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-the association of Glutathione S Transferase M1 (GSTM1) and Glutathione S Transferase T1 (GSTT1) gene polymorphism with Chronic Obstructive Airway Disease (COAD) patients in India
Cigarette smoking is the most important risk factor leading to development of COAD. Smoking accounts for as much as 80-85% cases of COAD while only 15% smokers develop clinically symptomatic COAD. One possible reason why only a small proportion of smokers develop COAD might be genetic variation in the enzymes that detoxify cigarette smoke products. We therefore studied the frequencies of genetic polymorphisms of GST M1 and GST T1 in patients with COAD and healthy subjects.
Methods: The study comprised of 120 COAD cases and 120 healthy controls. Men with COAD in age group 30-60 years were taken, diagnosis of COAD being made on the basis of history, clinical examination, radiological examination, ABG and pulmonary function tests (PFT). Blood samples were taken from both study and control groups. Analysis included genomic DNA extraction and PCR amplification of GSTM1 and GSTT1 gene to detect their null polymorphism in cases and controls along with control albumin gene.
Results: The mean age in the study group was 51.67Â± 8.9 and in the control group was 51.40 Â± 7.65. The age distribution between the cases and controls was not statistically significant (p=0.91). The average pack years in the case group was 20.77 Â± 4.754 whereas it was only 10.27 Â± 2.913 for the control group which was statistically significant (p<0.001). 16.7% (20/120) of COAD cases were GSTM1 null in comparison to 33.3% (40/120) in controls which was not statistically significant (p=0.135). A total of 40% (48/120) of the cases presented homozygous deletion of GSTT1 genotype as compared to controls 13.3% (16/120) which was statistically significant (p=0.019).
Conclusion: In summary, we have shown that GSTT1 null genotype might be associated with the pathogenesis of COAD.
Foremost, I would like to thank my supervisor, Dr. Amit Kumar Verma, who shared with me a lot of his expertise and research insight. He quickly became for me the role model of a successful researcher in the field of Clinical Research. He is not only a great Supervisor and Professor; he has also been a cornerstone in my professional development in the Clinical Research field.
I also like to express my gratitude to Dr. Sachin Manocha, whose thoughtful advice often served to give me a sense of direction during my M.Sc.in Clinical Research studies. And I am deeply grateful to the Institute of Clinical Research India (ICRI), Delhi, for the trust and support that they gave me in order to study in the UK.
It is difficult to overstate my appreciation to Dr. Devesh Gupta who has always guided me in suitable direction for my future career in the Clinical Research field.
I wish to thank everybody with whom I have shared experiences in life. From the people who first persuaded and got me interested into the study of Clinical Research, especially those who also played a significant role in my life.
I am tempted to individually thank all of my friends which, from my childhood until post graduate school, have joined me in the discovery of what is life about and how to make the best of it. However, because the list might be too long and by fear of leaving someone out, I will simply say thank you very much to you all.
Finally I wish to express special thanks to my parents and family members who have endured the process of the project with conspicuous fortitude and patience. I cannot finish without saying how grateful I am with my parents and other family members who have given me a loving environment where to develop. Lastly, and most importantly I wish to thank my parents and family members, they have always supported and encouraged me to do my best in all matters of life.
Statement Regarding Research:
This is to certify that thesis contains no material that has been accepted for the award of any degree or diploma of any university or other institution. Furthermore to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference is made in the text of the thesis.
Dr. Julfikar Rahaman
List of contents:
List of tables..............................................................................................................8
List of figures.............................................................................................................9
List of abbreviations................................................................................................10
Chapter- 1: Introduction and literature review....................................................11
Chapter- 2: Aims and objectives............................................................................27
Chapter- 3: Materials and methods.....................................................................13
Chapter- 4: Results..............................................................................................25
Chapter- 5: Discussion.........................................................................................33
Chapter- 6 Conclusions & Future Work...............................................................45
References (Literature cited)...................................................................................40
List of Tables:
Table-1: The age distribution between the study and control group..................25
Table-2: Grading of severity of COPDâ€¦â€¦â€¦â€¦..â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦..27
Table-3: Summary of Smoking habitâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦..â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.29
Table-4: Distribution of GSTM1 and GSTT1 genotypes in patients with COPD and
Table-5: Risk of both null genotypesâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦..â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦32
List of Figures:
Figure 1: JAEGER's SPIROMETER..â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦17
Figure 2: Blood Gas Analyzerâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦..19
Figure 3: Ethidium bromide stained DNA in 1% agarose gelâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.22
Figure 4: Representative multiplex PCR analysis of GSTM1 and GSTT1 gene products resolved by 3% Agarose gel electrophoresisâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦..24
Figure 5: Age distribution between the study and control groupâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.27
Figure 6: Grading of severity of COPD in study groupâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦29
Figure 7: Smoking summaryâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦.30
Figure 8: Representative multiplex PCR analysis of GSTM1 and GSTT1 gene products resolved by 3% Agarose gel electrophoresisâ€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦..â€¦â€¦â€¦â€¦â€¦â€¦â€¦â€¦32
List of Abbreviations:
COAD Chronic obstructive airway disease
Chronic obstructive airway disease (COAD) is one of the most important respiratory causes of chronic morbidity and mortality throughout the world1. Currently, COAD ranks fifth among the leading causes of death2, and further increases in its prevalence and mortality are exÂpected in the near future3. It is defined by the Global Initiative for Chronic Obstructive Lung Disease4 as a "disease state characterized by airflow limitation that is not fully reversible. The airflow limitation is usually both progressive and associated with an abnormal inflammatory response of the lungs to the noxious particles or gases".
Chronic obstructive Airway disease (COAD) comprises a mixed group of common respiratory disease united by the presence of persistent airflow limitation, arising usually after many years of tobacco smoking. Cigarette smoking is the most important risk factor leading to development of COAD. Smoking accounts for as much as 80-85% cases of COAD while only 15% smokers develop clinically symptomatic COAD. One possible reason why only a small proportion of smokers develop COAD might be genetic variation in the enzymes that detoxify cigarette smoke products. These enzymes include microsomal epoxide hydrolase (mEPHX), glutathione S-transferase (GST), and cytochrome p 450 1A1 (CYPA11).
GST is a complex supergene family of soluble isozymes which catalyse the nucleophilic attack of glutathione on a wide range of hydrophobic electrophiles5. Among the isoenzymes of GST, homozygous null GST M1 genotypes have been reported to have association with the pathogenesis of lung cancer6-8, bladder cancer9, pituitary adenoma10, and particularly, emphysema11. GSTT1 conjugates glutathione and various potential carcinogens including halomethanes12 which are present in cigarette smoke and its null type mutant has been suggested as a risk factor in colon cancer13, myelodysplastic syndrome14, meningioma and astrocytoma15. However, no association between COAD and the homozygous null genotype of GST T1 has been reported.
There is increasing evidence that several genes influence the development of COAD. When multiple genes are operating in the pathogenesis of diseases, the influence of each gene might be relatively weak. Multiple gene polymorphisms should therefore be investigated to determine the genetic markers that predict the risk for developing COAD. We therefore studied the frequencies of genetic polymorphisms of GST M1 and GST T1 in patients with COAD and healthy subjects to determine whether multiple polymorphisms of these genes are linked to a genetic susceptibility to COAD.
MATERIALS AND METHODS
The study was carried out in the Department of Medicine, Maulana Azad Medical College and associated Lok Nayak Hospital, New Delhi, India. The study comprised of 120 COAD cases. The control group comprised of 120 healthy individuals. All control subjects were male and were current smokers free from pulmonary disease with normal PFT. None of the control subjects had family members affected by COPD.
Subject selection: The study group comprised of 120 COAD cases from the medical out patient department and wards of LN Hospital, New Delhi. Men with COPD in age group 30-60 years were taken, diagnosis of COPD being made on the basis of history, clinical examination, radiological examination, ABG and pulmonary function tests (PFT). Pulmonary function tests were done in all cases and controls.
Patients with bronchial asthma
Patients with diabetes and hypertension
Evidence of any other associated primary neoplasm, or a history of it.
The purpose of tests was explained to each subject and the method of testing was demonstrated. The need of avoiding leaks around the nose clip and mouth piece was emphasized. All the questions of the subject were answered and every effort made to allay apprehension and promote cooperation.
Before each test, the nose clip was applied and patient instructed to blow or sniff through the nose with mouth closed to test for leaks.
Pulmonary function tests done
5. pO2, PCO2, pH
Criteria for the selection of COAD cases
Patients suffering from excertional dyspnea of varying severity throughout the year were taken for the study. History of cough with expectoration for more than 2 years, expectoration being mucoid or occasionally mucopurlent, present on most days for two consecutive years was selected in the study.
A. Clinical history
Patient's clinical history was taken carefully. The chief symptoms and their duration were noted. Following questions were asked from smokers.
1. Type of tobacco smoked: cigarette, beedi and Hukka
2. Duration of smoking
3. Average daily consumption
B. Each subject was given thorough physical examination and history was taken to exclude.
a) Any recent episode of respiratory infection or allergy.
b) Any chronic cardiopulmonary disease
c) Thoracic cage abnormality
d) General debility
e) Evidence of any neoplasm or history of it.
f) Bronchial asthma, hypertension and diabetes mellitus
Clinical examination: Presence of physical signs of emphysema.
a) Restricted expansion of the chest during respiration.
b) Increased artero-posterior diameter of the chest
c) Hyperresonance of the lung fields, obliteration of the cardiac and hepatic dullness to a variable extent.
d) Prolonged expiration, presence of rhonchi with or without moist sounds in the chest.
e) Absence of any of other cause for progressive dyspnoea like cardiac lesions etc.
C. Roentological examination (Criterion for diagnosis) and to exclude stent pulmonary disease/malignancy
a) Increased translucency of the lungs
b) Flat and straightened out domes of the diaphragm
c) Presence of air between the cardiac shadow and the domes of the diaphragm
d) Increased hilar markings
e) Prominent Pulmonary conus
f) Presence of bullae and cysts
D. Laboratory Investigations
The following routine laboratory investigations were done in all the cases.
1. Examination of blood
(i) Total WBC count
(ii) Differential WBC count
(iii) Peripheral smear examination
(iv) Hemoglobin estimation
(v) Serum electrolytes, blood urea, blood sugar etc.
2. Examination of the sputum for Gram Stain and acid fast bacilli
3. Radiology - postero anterior view of the chest was taken as a rule in all the patients.
E. Pulmonary function tests can help a doctor diagnose a range of respiratory diseases which might not otherwise be obvious to the doctor or the patient. The tests are important since many kinds of lung problems can be successfully treated if detected early.
The tests are also used to measure how a lung disease is progressing, and how serious the lung disease has become. Pulmonary function tests also can be used to assess how a patient is responding to different treatments. One of the most common of the pulmonary function tests is spirometry (from the Greco-Latin term meaning "to measure breathing").
Figure 1: JAEGER's SPIROMETER
This test, which can be given in a hospital or doctor's office, measures how much and how fast the air is moving in and out of the lungs.
The patient places a clip over the nose and breathes through the mouth into a tube connected to a machine known as a Spirometer. First the patient breathes in deeply, and then exhales as quickly and forcefully as possible into the tube. The exhale must last at least six seconds for the machine to work properly. Usually the patient repeats this test three times, and the best of the three results is considered to be the measure of the lung function. The results will help a doctor figure out which type of treatment to pursue.
E. PO2, pCO2, pH measurements were done with arterial blood obtained from the brachial artery. Apparatus used for this purpose was radiometer BMS3-MKZ blood microsystem.
Only males were included in the study to rule out sex difference.
Figure 2: Blood Gas Analyzer
F. DNA extraction from whole blood
1. 10 ml of heparanized blood was taken in 50ml falcon tube.
2. 30 ml of lysis solution (155mM NH4Cl), 10mM KHCO3 and 0.1mM EDTA) was added to it and kept for 30 min in ice.
3. Centrifuged at 2000g for 10 min at 4oC and discarded the supernatant.
4. Resuspended cell pellet in 10ml of 75mM NaCl and 20mM EDTA, (pH 8.0) ensuring that no cell clumps remains.
5. Added proteinase k to a final conc. Of 100Âµg/ml. Mixed well.
6. Added 2ml of 10% SDS solution.
7. Incubated at 37oC for 3-4 hrs.
8. Following digestion, phenol chloroform extraction was done. Equal volume of equilibrated phenol was added and mixed it thoroughly on overhead shaker for 45 min.
9. Centrifugation was done at 6000 rpm for 30min at 4oC.
10. Took out the supernatant in a fresh falcon tube with the help of a Pasteur pipette.
11. Added an equal volume of equilibrated phenol and chloroform isoamyl alcohol (1:1) to remove protein.
12. Mixed it thoroughly on an overhead shaker for 30 min and repeated the steps 9 and 10.
13. Took out the aqueous supernatant from step 11 and added 1/10th volume of chilled 3M sodium acetate solution and 2.5 volume of chilled absolute alcohol.
14. Left it at -20oC for overnight.
15. Next day, centrifuged at 6000 rpm at 0oC for 15 min.
16. Washed the DNA pellet in 70% ethanol and was left for air drying.
17. Resuspended the DNA pellet in 200Âµl of 1 x TE (Tris EDTA) and transferred to a small eppendorf tube.
G. DNA estimation.
1. Added 7Âµl of DNA solution from the final suspension to 700Âµl deionised distilled water in a 1.5ml microfuge tube.
2. Vortexed briefly and transferred to an appropriate spectrophotometer cuvette.
3. Read and recorded the A260 and A280 OD of each 1:100 dilution in spectrophotometer.
4. Calculated A260 /A280 ratio. The reading at 260 gives DNA and that at 280 gives protein. The ratio between DNA and protein should be within 2:1 (2DNA:1 protein).
5. if ratio was less than 1.6, the sample was re-extracted and reprecipitation of the sample was done.
DNA concentration was calculated as : OD at 260nm x dilution x 50 = Âµg/ml).
Figure 3: Ethidium bromide stained DNA in 1% agarose gel
H. Genotyping assays
The homozygous null polymorphisms of GSTMÂ1 and GSTT1 genotypes were determined by using three sets of primers to amplify a 215bp sequence of the GSTM1 gene, a 480bp sequence of GSTT1+ and a 350bp sequence of albumin gene fragment, which served as an internal positive control16. The PCR primers were as follows:
5'-GAA CTC CCT GAA AAG CTA AAG C-3' AND
5'-GTT GGG CTC AAA TAT ACG GTGG-3'
Polymerase chain reaction (PCR) was performed in 25Âµl reaction volume containing 50-100ng of genomic DNA. 50mM KcL, 2.5mM MgCl2, 200M Tris-HCl (pH8.4), 200mM of dNTPs, GSTM1 primers at 3Âµg each, GSTT, primers at 1Âµg each, albumin primers at 600ng/ml each and 1.5 units of DNA ampli taq (cetus) in a Perkin-Elmer thermal cycler. After an initial denaturation at 96oC for 5 min, amplification was carried out for 35 cycles at 94oC for 1 min, 56oC for 1 min and 72oC for 1 min, followed by final elongation at 72oC for 7 min.
The products of multiplex PCR (215 bp for GSTM1, 480 bp for GSTT1 and 350 bp for albumin) were separated by electrophoresis with ethidium bromide stained 3% agarose gel. The GSTM1 homozygous null was evidenced by the absence of 215bp fragment and of GSTT1 homozygous null by the absence of a 480 bp fragments. The presence of 350bp albumin fragment was indicator of a successful PCR (figure4).
Figure 4: Representative multiplex PCR analysis of GSTM1 and GSTT1 gene products resolved by 3% Agarose gel electrophoresis. A 350bp DNA fragment corresponding to albumin gene product provides an internal positive control, seen in all lanes. A 215bp product only seen in lanes 2,3,4,7,8 and 10 containing the GSTM1 gene, while a 480 bp product of GSTT1 gene is seen in lanes 4, 5, 8, 9 and 10. Both GSTM1 and GSTT1 genes are absent in lanes 1 and 6. M represents phi x Hae III digested marker.
This study was conducted on stable patients of Chronic Obstructive Airway Disease. Two forty patients were taken and of which 120 comprised of COAD cases and 120 were heathy individuals as control group. Patients of both group i.e. study and control group were matched for age. In all the patients of study and the control group the following parameters were measured: arterial blood gas analysis, pack years, pulmonary function test (FEV1, FVC, FEV1/FVC) and GSTM1 and GSTT1 genotype. The above mentioned parameters were then compared between the study and control group.
The age distribution between the study and control group was as follows:
Figure 5: Age distribution between the study and control group
As can be seen from the table 1, maximum number of patients are in 45-54 years age group. The minimum age was 32 years and the maximum was 70 years. The age distribution between the cases and controls was not statistically significant. (p=0.91)
Grading of severity of COPD in the study group based on forced expiratory volume in first second percentage (FEVÂ1%).
TABLE 2: Grading of severity of COPD
Figure 6: Grading of severity of COPD in study group
The minimum FEV1 in the study group was 23.2% and maximum was 65.5%. Most of the patients (50.0%) were in the moderate category (FEV1 between 40-59%) with only 33.3% patients in severe (FEV1<40%) and 16.7% in the mild category (60-80%).
Table 3: Summary of Smoking habit
Figure 7: Smoking summary
As seen in the table all patients in the study and control group were smokers. The average pack years in the case group was 20.77=4.754 whereas it was only 10.27 Â± 2.913 for the control group which was statistically significant (p<0.001).
8 7 6 5 4 3 2 1
Figure 8: Representative multiplex PCR analysis of GSTM1 and GSTT1 gene products resolved by 3% Agarose gel electrophoresis. A 350bp DNA fragment corresponding to albumin gene product provides an internal positive control, seen in all lanes. A 215bp product only seen in lanes 2, 3,4,5,7, and 8 containing the GSTM1 gene, while a 480 bp product of GSTT1 gene is seen in lanes 1,2,3,4, 5, and 8. Both GSTM1 and GSTT1 genes are absent in lanes 6.
TABLE 4: Distribution of GSTM1 and GSTT1 genotypes in patients with COPD and healthy subjects
GSTM1Â nonÂ null
GSTM1Â null &GSTT1null
Fisher exact test p = 0.35
TABLE 5: Risk of both null genotypes
Both null genotype
OD (95% CI)
OR (95% CI) smoking adjusted
Glutathione S-Transferases (GSTs) are members of a family of enzymes which play an important role ill detoxifying various aromatic hydro- carbons found in cigarette smoke. GSTs conjugate electrophilic substrates with glutathione and this facilitates further metabolism and excretion. GSTM1 is expressed in the liver and the lung. Homozygous deletion of the GSTM1 gene occurs in approximately 50% of Caucasians. Homozygous deficiency for GSTM1 was associated with emphysema in patients who had lung cancer (OR = 2.1)17 and severe chronic bronchitis in heavy smokers (OR = 2.8). However, in a Korean study there was no association between GSTMI and GSTT1 polymorphisms and COPD18. The present study was conducted in 120 stable patients of chronic obstructive airway disease and 120 controls. All subjects were males. These patients were subjected to thorough history and clinical examination including a detailed examination of the respiratory system and a detailed pre-structured proforma was filled along with taking the informed consent of the patient. All patients underwent spirometry and were divided into study and control group. The following parameters were assessed:
1. Age distribution in study and control group.
2. Classification of severity of COPD in study patients was done on basis of pulmonary function test.
3. The pack years of the study and control group was analyzed.
4. The distribution of GSTM1 and GSTT1 genotypes in patients with COPD and healthy subjects were compared.
Statistical analysis was done using the chi-square test for categorical variables and the student 't' test for continuous variables.
1. Age distribution of COPD patients
The mean age of patients in the study was 51.67 years while the mean in the control group was 51.40 years. The maximum number of patients were in the 45-54 years age group (51.4%) and then in the >55 years age group (52.9%).
Studies reported on COPD patients in western literature show a higher mean age of patients. A study by Pauwels et al19., on COPD patients in Europe showed that approximately half of the patients were in the age range of 60-69 years. Another study by Katherine Gary20 et al., showed a mean age of 63.1 years in study patients. Redelmeier21 et al., in their study had a mean age of 67 years in stable COPD patients. The National Health and nutrition Examination Survey III in USA also showed that maximum patients of COPD were > 60 years old. These results show that the mean age of the COPD patients in our study was less than the mean age of COPD patients in western literature. This may be explained on the basis of the genetic makeup of the Indian population, environmental factors, poor living conditions or smoking habits. The exact cause is still a matter of study and is not known.
2. Smoking summary
All the patients in both study and control group were smokers. The number of bidi smokers was more than the number of cigarette smokers. The mean number of pack years in the study group was 20.77 years as against 10.27 years in the control group which was statistically significant (p<0.001). An Indian study by Jindal et al22 showed that smoker is to non-smoker ratio was 82.3% and the prevalence of COPD amongst smokers was 8.3% which was significantly more than non-smokers (p value <0.01). Another Indian from CMC, Vellore by Ray et al23 showed that 63% of the patients were smokers. Pauwels et al19 showed that 50% of the patients were smokers and had an average of 39 packs of cigarette/bidi per years of smoking.
Association between smoking and COPD was also shown by Victor Sobradillo Pena24 et al who showed that men >60 years and >15 pack years smoking history had a higher probability of developing COPD. The prevalence of COPD was 15% in smokers as against 4.1% in non-smokers. This went up to 40.3% in patients with >30 pack years and >60 age. Thus it was seen that COPD developed earlier in the Indian population with a shorter pack year history. This can be due to factors as mentioned in relation to early age of onset. Siafaskas et al25 had shown that difference in nutrition could also play a role in protecting against oxidative stress from smoking and hence preventing COPD. Also the Indian population is different from Western population with regard to baseline lung function, smoking habits and living conditions. Although maximum patients in our study smoked bidi's, literature shows that this form of smoking is equally hazardous as others.
3. GSTM1 and GSTT1 genotyping in patients with COPD and healthy subjects.
There is no doubt that cigarette smoking is the major risk factor for COPD, and there is a genetic susceptibility to this complex genetic disorder26. Potential susceptibility genes include those regulating the protease-antiprotease and oxidant-antioxidant interactions. Although there have been many studies in experimental animals and humans that support a role for imbalances in oxidant-antioxidant systems in the pathogenesis of smoking induced COPD27, there have been relatively few studies that investigated the role of polymorphisms of antioxidant genes in COPD. In the present study, we have investigated whether polymorphisms of GSTM1 and GSTT1 antioxidant genes are associated with development of COPD. There are substantial differences in the baseline frequencies of null genotypes for GSTM1 and GSTT1 in different ethnic groups28. The prevalence of GSTM1 null genotype has been reported to vary between 39-62% in Europeans, 33-63% in East Asians and 23-45% in Africans29. The highest frequencies have been reported in studies involving small number of subjects from parts of the South Pacific i.e. 64-100%30. The prevalence of GSTM1 null genotype has been reported to be 17% and 24% in Indian population from Bombay and Trivandrum regions, respectively31,32. The prevalence of GSTM1 null genotype was 16% in Indians from Malaysia and Singapore, while in Asian Indians from Los Angeles or Malaysia it was 36 and 33% respectively33. The prevalence of GSTMÂ1 null genotype was 33.3% and GSTT1 null genotype was 12.5% in controls from Delhi in a study conducted by Sharma et al34. The frequencies of the GSTM1 null genotypes among controls in the present study were comparable with data that has been reported in various studies from different ethnic groups. We observed the prevalence of 33.3% for GSTM1 null genotype which was relatively lower than in Caucasian populations (50%)35 and higher than Indian population from Bombay (17%) and Trivandrum (24%)31,32. In our study the prevalence of GSTT1 null genotype was found to be 13.3% in Indian population. The presence of GSTT1 null genotype was less in Indian population as compared to GSTM1 null. Our results are comparable to those of others31,32 reported by other studies, 12.3%, 22% for GSTT1 null genotypes in controls. Studies of GSTT1 null genotype from various geographical regions have demonstrated the range of frequencies from 16% to 64% in Asia, 44% or higher in China and Japan28. It has been suggested that in Asian countries the frequency of GSTT1 null is similar to that of GSTM1, whereas in Africans, African-Americans and white populations, the frequency of GSTT1 null genotype is lower than for GSTM1 null genotype. Nair et al32 did not find any subject with homozygous null genotype for both GSTM1 and GSTT1 in 82 controls in Indian population from Trivandrum, suggesting that this combination is rare in Indian ethnic population, but we have found 13.33% subjects with null genotype for both GSTM1 and GSTT1 in controls. Till now, there are not many studies on the role of GSTs in COPD. This is amongst the few report from Indian population on the role of GSTs in COPD.
In the present study, 16.7% of COPD cases were GSTM1 null in comparison to 33.3% in controls which was not statistically significant (p=0.135). A total of 40% of the cases presented homozygous deletion of GSTT1 genotype as compared to controls 13.3% which was statistically significant (p=0.019). An attempt was made to evaluate the proportion of the cases that were null for both genotypes GSTM1 and GSTT1. It was observed that COPD cases had marginally higher proportion of subjects who had the homozygous null genotypes of both GSTM1 and GSTT1 as compared to controls. However, differences were not statistically significant (p=0.35).
This was in contrast to other studies that have been conducted to test for associations of GST polymorphisms and COPD. A weak association has been reported to exist between null GSTM1 and emphysema in combination with lung cancer (OR=2.1)36 and severe chronic bronchitis in heavy smokers (OR=2.8)37 in whites. However, this association between GSTM1 and COPD was not confirmed in a Korean population and no association between GSTT1 and COPD was found in a study conducted by Ishii et al38. In their study the genotypes of 83 patients with COPD and 76 healthy smoking controls were determined by multiplex PCR for GSTM1 and GSTT1 genes. They concluded that the frequency of null genotypes of both GSTM1 and GSTT1 in patients with COPD (34%) was not very different from that of control group (38%).
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. This association could be explained by the fact that tobacco smoke is known to contain multiple substrates for GSTM, GSTT and GSTP1. Individuals having a defective genotype for more than one of these genes would therefore be at greater risk for smoking induced decline in lung function than those having defective genotype for only one gene.
The limitations of the study were related to the fact that controls were younger than the patients with COPD. This could have reduced the effects of polymorphic genotypes of this enzyme in the pathogenesis of COPD as some of the control subjects could be destined to develop COPD in the future. Moreover, only a few genes involved in the detoxification of smoke products were studied.
In summary, we have shown that GSTT1 null genotype might be associated with the pathogenesis of COPD. In terms of the number of subjects examined, this study is a preliminary work and a further study using a larger population is needed to clarify the association of GSTT1 null genotype and individual susceptibility to development of COPD. Furthermore, investigation of the function of other xenobiotic enzymes such as GSTP1 may provide more insight to the pathogenesis of COPD in smokers. Hence, the data presented here indicates that the incidence of homozygous GSTT1 null genotype is significant in COPD cases as compared to controls, but GSTM1 null genotype results showed that this is not a critical factor in COPD development. Further studies are needed to investigate more number of cases and also examine polymorphisms of other detoxifying agents.