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Research Article

Naushad Ahmad
Association of Xenobiotic Metabolizing
Khan1,2, Naresh Gene Polymorphisms and Chronic
Kumar3, Syed Akhtar
Husain4, Mradul Kumar Obstructive Pulmonary Disease in Indian
Daga5
1,3,5
Department of Medicine,
Population
Maulana Azad Medical
College, New Delhi-110002. Abstract
2,4
Department of Background and Aims: Genetic susceptibility to the development of chronic
Biosciences, Jamia Millia
Islamia University, New Delhi
obstructive pulmonary disease might depend on variation in the activities of enzymes
-110025 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
Correspondence to: Dr.
Indian patients and controls.
Naushad Ahmad Khan,
Maulana Azad Medical
Material and Methods: A total of 186 patients with COPD and 160 healthy controls
College, New Delhi-110002.
were included in the study. The frequencies of GSTP1, GSTM1 alleles were
E-mail Id: determined by using conventional multiplex PCR and GSTP1 by polymerase chain
naushadkhan82@gmail.com
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
How to cite this article: in the global burden of disease, but has been predicted to rise to fifth highest burden
Khan NA, Kumar N, Husain
by 2020.2 COPD is characterized by irreversible airflow limitation in the lungs.3 It is
SA et al. Association of
Xenobiotic Metabolizing generally accepted that cigarette smoking is the most important risk factor for COPD.
Gene Polymorphisms and Nevertheless, only 10-20% of chronic smokers develop the severe impairment of
Chronic Obstructive pulmonary function associated with COPD.4,5 This indicates the possible contribution
Pulmonary Disease in Indian of environmental or genetic cofactors to the development of COPD. Although
Population. J Adv Res Med cofactors, such as childhood viral infection and environmental and occupational
2016; 3(1): 5-14.
pollution play important roles in pathogenesis,6 genetic susceptibility may be a factor
ISSN: 2349-7181 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

© ADR Journals 2016. All Rights Reserved.


Khan NA et al. J. Adv. Res. Med. 2016; 3(1)

The only established genetic risk factor for COPD is GSTT1 and GSTP1 genes polymorphism in Indian
homozygosity of the Z allele of the a1-antitrypsin (a1- population and to assess the correlation of GSTP1
AT) gene. Patients with genetic a1-AT deficiency have a genotype with clinical and functional parameters of
very high risk of developing emphysema at an early age COPD.
if they smoke. However, these patients account for only
a small proportion of all patients with emphysema.9 Materials and Methods
Recent studies reported that genetic variations in the
enzymes that detoxify cigarette smoke products might Setting and Inclusion Criteria
be associated with the development of COPD. These
There were 186 patients with COPD and 160 controls all
enzymes include microsomal epoxide hydrolase
unrelated in this case-control study. The patients were
(mEPHX), glutathione S-transferase (GST), and
recruited from LN Hospital (New Delhi, India) from both
cytochrome p450 1A1. The genes coding for xenobiotic-
the outpatient department as well as from inpatient
metabolizing enzymes have attracted attention because
facility. All genotyping assays were performed at
of their function in detoxifying of cigarette smoke
molecular genetics laboratory, department of
products.
biosciences, Jamia Millia Islamia University (New Delhi,
GSTs are a superfamily of enzymes involved in phase-II India). The demographic characteristics of the study
of xenobiotic-metabolism catalyzing the conjugation of subjects are summarized in Table 1. All patients satisfied
a wide range of electrophilic substances with reduced the clinical criteria of COPD set down in the Global
glutathione, thereby facilitating detoxification and Strategy for Obstructive Lung Disease (GOLD 2006)
further metabolism and excretion. Most of GSTs are guidelines.23
polymorphic enzymes and it is shown that the gene
The inclusion criteria for COPD were as follows: age
variations affect the enzyme activity and/ or gene
higher than 30 years; chronic airways symptoms and
expression.10-12
signs such as coughing, breathlessness wheezing, and
GSTs are separated into the following classes: alpha, mu chronic airway obstruction defined as forced expiratory
(GSTM), pi (GSTP), theta (GSTT), sigma and kappa. The volume in 1 sec. FEV1/ forced vital capacity (FVC) of
GST M1, T1 and P1 genes are located on chromosomes <70% and an FEV1 of <80% of predicted values from
1p13, 22q11.2 and 11q13, respectively. Among the spirometric data and FEV1 reversibility after inhalation
isoenzymes of GST, the homozygous GSTM1-null of 200 mcg of salbutamol of less than 12% pre-
genotype has been reported to show some association bronchodialator of FEV1. Patients with bronchial asthma
with the pathogenesis of lung cancer13,14 bladder were excluded on the basis of reversibility of airflow
cancer15 and, especially, emphysema16. The GSTT1-null obstruction patients with respiratory disorders other
mutant has also been suggested as a risk factor in many than COPD such as interstitial lung disease, lung cancer,
diseases.17-20 Polymorphisms of GSTP1 genotypes have tuberculosis, bronchiectasis, previous medical record of
been reported, including isoleucine (Ile) 105 to valine bronchial asthma, thoracic surgery in the past, and
(Val) mutation in exon 5 and alanine 114 to Val (Val) patients unable to perform spirometry to confirm the
mutation in exon 6. Individuals with the Val105 (mutant) diagnosis were also excluded.
allele have a higher risk of developing lung cancer than
those with the Ile105 (wild-type) allele.21 In addition, Pulmonary Function Test
GSTP1 is expressed more abundantly in respiratory
The subjects were instructed about the proper
tissues than other kinds of GST.22
technique of performing the spirometry and technique
There is a paucity of information with respect to the was also demonstrated to each patient before starting
influence of genetic polymorphism of GSTM1, T1 and P1 the test. Nose clip was used to prevent air entry or exit
in cases with or without COPD in India. Also, the current through the nose. At least three acceptable spirograms
data on the potential associations between an increased were obtained from a minimum of five forced
COPD risk and genes encoding the enzymes expirations. Lung function was assessed by a trained
metabolizing xenobiotic substances are inconsistent. technician (research assistant). Subjects showing an
obstruction on pulmonary function test were subjected
Hence, the aim of our study was to analyze the relation to a repeat test after bronchodilation with 200 mcg of
between COPD and gene polymorphisms of GSTM1, Salbutamol to test the reversibility of obstruction.

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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


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

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72°C for 5 min. the products of the multiplex PCR groups. The rate of literacy was primary school literates/
containing fragments of 215 bp (indicating the presence illiterates 49.4%, high school 33.3% and professional or
of GSTM1), 480 bp (GSTT1), and 350 bp (albumin), were post graduate degree 17.2%. There was no significant
separated by electrophoresis with ethidium bromide- difference in alcohol consumption and educational
stained 3% agarose gel and visualized by UV detection. status across the groups.

Statistical Analysis The number of individuals smoking bidi was greater


than of cigarette. Cigarette/ bidi smoking and pack-
The SPSS for Windows version 12.0 software (SPSS Inc. years were the only variables that differed significantly
Chicago, IL, and USA) was used in all analyses. Hardy- between controls and those with COPD. The mean
Weinberg equilibrium of genotype distribution in cases number of pack-years in the study group was 24.8±8.2
and controls was evaluated by calculating χ2 and P years as against 15.2±3.9 years in the control group
values. Differences in allele distribution and allele which was statistically significant (P<0.001) (Table 1).
frequencies among the groups were examined for
statistical significance by the Pearson χ2 test. Odds Pulmonary Function Test and Grading of
ratios (OR) with their corresponding 95% confidence Severity of COPD Patients
intervals (CI) were calculated. The χ2 test was again
used to compare gender, smoking status between the The spirometric characteristics of the patients are
two groups. Age, smoking index expressed as pack-years presented in Table 2. The mean FVC %, FEV1% and
(number of cigarettes smoked per day×number of years FEV1/ FVC% values in patients with COPD were 62 ±
smoked/20) were compared using the unpaired Student 16.7, 36.2 ± 12.2, and 42.2 ± 12.4 those in controls were
t-test. Analysis of variance was carried out to evaluate 98.2 ± 12.8, 98.8 ± 14, and 78.2 ± 3.8 respectively. The
clinical and functional characteristics of patients lung function values in patients with COPD were
according to different genotypes. Logistic regression significantly different from those in controls (p<0.001).
was performed to estimate OR and 95% CI of COPD risk Patients with COPD were classified into three subgroups
factors. P<0.05 was considered to be statistically according to severity of the disease-mild, moderate and
significant. severe. Out of 186 patients, 40 (21.5%) were in the mild
category (FEV1 between 60 and 80%), 88 (47.3%) in the
Results moderate category (FEV1 between 40 and 69%), and 58
(31.1%) in the severe category (FEV1<40%). Hardy-
Baseline Clinical Characteristics of Study Subjects Weinberg equibillirium was tested for all
polymorphisms and no obvious deviation was observed.
The demographic characteristics of the COPD patients In our findings, GST M1 null genotype was found to be
and healthy controls matched for age and pack-years significantly associated with increased risk of COPD on
are shown in Table 1. A total of 186 COPD patients and multivariate analysis. The odd ratio was 2.4 (95% (CI)
160 healthy controls were enrolled in this case-control 1.2-3.8).
study. 18 (9.76%) of the 186 COPD patients and 5 (3.1%)
of the 160 healthy controls were females. There was no The frequency of GSTM1 genotype was significantly
significant difference observed between the patients higher in the patients of COPD than in the control
and controls group in terms of age (mean age 55.9 ± 9.2 subjects (61.8% vs. 55.0%). However, no significant
vs 51.4 ± 7.3), gender (168:18 vs 155:5), as well as BMI. association was found between GST M1 and COPD after
The proportion of current smokers was less in COPD adjustment for age, sex, and smoking status using
Patients. BMI was almost similar in both logistic regression analysis (P>0.05, data not shown).

Table 2.Spirometric Characteristics of the Study Subjects according to GOLD Classification Criteria
Controls (N = 160) COPD Patients (N = 186)
Spirometry
FEV1% (predicted) 98.8 ± 14 36.2 ± 12.2*
FVC% (predicted) 98.2 ± 12.8 62 ± 16.7*
FEV1/FVC% 78.2 ± 3.8 42.2 ± 12.4*
Severity of the Disease
Mild (60-80) 40 (21.5%)
Moderate (40-59) 88 (47.3%)
Severe (<40) 58 (31.1%)
*Statistically significant

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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

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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


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

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J. Adv. Res. Med. 2016; 3(1) Khan NA et al.

antioxidant imbalance, are believed to play an Smolonska and colleagues29 reported that GSTP1
important role in the pathogenesis of COPD.6-8 Indeed, it Ile105Val polymorphism was protective against COPD in
is well known that chronic tobacco smoking is a major only Asian populations (OR=0.69; 95% CI=0.56-0.85). A
risk factor for the development of COPD, and a defect in protective effect of the Val/Val genotype against asthma
the detoxification of reactive species produced by was reported by Haneni and co-authors30 in 105
cigarette smoke may predispose smokers to airflow asthmatic children and 112 control individuals from
obstruction and emphysema.9 Tunisian population. Similar finding was repeated by
Tamer et al.31 who reported that the GSTP1 Val/Val was
Glutathione S-tranferase plays a protective role as an more prevalent among asthmatic subjects than in the
antioxidant in the lung.10-12 In a complex polygenic control group. One study involving Turkish patients32
disease such as COPD, it is likely that genetic demonstrated that the Val allele of GSTP1 may have a
susceptibility is dependent on the action of several gene protective effect against the development of COPD
polymorphisms operating in concert. Polymorphisms in which is consistent with our findings.
individual gene may impart only a small relative risk of
COPD and it is likely that the cumulative effect of many We further analyzed the relationship between GSTM1
polymorphisms will be important in its pathogenesis. and GST T1 with COPD. The GSTM1 null genotype
exhibited a significant association. (p=0.04) on
The study provides a new genotyping data of GSTP1, comparison between COPD patients and healthy
GSTM1, GSTT1 polymorphisms and their association controls. The occurrence of GST M1 null genotype was
with susceptibility to chronic obstructive pulmonary higher in the patients than in the controls (61.8% vs
disease in Indian population. When compared, the 55.0%; OR: 2.4 CI: 1.2-3.8; P value=0.04). Similar higher
frequencies of exon 5’ GSTP1 polymorphism in COPD frequency of GSTM1 null genotype was reported by
patients differed significantly from the control group. Lakhdar et al.33 However, our findings are in
Our results demonstrated that the homozygous mutant contradiction to the findings of Malhotra et al.34 from
genotype Val/Val was two times higher in patients than India where only 30 cases and equal controls patients
in controls 18.2% vs. 8.1%). We also found that the were recruited in the study. The association between
frequency of the Val allele was higher in the patient GSTM1 and COPD was not significant after adjustment
group (OR: 1.8; CI: 1.4-4.2; P Value=0.001), thus of age, sex, and pack-years BMI, smoking status, using a
suggesting that the Val/Val genotype plays an important logistic regression model; therefore the GSTM1 does not
role in predisposition to COPD in the Indian population. seem to be an independent risk factor for the disease.
Our findings relate to the numerous studies that report
Published studies indicate contradictory results for GST
a non-significant association between GSTM11 deletion
P1 polymorphism. Harries and colleagues13 reported a
and COPD.18,36,37 However, Cheng et al.38 reported that
non-significant increase in the proportion of
GSTM1 null genotype is an independent risk factor for
homozygous 105Val status among British patients with
developing COPD. Our analysis of combined GSTM1 and
COPD as compared to healthy control subjects. This
GST T1 null alleles showed no significant association
finding was confirmed by Rodríguez et al.25 in a Spanish
between the double deletion and an increased risk of
population, but the authors reported that the frequency
COPD.
of the GSTP1 105Val polymorphism is increased in COPD
patients with AAT deficiency, which is strongly Our results suggest an absence of significant association
associated with an increased risk of developing the between GSTT1 null genotypes between COPD patients
disease. 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
A combination of oxidative attack and changes in
studies from previous researchers investigating COPD.
antiprotease activity could amplify the lung tissue
There is no association reported between COPD and null
damage in COPD. So the interactions between genes as
homozygous genotype of GSTT1 in previous studies.33
well as environmental factors may play an important
The null GST T1 has been suggested as a risk factor in
role in the development of this pathogenesis. Ishii et
many diseases.18 Furthermore, several studies have
al.26 found that the wild-type variant Ile/Ile is related to
reported that individuals with GSTT1 null genotype may
the development of COPD in the Japanese population,
be more susceptible to genotoxic damage and lung
however the number of subjects in each group was
diseases than individuals with the GSTT1 gene.38,39 In
small (around 50) and the 105Ile allelic frequency
our study, the frequencies of genotype in exon 3 of GST
observed was lower than that reported in two previous
T1 were significantly different from those found in
studies27,28 in healthy Japanese volunteers.
western countries. The frequency of the null type GST

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T1 in our control group was also higher than in carriers of the combined GSTM1, GSTP1 and GSTT1
Caucasians (50.6% vs. 20.4%).24 genotype in the Indian population.

The most abundant mammalian GSTs are in alpha, mu, In the present study, several limitations remain. First the
and pi classes. Regulation and expression of these number of males and females were not balanced and
detoxifying enzymes differ among tissues. Varying relatively small population was recruited. In India the
expressions and distribution of GST enzymes between majority of smokers and COPD patients are male so it is
the lung compartments could be relevant in the quite difficult to find the balanced set of female subjects
contribution of each gene in pulmonary tract. GSTP1 is for genotyping studies. Secondly, there was a difference
more abundantly expressed than other GSTs in alveoli, in mean age between cases and controls. In fact,
alveolar macrophages and respiratory bronchioles21 and younger controls were more likely to provide a blood
thus may play a crucial role in the respiratory tract. sample than older controls, and were thereby over
sampled. The fact that the controls were younger than
A decrease or loss of GSTM1 expression in distal lung the patients with COPD in this study could have reduced
has been found.21 It is suggested that GSTT1 is less the effects of polymorphic genotypes of the enzymes in
expressed in the lung tissues than other tissues and also the development of COPD. Thirdly, the cases and
less than GSTM1 and GSTP1.40 Considering this fact, the control subjects were recruited from a tertiary hospital.
role of GSTT1 in protecting lungs against toxic products This kind of hospital-based recruitment of patients may
is of less importance than GSTM1 and GSTP1 and cause selection bias. Moreover, information regarding
therefore it should be of much importance in the environmental pollution or inhaled irritants other than
pathogenesis of COPD. cigarette smoking is not available. Thus, it is difficult to
quantify their contribution to airway obstruction.
The distribution patterns of GST genotypes are complex
among ethnic groups. So contradictory results regarding In conclusion, we found an association between GSTP1
the association of COPD and gene polymorphism in homozygous mutant genotype (105Val/Val) and
different studies, may be explained by different allele increased risk of COPD in Indians. We have also
frequencies among races. Ethnic Differences could demonstrated that the genetic polymorphism of GSTM1
therefore be accounted for by the differing frequencies was not independently associated to the development
of genes relevant to pathogenesis. of COPD moreover, no significant association was found
regarding the GST1 null genotype. It is also observed
Studies in the western population have reported a
that COPD developed in the early age and with a shorter
higher mean age of patients with mean age between 60
pack-year history in Indian population as compared to
and 69 years.41,42 whereas in the present study the
western populations. In terms of the limitation in the
mean age was 55.9±9.2 years. This difference may be
number of subjects examined, the present study is a
attributed to the genetic makeup of Indian populations,
preliminary work and further studies using a larger
environmental factors, early smoking habits and poor
population becomes indispensible to confirm these
living conditions.
findings. Furthermore, the study of more candidate
Associations between smoking and COPD have been genes, such as those of xenobiotic phase I and II
reported in many studies. Pauwels et al.42 have reported metabolizing enzymes, remains necessary in order to
50% of their study subjects being smokers, with an elucidate the genetic pathogenesis of chronic
average of 39 pack-years and individuals had a higher obstructive pulmonary disease as a complex polygenic
probability of developing COPD if they were living in disease.
urban areas, were male genders, were >60 years of age,
had higher educational levels, had >15 pack-year
Acknowledgments
smoking history, or had symptoms of chronic bronchitis. The authors thank Mr. Govind Mawari for his help to
But in our study we have observed that COPD develops collect blood, for genotyping assays and for assistance in
earlier in the Indian population with a shorter pack-year spirometry; Dr. Mohammad Asim, Istaq Ahmed for their
history. skillful technical assistance in genotyping assays
statistical analysis and their suggestions and helpful
Most of the patients enrolled in the study smoked bidis;
this form of smoking is more hazardous than cigarette comments.
smoking. To, our knowledge our study is the first to
Conflict of Interest: None
identify an increased risk of development of COPD in the

ISSN: 2349-7181 12
J. Adv. Res. Med. 2016; 3(1) Khan NA et al.

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