The Effects Of Smoking And Genes On Coad Biology Essay

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Most important risk factor is the cigarette smoking which is 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 51.67 8.9 and in the control group was 51.40  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  4.754 whereas it was only 10.27  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).

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.

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 & heavy smokers having severe chronic bronchitis (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.

It could be accepted without any doubt that cigarette smoking is the major risk factor for COPD and genetic susceptibility is also there for the complicated disorder26of gene. Protease-antiprotease and oxidant-antioxidant are the potential susceptible genes. Instead of having lots of experimental studies in animal beings as well as in human beings which is supporting the role of imbalanced oxidant- antioxidant systems for the pathogenesis of COPD27 resulting from smokingbut few relative studies are there which have been done to investigate the role of polymorphisms of antioxidant genes with COPD For this current study ,polymorphisms of GSTM1 and GSTT1 antioxident genes have been investigated by us to know whether they 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 GSTM­1 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 was conducted for testing of association of COPD and GST polymorphism. The association was weak which was reported to exit between emphysema and null GSTM1 in the combination with the lung cancer (OR-2.1))36 and chronic bronchitis which was severe in nature with heavy smokersOR=2.8)37 among whites population.. But among the Korean population, there was no confirmation about the association between GSTM1 and COPD and could not found any association between GSTM1 and COPD in the 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 case of complicated polygenic disease like COPD, there is a possibility of genetic susceptibility which is dependent on the activity of various genepolymorphisms operating in concert.Polymorphisms in case of individual gene might impart a relatively low risk of COPD and that is the possibility that the cumulative effect of more polymorphisms would be important in it's pathogenesis. . The explanation of the association could be done by the fact of tobacco smoking which is known to have many substraces for GSTM, GSTT and GSTP1. GSTM, GSTT and GSTP1. There will be greater risk of smoke induced decline of function of the lung in comparative with them who is only having one defective genotype.for those individuals who have more than one defective genotype

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.

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