genotypes are at higher risk of COPD and are significant indicators of
susceptibility to chronic obstructive pulmonary disease in Indian population.
Keywords: Chronic obstructive pulmonary disease, Genetic polymorphism,
Polymerase chain reaction, Gluthathione S-transferase.
Introduction
Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and
mortality worldwide, affecting over 44 million people.1 COPD currently ranks twelfth
in the global burden of disease, but has been predicted to rise to fifth highest burden
by 2020.2 COPD is characterized by irreversible airflow limitation in the lungs.3 It is
generally accepted that cigarette smoking is the most important risk factor for COPD.
Nevertheless, only 10-20% of chronic smokers develop the severe impairment of
pulmonary function associated with COPD.4,5 This indicates the possible contribution
of environmental or genetic cofactors to the development of COPD. Although
cofactors, such as childhood viral infection and environmental and occupational
pollution play important roles in pathogenesis,6 genetic susceptibility may be a factor
of major importance.7 During the last three decades, research studies reported that
the imbalance of the protease-antiprotease and the oxidant-antioxidant systems is
the major factor causing emphysema and COPD.8
Khan NA et al.
The only established genetic risk factor for COPD is
homozygosity of the Z allele of the a1-antitrypsin (a1-
AT) gene. Patients with genetic a1-AT deficiency have a
very high risk of developing emphysema at an early age
if they smoke. However, these patients account for only
a small proportion of all patients with emphysema.9
Recent studies reported that genetic variations in the
enzymes that detoxify cigarette smoke products might
be associated with the development of COPD. These
enzymes include microsomal epoxide hydrolase
(mEPHX), glutathione S-transferase (GST), and
cytochrome p450 1A1. The genes coding for xenobiotic-
metabolizing enzymes have attracted attention because
of their function in detoxifying of cigarette smoke
products.
GSTs are a superfamily of enzymes involved in phase-II
of xenobiotic-metabolism catalyzing the conjugation of
a wide range of electrophilic substances with reduced
glutathione, thereby facilitating detoxification and
further metabolism and excretion. Most of GSTs are
polymorphic enzymes and it is shown that the gene
variations affect the enzyme activity and/ or gene
expression.10-12
GSTs are separated into the following classes: alpha, mu
(GSTM), pi (GSTP), theta (GSTT), sigma and kappa. The
GST M1, T1 and P1 genes are located on chromosomes
1p13, 22q11.2 and 11q13, respectively. Among the
isoenzymes of GST, the homozygous GSTM1-null
genotype has been reported to show some association
with the pathogenesis of lung cancer13,14 bladder
cancer15 and, especially, emphysema16. The GSTT1-null
mutant has also been suggested as a risk factor in many
diseases.17-20 Polymorphisms of GSTP1 genotypes have
been reported, including isoleucine (Ile) 105 to valine
(Val) mutation in exon 5 and alanine 114 to Val (Val)
mutation in exon 6. Individuals with the Val105 (mutant)
allele have a higher risk of developing lung cancer than
those with the Ile105 (wild-type) allele.21 In addition,
GSTP1 is expressed more abundantly in respiratory
tissues than other kinds of GST.22
There is a paucity of information with respect to the
influence of genetic polymorphism of GSTM1, T1 and P1
in cases with or without COPD in India. Also, the current
data on the potential associations between an increased
COPD risk and genes encoding the enzymes
metabolizing xenobiotic substances are inconsistent.
Hence, the aim of our study was to analyze the relation
between COPD and gene polymorphisms of GSTM1,
ISSN: 2349-7181
J. Adv. Res. Med. 2016; 3(1)
GSTT1 and GSTP1 genes polymorphism in Indian
population and to assess the correlation of GSTP1
genotype with clinical and functional parameters of
COPD.
Materials and Methods
Setting and Inclusion Criteria
There were 186 patients with COPD and 160 controls all
unrelated in this case-control study. The patients were
recruited from LN Hospital (New Delhi, India) from both
the outpatient department as well as from inpatient
facility. All genotyping assays were performed at
molecular genetics laboratory, department of
biosciences, Jamia Millia Islamia University (New Delhi,
India). The demographic characteristics of the study
subjects are summarized in Table 1. All patients satisfied
the clinical criteria of COPD set down in the Global
Strategy for Obstructive Lung Disease (GOLD 2006)
guidelines.23
The inclusion criteria for COPD were as follows: age
higher than 30 years; chronic airways symptoms and
signs such as coughing, breathlessness wheezing, and
chronic airway obstruction defined as forced expiratory
volume in 1 sec. FEV1/ forced vital capacity (FVC) of
<70% and an FEV1 of <80% of predicted values from
spirometric data and FEV1 reversibility after inhalation
of 200 mcg of salbutamol of less than 12% pre-
bronchodialator of FEV1. Patients with bronchial asthma
were excluded on the basis of reversibility of airflow
obstruction patients with respiratory disorders other
than COPD such as interstitial lung disease, lung cancer,
tuberculosis, bronchiectasis, previous medical record of
bronchial asthma, thoracic surgery in the past, and
patients unable to perform spirometry to confirm the
diagnosis were also excluded.
Pulmonary Function Test
The subjects were instructed about the proper
technique of performing the spirometry and technique
was also demonstrated to each patient before starting
the test. Nose clip was used to prevent air entry or exit
through the nose. At least three acceptable spirograms
were obtained from a minimum of five forced
expirations. Lung function was assessed by a trained
technician (research assistant). Subjects showing an
obstruction on pulmonary function test were subjected
to a repeat test after bronchodilation with 200 mcg of
Salbutamol to test the reversibility of obstruction.
6
J. Adv. Res. Med. 2016; 3(1) Khan NA et al.

Table 1.Demographic Characteristics of the Study Subjects

COPD Patients N=186 Healthy Controls N=160 P Value
Age (yrs) 55.9 ± 9.2 51.4 ± 7.3 0.16
Gender (M:F) 168:18 155:5 0.87
BMI 23.2 ± 6.8 21.8 ± 5.7 0.52
Smoking Status
Never 15 (8.06%) 25 (15.6%)
Ex 89 (47.8%) 40 (25%) 0.14
Current 82 (44.0%) 95 (59.3%)
Cigarette/ Bidi Smoking
Non Smoker 15 (8.06%) 25 (15.6%)
<10/day 50 (26.8%) 70 (43.7%) <0.001*
11-20/day 80 (43.01%) 40 (25.0%)
>20/day 41 (22.04%) 25 (15.6%)
Pack years 24.8 ± 8.2 15.2 ± 3.9 < 0.001*
Educational Level
Illiterate and elementary school 92 (49.4%) 76 (47.5%) 0.70
High School 62 (33.3%) 50 (31.2%)
Senior secondary and above 32 (17.2%) 34 (21.2%)
Alcohol Consumption
Non-drinking 142 (76.3%) 136 (85%) 0.15
Drinking 44 (23.6%) 24 (15%)
Data are presented as (n%) or mean + standard deviation; persons who reported smoking 1 cigarette/bidi per day for at least 12 months
were defined as cigarette/bidi smokers; persons who reported drinking beer, wine, more than thrice per month for at least six months
were defined as alcoholic drinkers. COPD = chronic obstructive pulmonary disease
*Statistically significant

Methods for 10 min. The PCR products were subsequently

5 mL of venous blood was collected in heparnised tubes
and stored at 20 °C until DNA extraction was carried
out. Genomic DNA was isolated from whole blood
applying the conventional proteinase K digestion
followed by protein precipitation with over-saturated
solution of NaCl, and deposit of genomic DNA with
absolute ethanol (Phenol chloroform method).24
Genotyping Assays
Polymorphism of GSТР1 was analyzed by the
polymerase chain reaction (PCR)-based restriction
fragment length polymorphism (RFLP) technique as
described by Watson et al.25 with a single modification.
PCR assay for exon 5 used the primer pairs 5-
GTAGTTTGCCCAAG GTCAAG-3 and 5-AGCCACCT
GAGGGGTAAG-3. PCRs were carried out in a thermal
cycler (PTC-100TM MJ Research, Inc. USA) in 20-µL
reaction mixture containing 1.5 mM MgCl2, 1 μM of
each primer, 0.2 mM deoxyribonucleoside triphosphate
(dNTP) and 0.5 IU GoTaq® Flexi DNA polymerase
(Promega, USA). Following an initial step of
denaturation at 95 °C for 12 minutes, samples were
subjected to 35 cycles of denaturation at 95 °C for 30 s,
annealing at 58 °C for 30 s and extension at 72 °C for 1
min. A final step of elongation was performed at 72 °C
digested with 5 U of Alw26I (Fermentas, Germany) at 37
°C for 2 h. The homozygous wild-type genotype yielded
two bands, 329 and 104 bp, while homozygous mutant-
type genotype yielded two bands of 222 and 107 bp.
The fragments were resolved using 2% agrose gel
(Amresco, 30175 Lot#3056B019) stained with ethidium
bromide and transilluminated with UV light. The
homozygous null polymorphisms of GSTM1 and GSTT1
were assessed using a simultaneous amplification of
genes of interest by conventional multiplex polymerase
chain reaction method.18 PCR for albumin was also
performed as an internal control. The primer pairs for
GSTM1 gene were 5’-GAACTCCCTGAAAA GCTAAAGC-3’
and 5’-GTTGGGCTCA AATATACGGTGG-3’. The primer
pairs for GSTT1 gene were 5’-TTCCTTACTGGTCCCACA
TCTC-3’and 5’-TCACCGGA TCATGGCCAGCA-3’. Albumin
5’-GCCCTCTGCTAACAAGT CCTAC-3’ and 5’GCCCTAAAA
AGAAAATCGCCAATC-3’. The PCR contained 50-100 ng
DNA, 1.5 Mm MgCl2, 0.2 Mm of dNTP mix, GSTM1
primers at 2 mg/mL each, GSTTI primers at 1 mg/mL
each and albumin primers at 1 mg/mL each, and 1.5U
DNA Ampli Taq DNA polymerase (Fermentas) a Perkin
Elmar thermal cycler (Norwalk, CT, USA). After an initial
denaturation at 95 °C for 5 min, amplification was
carried out for 35 cycles at 94 °C for 1 min, 56 °C for 1
min and 72 °C for 1 min. followed by a final extension at

7 ISSN: 2349-7181
Khan NA et al.
72°C for 5 min. the products of the multiplex PCR
containing fragments of 215 bp (indicating the presence
of GSTM1), 480 bp (GSTT1), and 350 bp (albumin), were
separated by electrophoresis with ethidium bromide-
stained 3% agarose gel and visualized by UV detection.
Statistical Analysis
The SPSS for Windows version 12.0 software (SPSS Inc.
Chicago, IL, and USA) was used in all analyses. Hardy-
Weinberg equilibrium of genotype distribution in cases
and controls was evaluated by calculating χ2 and P
values. Differences in allele distribution and allele
frequencies among the groups were examined for
statistical significance by the Pearson χ2 test. Odds
ratios (OR) with their corresponding 95% confidence
intervals (CI) were calculated. The χ2 test was again
used to compare gender, smoking status between the
two groups. Age, smoking index expressed as pack-years
(number of cigarettes smoked per day×number of years
smoked/20) were compared using the unpaired Student
t-test. Analysis of variance was carried out to evaluate
clinical and functional characteristics of patients
according to different genotypes. Logistic regression
was performed to estimate OR and 95% CI of COPD risk
factors. P<0.05 was considered to be statistically
significant.
Results
Baseline Clinical Characteristics of Study Subjects
The demographic characteristics of the COPD patients
and healthy controls matched for age and pack-years
are shown in Table 1. A total of 186 COPD patients and
160 healthy controls were enrolled in this case-control
study. 18 (9.76%) of the 186 COPD patients and 5 (3.1%)
of the 160 healthy controls were females. There was no
significant difference observed between the patients
and controls group in terms of age (mean age 55.9 ± 9.2
vs 51.4 ± 7.3), gender (168:18 vs 155:5), as well as BMI.
The proportion of current smokers was less in COPD
Patients. BMI was almost similar in both
Table 2.Spirometric Characteristics of the Study Subjects according to GOLD Classification Criteria
Controls (N = 160)
Spirometry
FEV1% (predicted) 98.8 ± 14
FVC% (predicted) 98.2 ± 12.8
FEV1/FVC% 78.2 ± 3.8
Severity of the Disease
Mild (60-80)
Moderate (40-59)
Severe (<40)
*Statistically significant
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J. Adv. Res. Med. 2016; 3(1)
groups. The rate of literacy was primary school literates/
illiterates 49.4%, high school 33.3% and professional or
post graduate degree 17.2%. There was no significant
difference in alcohol consumption and educational
status across the groups.
The number of individuals smoking bidi was greater
than of cigarette. Cigarette/ bidi smoking and pack-
years were the only variables that differed significantly
between controls and those with COPD. The mean
number of pack-years in the study group was 24.8±8.2
years as against 15.2±3.9 years in the control group
which was statistically significant (P<0.001) (Table 1).
Pulmonary Function Test and Grading of
Severity of COPD Patients
The spirometric characteristics of the patients are
presented in Table 2. The mean FVC %, FEV1% and
FEV1/ FVC% values in patients with COPD were 62 ±
16.7, 36.2 ± 12.2, and 42.2 ± 12.4 those in controls were
98.2 ± 12.8, 98.8 ± 14, and 78.2 ± 3.8 respectively. The
lung function values in patients with COPD were
significantly different from those in controls (p<0.001).
Patients with COPD were classified into three subgroups
according to severity of the disease-mild, moderate and
severe. Out of 186 patients, 40 (21.5%) were in the mild
category (FEV1 between 60 and 80%), 88 (47.3%) in the
moderate category (FEV1 between 40 and 69%), and 58
(31.1%) in the severe category (FEV1<40%). Hardy-
Weinberg equibillirium was tested for all
polymorphisms and no obvious deviation was observed.
In our findings, GST M1 null genotype was found to be
significantly associated with increased risk of COPD on
multivariate analysis. The odd ratio was 2.4 (95% (CI)
1.2-3.8).
The frequency of GSTM1 genotype was significantly
higher in the patients of COPD than in the control
subjects (61.8% vs. 55.0%). However, no significant
association was found between GST M1 and COPD after
adjustment for age, sex, and smoking status using
logistic regression analysis (P>0.05, data not shown).
COPD Patients (N = 186)
36.2 ± 12.2*
62 ± 16.7*
42.2 ± 12.4*
40 (21.5%)
88 (47.3%)
58 (31.1%)
8
J. Adv. Res. Med. 2016; 3(1) Khan NA et al.

In contrast to GSTM1 in the analysis for GSTT1 null compared to heterozygote variants Ile/Val (OR: 1.28 CI:
polymorphism, we did not find significant prevalence of 0.7-2.14). In a population of 186 patients, the
the homozygous null genotype in the patient population distribution of the GSTP1 genotypes among COPD
compared to the controls (54.8% vs. 50.6% OR: 1.26; CI patients was as follows: 94 (50.5%) (A/A, Ile/Ile), 58
(0.87-1.84) P value=0.82). We found a higher frequency (31.1%) (A/G, Ile/Ile) and 34 (18.2%) (G/G, Val/Val).
of null homozygous genotype in patient group than in Among 160 controls, 91 (56.8%) were homozygous for
control group on analysis of combination of both null wild GSTP1 allele (A/A, Ile/Ile), 56 (35.0%) controls were
polymorphisms of GSTM1 and T1 (24.1% vs. 16.2%). heterozygous and rest of 13 (8.1%) were homozygous
However, this association was not statistically significant for the variant GSTP1 allele (G/G, Val/Val). It was also
(P value = 0.76) and hence, no association with the found that the mutant allele frequency (Val) was
pathogenesis of the disease was found. significantly higher in patients as compared to controls
and the difference was found to be statistically
As for GSTP1 exon 5 polymorphism, we found that significant (OR: 1.8 CI: 1.4-4.2; P Value=0.001) (Table 3)
subjects homozygous Val/Val variants were at increased (Figs. 1 and 2).
risk of developing COPD (OR: 2.58; CI: 1.2-4.8) as
Table 3.Distribution of Genotypic Frequencies of the GSTP1, GSTM1 and GSTT1 Genes in Patients of Chronic
Obstructive Pulmonary Disease and Healthy Controls in Indian Population
Genotype COPD N=186 (%) Controls N=160 (%) OR (95% CI) p-value
GSTM1
Non-null genotype 66 (35.4) 72 (45.0) 1 (Reference) 0.04*
Null genotype 120 (61.8) 88 (55.0) 2.4 (1.2-3.8)
GSTTI
Non null genotype 84 (45.1) 79 (49.3) 1 (Reference) 0.82
Null genotype 102 (54.8) 81 (50.6) 1.26 (0.87-1.84)
Combined GSTM1 and GSTT1
Null genotype 45 (24.1) 26 (16.2) 1.34 (0.4-2.84) 0.07
GSTP1
A/A (Ile/Ile) 94 (50.5) 91 (56.8) 1 (Reference) 0.76
A/G (Ile/Val) 58 (31.1) 56 (35.0) 1.28 (0.7-2.14) 0.001*
G/G (Val/Val) 34 (18.2) 13 (8.1) 2.58 (1.2-4.8)
GST P1 (Allelic frequency)
A (Ile) 236 (52.2) 212 (61.2) 1 (Reference) 0.001*
G (Val) 184 (41.2) 94 (28.4) 1.8 (1.4-4.2)
Data are represented as N (%) unless otherwise indicated. OR: Odd ratio; CI: Confidence interval; GST: gluthathione S-Transferase; Ile:
Isoleucine; Val: Valine
* Statistically significant

Figure 1.Representative Gel Photograph Showing PCR-RFLP Analysis of GSTP1

Lane 1: 100 bp DNA Ladder

Lanes 2, 5-8: 222 bp & 329 bp Wild homozygous genotype
Lane 3: Mutant homozygous genotype (not restricted)
Lanes 4, 9, 10, 11: Heterozygous genotype
Lane 12: Negative control

9 ISSN: 2349-7181
Khan NA et al. J. Adv. Res. Med. 2016; 3(1)

Figure 2.Representative Gel Photograph Showing Amplification of GSTT1, GSTM1 and Albumin Gene Products

Lane 1: 100 bp marker

Lane 2 and 5, 6: Positive reaction of GSTT1 and GSTM1 in COPD cases
Lane 3 & 4: Positive reaction of GSTM1 in COPD cases
Lane 7 & 8: Positive reaction of GSTT1 and GSTM1 in control cases
Lane 1-8: Positive reaction of Albumin (internal control) in all the cases

Out of 186 patients, severity of COPD was classified as The distribution of GSTM1 null genotypes occurred
mild/ moderate (FEV1>35% PRED) in 146 (78.4%) and significantly and more frequently than non-null
severe in 58 (31.1%) patients (FEV1<35% PRED). The genotypes in patients with severe COPD (79.3% vs.
grading of severity was done according to the guidelines 56.1%).
of American Thoracic Society (ATS).43
As for others, the distributions of GST P1 and GSTT1
There were significant differences observed in the were found not to be associated with COPD severity
distribution of GSTM1 genotypes and COPD severity. with an odd ratio of 4.8; CI 1.8-12.4 (Table 4).
Table 4.Interaction between Genetic Polymorphisms of GST Genes and Their Association with Severity in COPD Patients
FEV1 OR (95% CI)
<35% PRED (%) >35% PRED (%)
No. of Subjects 58 (31.1) 146 (78.4)
Polymorphism
GSTM1
Wild Type 12 (20.6) 64 (43.8) 1 (Reference)
Null Type 46 (79.3) 82 (56.1) 4.8 (1.8-12.4)*
GSTT1
Wild type 26 (44.8) 64 (43.8) 1 (Reference)
Null type 32 (55.17) 82 (56.1) 0.7 (0.2-2.0)
GSTP1
G/G (Val/Val) 02 (3.4) 05 (03.4) 1 (Reference)
A/A (Ile/Ile) 34 (58.6) 79 (54.1) 0.8 (0.1-2.8)
A/G (Ile/Val) 22 (37.9) 62 (42.4) 0.3 (0.1-2.2)
Data are represented as N(%) unless otherwise indicated. FEV1: forced expiratory volume in one second; % pred: percentage of
predicated value; OR: Odd ratio; CI: Confidence interval; GST: Gluthione S transferase.
Odd ratio was adjusted age, sex, and bidi/cigrattee consumption.
*Statistically significant

Discussion susceptibility to disease depends on the coincident

COPD is a complex multifactorial disease and it has been
suggested that a complicated interplay between
environmental and genetic factors is likely to be
involved in its development.1-3 It is likely that the
operation of multiple genes is necessary and that
actions of several genetic events due to polymorphisms.
Polymorphism of each gene may impart only a small
relative risk of COPD, and therefore it is reasonable to
speculate that the existence of several polymorphisms is
important in the pathogenesis of COPD.4,5 Oxidant stress
and reactive oxygen species, resulting from an oxidant/

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J. Adv. Res. Med. 2016; 3(1)
antioxidant imbalance, are believed to play an
important role in the pathogenesis of COPD.6-8 Indeed, it
is well known that chronic tobacco smoking is a major
risk factor for the development of COPD, and a defect in
the detoxification of reactive species produced by
cigarette smoke may predispose smokers to airflow
obstruction and emphysema.9
Glutathione S-tranferase plays a protective role as an
antioxidant in the lung.10-12 In a complex polygenic
disease such as COPD, it is likely that genetic
susceptibility is dependent on the action of several gene
polymorphisms operating in concert. Polymorphisms in
individual gene may impart only a small relative risk of
COPD and it is likely that the cumulative effect of many
polymorphisms will be important in its pathogenesis.
The study provides a new genotyping data of GSTP1,
GSTM1, GSTT1 polymorphisms and their association
with susceptibility to chronic obstructive pulmonary
disease in Indian population. When compared, the
frequencies of exon 5’ GSTP1 polymorphism in COPD
patients differed significantly from the control group.
Our results demonstrated that the homozygous mutant
genotype Val/Val was two times higher in patients than
in controls 18.2% vs. 8.1%). We also found that the
frequency of the Val allele was higher in the patient
group (OR: 1.8; CI: 1.4-4.2; P Value=0.001), thus
suggesting that the Val/Val genotype plays an important
role in predisposition to COPD in the Indian population.
Published studies indicate contradictory results for GST
P1 polymorphism. Harries and colleagues13 reported a
non-significant increase in the proportion of
homozygous 105Val status among British patients with
COPD as compared to healthy control subjects. This
finding was confirmed by Rodríguez et al.25 in a Spanish
population, but the authors reported that the frequency
of the GSTP1 105Val polymorphism is increased in COPD
patients with AAT deficiency, which is strongly
associated with an increased risk of developing the
disease.
A combination of oxidative attack and changes in
antiprotease activity could amplify the lung tissue
damage in COPD. So the interactions between genes as
well as environmental factors may play an important
role in the development of this pathogenesis. Ishii et
al.26 found that the wild-type variant Ile/Ile is related to
the development of COPD in the Japanese population,
however the number of subjects in each group was
small (around 50) and the 105Ile allelic frequency
observed was lower than that reported in two previous
studies27,28 in healthy Japanese volunteers.
11
Khan NA et al.
Smolonska and colleagues29 reported that GSTP1
Ile105Val polymorphism was protective against COPD in
only Asian populations (OR=0.69; 95% CI=0.56-0.85). A
protective effect of the Val/Val genotype against asthma
was reported by Haneni and co-authors30 in 105
asthmatic children and 112 control individuals from
Tunisian population. Similar finding was repeated by
Tamer et al.31 who reported that the GSTP1 Val/Val was
more prevalent among asthmatic subjects than in the
control group. One study involving Turkish patients32
demonstrated that the Val allele of GSTP1 may have a
protective effect against the development of COPD
which is consistent with our findings.
We further analyzed the relationship between GSTM1
and GST T1 with COPD. The GSTM1 null genotype
exhibited a significant association. (p=0.04) on
comparison between COPD patients and healthy
controls. The occurrence of GST M1 null genotype was
higher in the patients than in the controls (61.8% vs
55.0%; OR: 2.4 CI: 1.2-3.8; P value=0.04). Similar higher
frequency of GSTM1 null genotype was reported by
Lakhdar et al.33 However, our findings are in
contradiction to the findings of Malhotra et al.34 from
India where only 30 cases and equal controls patients
were recruited in the study. The association between
GSTM1 and COPD was not significant after adjustment
of age, sex, and pack-years BMI, smoking status, using a
logistic regression model; therefore the GSTM1 does not
seem to be an independent risk factor for the disease.
Our findings relate to the numerous studies that report
a non-significant association between GSTM11 deletion
and COPD.18,36,37 However, Cheng et al.38 reported that
GSTM1 null genotype is an independent risk factor for
developing COPD. Our analysis of combined GSTM1 and
GST T1 null alleles showed no significant association
between the double deletion and an increased risk of
COPD.
Our results suggest an absence of significant association
between GSTT1 null genotypes between COPD patients
and healthy controls (54.8% vs. 50.6%; OR: 1.26 CI: 0.87-
1.84 P value: 0.82). These findings are well supported by
studies from previous researchers investigating COPD.
There is no association reported between COPD and null
homozygous genotype of GSTT1 in previous studies.33
The null GST T1 has been suggested as a risk factor in
many diseases.18 Furthermore, several studies have
reported that individuals with GSTT1 null genotype may
be more susceptible to genotoxic damage and lung
diseases than individuals with the GSTT1 gene.38,39 In
our study, the frequencies of genotype in exon 3 of GST
T1 were significantly different from those found in
western countries. The frequency of the null type GST
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Khan NA et al.
T1 in our control group was also higher than in
Caucasians (50.6% vs. 20.4%).24
The most abundant mammalian GSTs are in alpha, mu,
and pi classes. Regulation and expression of these
detoxifying enzymes differ among tissues. Varying
expressions and distribution of GST enzymes between
the lung compartments could be relevant in the
contribution of each gene in pulmonary tract. GSTP1 is
more abundantly expressed than other GSTs in alveoli,
alveolar macrophages and respiratory bronchioles21 and
thus may play a crucial role in the respiratory tract.
A decrease or loss of GSTM1 expression in distal lung
has been found.21 It is suggested that GSTT1 is less
expressed in the lung tissues than other tissues and also
less than GSTM1 and GSTP1.40 Considering this fact, the
role of GSTT1 in protecting lungs against toxic products
is of less importance than GSTM1 and GSTP1 and
therefore it should be of much importance in the
pathogenesis of COPD.
The distribution patterns of GST genotypes are complex
among ethnic groups. So contradictory results regarding
the association of COPD and gene polymorphism in
different studies, may be explained by different allele
frequencies among races. Ethnic Differences could
therefore be accounted for by the differing frequencies
of genes relevant to pathogenesis.
Studies in the western population have reported a
higher mean age of patients with mean age between 60
and 69 years.41,42 whereas in the present study the
mean age was 55.9±9.2 years. This difference may be
attributed to the genetic makeup of Indian populations,
environmental factors, early smoking habits and poor
living conditions.
Associations between smoking and COPD have been
reported in many studies. Pauwels et al.42 have reported
50% of their study subjects being smokers, with an
average of 39 pack-years and individuals had a higher
probability of developing COPD if they were living in
urban areas, were male genders, were >60 years of age,
had higher educational levels, had >15 pack-year
smoking history, or had symptoms of chronic bronchitis.
But in our study we have observed that COPD develops
earlier in the Indian population with a shorter pack-year
history.
Most of the patients enrolled in the study smoked bidis;
this form of smoking is more hazardous than cigarette
smoking. To, our knowledge our study is the first to
identify an increased risk of development of COPD in the
ISSN: 2349-7181
J. Adv. Res. Med. 2016; 3(1)
carriers of the combined GSTM1, GSTP1 and GSTT1
genotype in the Indian population.
In the present study, several limitations remain. First the
number of males and females were not balanced and
relatively small population was recruited. In India the
majority of smokers and COPD patients are male so it is
quite difficult to find the balanced set of female subjects
for genotyping studies. Secondly, there was a difference
in mean age between cases and controls. In fact,
younger controls were more likely to provide a blood
sample than older controls, and were thereby over
sampled. The fact that the controls were younger than
the patients with COPD in this study could have reduced
the effects of polymorphic genotypes of the enzymes in
the development of COPD. Thirdly, the cases and
control subjects were recruited from a tertiary hospital.
This kind of hospital-based recruitment of patients may
cause selection bias. Moreover, information regarding
environmental pollution or inhaled irritants other than
cigarette smoking is not available. Thus, it is difficult to
quantify their contribution to airway obstruction.
In conclusion, we found an association between GSTP1
homozygous mutant genotype (105Val/Val) and
increased risk of COPD in Indians. We have also
demonstrated that the genetic polymorphism of GSTM1
was not independently associated to the development
of COPD moreover, no significant association was found
regarding the GST1 null genotype. It is also observed
that COPD developed in the early age and with a shorter
pack-year history in Indian population as compared to
western populations. In terms of the limitation in the
number of subjects examined, the present study is a
preliminary work and further studies using a larger
population becomes indispensible to confirm these
findings. Furthermore, the study of more candidate
genes, such as those of xenobiotic phase I and II
metabolizing enzymes, remains necessary in order to
elucidate the genetic pathogenesis of chronic
obstructive pulmonary disease as a complex polygenic
disease.
Acknowledgments
The authors thank Mr. Govind Mawari for his help to
collect blood, for genotyping assays and for assistance in
spirometry; Dr. Mohammad Asim, Istaq Ahmed for their
skillful technical assistance in genotyping assays
statistical analysis and their suggestions and helpful
comments.
Conflict of Interest: None
12
J. Adv. Res. Med. 2016; 3(1)
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Date of Submission: 22nd Apr. 2016
Date of Acceptance: 02nd May 2016
14