- Email: [email protected]
Genetic polymorphism of GSTT1 and GSTM1 and susceptibility to chronic obstructive pulmonary disease (COPD)
ICCC 2009 Meeting Abstracts [15] Eller T, Aluoja A, Vasar V, et al. Symptoms of anxiety and depression in Estonian medical students with sleep problems. Depress Anxiety 2006;23(4):250-6. [16] Aniebue PN, Onyema GO. Prevalence of depressive symptoms among Nigerian medical undergraduates. Trop Doct 2008;38(3):157-8. [17] Adomas B, Arune K, Robertas B. Symptoms of anxiety and depression in medical students and in humanities students: relationship with bigfive personality dimensions and vulnerability to stress. Int J Soc Psychiatry 2008;54(6):494-501. [18] Firth J. Levels and sources in medical students. BMJ 1986;292: 1177-80. [19] Chan DW. Depressive symptoms and depressed mood among Chinese medical students in Hong Kong. Compr Psychiatry 1991;32(2): 170-80. [20] Ahmadi J, Kamel M, Galal Ahmed M, et al. Dubai Medical College students' scores on the Beck Depression Inventory. IRCMJ 2008;10(3): 169-72. [21] Chandavarkar U, Azzam A, Mathews CA. Anxiety symptoms and perceived performance in medical students. Depress Anxiety 2006;24 (2):103-11. [22] Tyseen R, Vaglum P, Gronvold NT, et al. Factors in medical school that predict postgraduate mental health problems in need of treatment. A nationwide and longitudinal study. Med Educ 2001; 35:110-20. [23] Toyry S, Räsänen K, Kujala S, et al. Self-reported health, illness, and self-care among Finnish physicians: a national survey. Arch Fam Med 2000;9:1079-85. [24] Rosvold EO, Bjertness E, Alcorta A, et al. Illness behavior among Norwegian physicians. Scand J Public Health 2002;30:125-32.
e7 [25] Tyssen R, Rovik JO, Vaglum P, et al. Helpseeking for mental health problems among young physicians: is it the most ill that seeks help?— A longitudinal and nationwide study. Soc Psychiatry Psychiatr Epidemiol 2004;39:989-93. [26] Chew-Graham CA, Rogers A, Yassin N. “I wouldn't want it on my CV or their records”: medical students' experiences of help-seeking for mental health problems. Med Educ 2003;37:873-80. [27] Hooper C, Meakin R, Jones M. Where students go when they are ill: how medical students access health care. Med Educ 2005;39:588-93. [28] Center C, Davis M, Detre T, et al. Confronting depression and suicide in physicians. JAMA 2003;289(23). [29] Patti E, Acosta J, Chavda A, et al. Prevalence of anxiety and depression among emergency department staff. N Y Med J 2007;2(2). [30] Tjia J, Givens JL, Shea JA. Factors associated with undertreatment of medical student depression. J Am Coll Health 2005;53(5):219-24. [31] Levine RE, Litwins SD, Frye AW. An evaluation of depressed mood in two classes of medical students. Acad Psychiatry 2006;30(3):235-7. [32] Mehmet Aktekin, Taha Karaman, Yesim Yigiter Senol, et al. Anxiety, depression and stressful life events among medical students: a prospective study in Antalya, Turkey. Med Educ 2001;35(1):12-7. [33] Inam SNB, Saqib A, Alam E. Prevalence of anxiety and depression among medical students of private university. J Pak Med Assoc 2003; 53(2):44-7. [34] Smith CK, Peterson DF, Degenhardt BF, et al. Depression, anxiety, and perceived hassles among entering medical students. Psychol Health Med 2007;12(1):31-9.
doi:10.1016/j.jcrc.2009.06.003
Genetic polymorphism of GSTT1 and GSTM1 and susceptibility to chronic obstructive pulmonary disease (COPD) Mona M. Faramawy a , Tasnim O. Mohammed a , Aysheh M. Hossaini b , Reem A. Kashem b , Rania M. Abu Rahma c a
Second-year Medical student of Dubai Medical College for Girls, Dubai, United Arab Emirates Third-year Medical student of Dubai Medical College for Girls, Dubai, United Arab Emirates c Fourth-year Medical student of Dubai Medical College for Girls, Dubai, United Arab Emirates b
Abstract Background: Chronic obstructive pulmonary disease (COPD) represents a major public health care problem worldwide due to its increasing prevalence, morbidity and mortality. Chronic obstructive pulmonary disease is known to be the fourth leading cause of death and the only cause of death, which is increasing. It is generally accepted that cigarette smoking is the most important risk factor for COPD. Nevertheless, only 10% to 20% of chronic smokers develop the severe impairment of pulmonary functions associated with COPD. This indicates the presence of genetic predisposing factors in its pathogenesis. Objective: To test the hypothesis that genetic polymorphism of glutathione S-transferase θ 1 (GSTT1) and/or glutathione S-transferase μ 1 (GSTM1) is associated with COPD in smokers. Materials and Methods: A case-control study was done on 34 patients with COPD and 34 matched controls. DNA was extracted from white blood cells by salting out method. GSTT1 and GSTM1 genotypes were amplified by polymerase chain reaction. The fragments were then analyzed by agarose gel electrophoresis. Statistical analysis was done using SPSS program.
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I. Ahmed et al.
Results: The frequency of carriers of null GSTT1 genotype was 50% among cases compared to 44.1% in the control group. Carriers of null GSTT1 were at minor risk of developing COPD when compared with carriers of the wild GSTT1 genotype (OR, 1.3; 95% CI, 0.5-3.3). In case of GSTM1, the frequency of carriers of null GSTM1 genotype was 52.9% among cases compared to 26.5% in controls. Carriers of null GSTM1 were at much higher risk of developing COPD (OR, 3.13; 95% CI, 1.1-8.6). Furthermore, the risk of developing COPD was increased among carrier of null GSTT1 & GSTM1 haplotype (OR, 3.6; 95% CI, 1.1-11.6). Conclusion: Carriers of null GSTM1 genotype were at high risk of developing COPD especially when they were null GSTT1 and GSTM1 haplotype.
1. Introduction
2. Materials and methods
Chronic obstructive pulmonary disease (COPD) represents a major public health care problem worldwide due to its increasing prevalence, morbidity, and mortality [1]. Although COPD is now the 12th largest burden in the world, it is estimated that it will rise to be the fifth largest burden by 2020 [2]. Chronic obstructive pulmonary disease is known to be the fourth leading cause of death and the only cause of death, which is increasing [3]. Chronic obstructive pulmonary disease is characterized by airflow limitation that is not fully reversible, which usually progresses, together with an abnormal inflammatory response to noxious particles or gases. Patients may have chronic bronchitis, emphysema, small airways diseases, or a combination of these, with or without systemic manifestations of the disease [4]. It is generally accepted that cigarette smoking is the most important risk factor for COPD. Nevertheless, only 10% to 20% of chronic smokers develop the severe impairment of pulmonary functions associated with COPD. This indicates the possible contribution of environmental cofactors to the development of COPD [5]. Another possible reason why only a small proportion of smokers develop COPD might be genetic variation in the enzymes that detoxify cigarette smoke products. These enzymes include mEPHX, GST, and CYP1A1, and this indicates the presence of genetic predisposing factors in its pathogenesis [3]. GSTs are a super family of enzymes involved in the conjugation of a wide range of electrophilic substances with glutathione, thereby, facilitating detoxification and further metabolism and excretion. Among the isoenzymes of GST, the homozygous GSTM1-null genotype has been reported to show some association with the pathogenesis of lung cancer and, especially, emphysema [5]. GSTT1 conjugates glutathione and various potential carcinogens, which are present in cigarette smoke, and its null type mutant has been suggested as a risk factor for many diseases. There is increasing evidence that several genes influence the development of COPD [3].
The study group consisted of 34 patients with 10 or more years of smoking-related COPD recruited from Ain Shams Hospital, Cairo, Egypt. Chronic obstructive pulmonary disease was diagnosed on the basis of medical history, chest radiographic findings, physical examination, and spirometric data, according to American Thoracic Society guidelines [6]. The control group included 34 asymptomatic smokers or ex-smokers with smoking history of 10 or more pack-years without clinical or laboratory evidence of COPD. All control subjects exhibited normal pulmonary function (FEV1/FVCN 70% and FEV1N 80% pred) [7]. Demographic data for cases and control groups are shown in Table 1. Ethical approval and informed consent were obtained.
1.1. Aim To test the hypothesis that genetic polymorphism of GSTT1 and/or GSTM1 is associated with COPD in smokers.
2.1. Blood collection Blood sample from cases and controls were collected by venipuncture, into an EDTA-containing tube.
2.2. DNA extraction DNA was extracted from white blood cells by a salting out method [8]. The DNA purity and concentration were determined by spectrophotometer measurement of absorbance at 260 and 280 nm.
2.3. Analysis of GSTT1 and GSTM1 genotype For genotype analysis, the GSTT1 and GSTM1 were amplified by using multiplex polymerase chain reaction (PCR) protocol as described in the literature[9-11]. Polymerase chain reaction was done on 96-well Amp PCR System 9700 Thermocycler (Applied Biosystems). Primer Table 1
Demographic data of cases and controls
Subject
Control (n = 34)
Case (n = 34)
Mean age ± SD Smoking pack/y FEV1 % pred FEV1/FVC
55.1 ± 7.8 y 35.0 ± 7.4 73.9 ± 2.2 83.5 ± 2.1
54.6 ± 7.7y 35.3 ± 7.0 67.4 ± 7.4 56.4 ± 14.0
ICCC 2009 Meeting Abstracts
e9
3. Results The frequency of carriers of null GSTT1 genotype was 50% among cases in the COPD group compared to 44.1% in the control group. Carriers of null GSTT1 were at minor risk of developing COPD when compared with carriers of the wild GSTT1 genotype (OR, 1.3; 95% CI, 0.5-3.2). In case of GSTM1, the frequency of carriers of null GSTM1 genotype was 52.9% among cases compared to 26.5% in controls. Carriers of null GSTM1 were at much higher risk of developing COPD (OR, 3.1; 95% CI, 1.1-8.6). Furthermore, the risk of developing COPD was increased among carrier of null GSTT1 and GSTM1 haplotype (OR, 3.6; 95% CI, 1.111.6). All results are shown in Table 2. Fig. 1 Patterns of multiplex PCR for GSTM1 and GSTT1 genes. CYP1A1 gene was used as an internal positive control. Lane 2 DNA marker. Lane 5 wild genotype for both. Lanes 1, 3, 6, 7, and 8 represent null genotype for GSTM1 gene and wild for GSTT1. Lane 4 null for both.
sequences and PCR conditions were as follows (oligonucleotides were synthesized by Sigma Aldrich, Germany). For detection of GSTM1 polymorphism, the forward primer was 5′-GAACTCCCTGAAAAGCTAAAGC-3′ and reverse primer was 5′-GTTGGGCTCAAATATACGGTGG-3′. For detection of GSTT1 polymorphism, the forward primer was 5′-TTCGTTACTGGTCCTCACATCTC-3′ and reverse primer was 5′-TCACGGGATCATGGCCAGCA-3′. To confirm that the PCR had worked in subjects homozygous for GSTM1 or GSTT1 gene deletion, one pair of primers was used as an internal control to amplify a 312 bp fragment of CYP1A1 gene: the forward primer was 5′GAACTGCCACTTCAGCTGTCT-3′, and the reverse primer was 5′CAGCTGCATTTGGAAGTGCTC-3′. Multiplex PCR mixture was carried out in a 50-μL reaction volume containing 100 ng of genomic DNA, 0.2 μmol/L of each primer, 0.8 mmol/L dNTPs, 2.0 mmol/L MgCl2 in10% PCR buffer, and 1.5 U of DNA polymerase (Promega, UK). PCR involved an initial 2-minute denaturation at 95°C, 30 cycles of denaturing at 94°C for 1 minute, annealing at 64°C for 1 minute, and extension at 72°C for 1 minute, with a final extension at 72°C for 5 minute. Detection of different genotypes were done by 1.5% agarose gel electrophoresis, the presence of band 480 bp indicates the presence of GSTT1 while the presence of band of 215 bp indicates the presence of GSTM1 (Fig. 1).
2.4. Statistical analysis Statistical analysis was done with SPSS software version 11.0 (SPSS Inc, Chicago, Ill). Difference in genotype prevalence and association between case and control group were assessed by the χ2 test. Odds ratio (OR) and 95% confidence interval (CI) were used to describe the strength of association.
4. Discussion Chronic obstructive pulmonary disease 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[12-14]. Cigarette smoking has been generally accepted as one of the most important environmental factors[12,13,15]. However, only a relatively small proportion of heavy smokers develop COPD, which indicates that there is genetic basis for COPD. Detoxification genes are potential candidates in the susceptibility of patients with COPD. Polymorphisms in these genes alter the metabolism of xenobiotics, such as polycyclic aromatic hydrocarbons present in cigarette smoke [16]. In the present work, we reported our preliminary results from a case-control study, which showed that the frequency of null GSTM1 was more in cases compared to control. And by statistical analysis, it was clear that carrier of null GSTM1 were at much higher risk of developing COPD (OR, 3.1; 95% CI, 1.1-8.6). Three studies were done in the same field,
Table 2
Frequency and odds among cases and control
Genotype
Cases
Control
n = 34
n = 34
OR
95%CI
χ2
GSTM1 Null Present
18 (52.9%) 9 (26.5%) 3.13 1.1-8.6 16 (47.1%) 25 (73.5%)
4.97⁎
GSTT1 Null Present
17 (50.0%) 15 (44.1%) 1.3 17 (50.0%) 19 (55.9%)
0.24
Null GSTT1 and GSTM1 Null both 13(38.2%) 5 (14.7%) 3.6 Other 21 (61.8%) 29 (85.3%) haplotypes ⁎ P b .05.
0.5-3.2
1.1-11.6 4.83⁎
e10 agree with our results [17-19]. They reported similar odds ratio (OR, 2.8 and 2.2 and 2.1, respectively). While 2 other studies did not show a significant result and with much less OR, that of He et al [20] and Gaspar et al [21] (with OR = 0.95 and 0.6, respectively) did. In our study, the analysis of the association of null GSTT1 with COPD revealed that carriers of null GSTT1 were at minor risk of developing COPD (OR, 1.3). These data are compared with other similar analyses in COPD patients in other studies as Cheng [18] et al, He [20] et al, and Gaspar et al [21] with insignificant result and even less OR (0.9, 1.23, and 0.9, respectively). The combined analysis of GSTM1 and GSTT1 polymorphism showed that individuals who were carrier of GSTM1 and GSTT1 haplotype were at much higher risk of developing COPD (OR, 3.6; 95% CI, 1.1-11.6). This agree with results of He and coworkers [20], who reported that carrier of null GSTM1, null GSTT1, and GSTP1 wild-type were at a risk of developing COPD (OR, 2.8; P b .05).
5. Conclusion Our data provide evidence that smokers with null genotype of GSTM1 were more susceptible for developing COPD. Furthermore, they are usually more susceptible for rapid decline in lung function. Novel genes would allow the assessment of new mechanisms and pathways in disease and provide new therapeutic opportunities. At-risk individuals could be identified by screening and strongly advised to abstain from smoking and avoid occupations where there are high loads of environmental dusts.
Acknowledgments This study was done under kind supervision of Dr Neveen Salah El Din (biochemistry department) and Dr Marwa (community medicine department). Thanks to Dr Sahar Elhannan and Mr Khan for their technical support.
References [1] Židzik J, Slabá1 E, Joppa P, et al. GSTM1, GSTT1, and EPHX1 genes and chronic obstructive pulmonary disease. Croat Med J 2008;49: 182-91.
I. Ahmed et al. [2] Ishii T, Matsuse T, Teramoto S, et al. Glutathione S-transferase P1 (GSTP1) polymorphism in patients with chronic obstructive pulmonary disease. Thorax 1999;54:693-6. [3] Yim JJ, Park GY, Lee CT, et al. Morality patterns (1997) preliminary data, United States. MMWR 1996;46:941-8. [4] Wood AM, Stockley RA. The genetics of chronic obstructive pulmonary disease. Respir Res 2006;7:130-44. [5] Cheng SL, Yu CJ, Chen CJ, et al. Genetic polymorphism of epoxide hydrolase and glutathione S-transferase in COPD. Eur Respir J 2004; 23:818-24. [6] Fletcher C, Peto R, Tinker C, et al. The natural history of chronic bronchitis and emphysema: an eight-year follow-up study of working men in London. Oxford: Oxford University Press; 1979. p. 70-105. [7] Wu MC. Study on maximal expiratory flow and volume in Chinese. I. Normal nonsmoking adults. J Formos Med Assoc 1981;80:19-29. [8] Joseph S, David WR, Nina I, et al. J. molecular cloning: rapid isolation of genetic polymorphism in cytochrome p450 2E1 and glutathione Stransferase M1, T1, and P1 on susceptibility to esophageal cancer among high-risk individuals in China. Cancer Epidemiol Biomarkers Prev 2002;9:551-6. [9] Abdel-Rahman SZ, Anwar WA, Abdel-Aal, et al. GSTM1 and GSTT1 genes are potential risk modifiers for bladder cancer. Cancer Detect Prev 1998;22:129-38. [10] Joseph S, David WR. Gel electrophoresis of DNA, 3rd ed., vol. 5. New York: Cold Spring Harbor Laboratory Press; 2001. p. 5.14-7. [11] Devi MR, Srivastava DSL, Mandhani A, et al. Polymorphism of GSTM1 and GSTT1 genes in prostate cancer: a study from north India. Ind J Cancer 2004;41:115-9. [12] Mannino DM, Braman S. The epidemiology and economics of chronic obstructive pulmonary disease. Proc Am Thorac Soc 2007;4:502-6. [13] Teramoto S. COPD pathogenesis from the viewpoint of risk factors. Intern Med 2007;46:77-9. [14] Sandford AJ, Joos L, Pare PD. Genetic risk factors for chronic obstructive pulmonary disease. Curr Opin Pulm Med 2002;8:87-94. [15] Doll R, Peto R, Boreham J, et al. Mortality in relation to smoking: 50 years' observations on male British doctors. BMJ 2004;328:32. [16] Boström CE, Gerde P, Hanberg A, et al. Cancer risk assessment, indicators, and guidelines for polycyclic aromatic hydrocarbons in the ambient air. Environ Health Perspect 2002;11:451-548. [17] Baranova H, Perriot J, Albuisson E, Ivaschenko, et al. Peculiarities of the GSTM1 0/0 genotype in French heavy smokers with various types of chronic bronchitis. Hum Genet 1997;99:822-6. [18] Cheng SL, Yu CJ, Chen CJ, et al. Genetic polymorphism of epioxide hydrolase and glutathione S-transferase in COPD. Eur Respir J 2004; 23:818-24. [19] Harrison DJ, Cantlay AM, Rae. Frequency of glutathione S-transferase M1 deletion in smokers with emphysema and lung cancer. Hum Exp Toxicol 1997;16:356-60. [20] He JQ, Ruan J, Connett JE, et al. Antioxidant gene polymorphisms and susceptibility to a rapid decline in lung function in smokers. Am J Respir Crit Care Med 2002;166:323-8. [21] Gaspar P, Moreira J, Kvitko K. CYP1A1, CYP2E1, GSTM1, GSTT1, GSTP1 and TP53 polymorphisms: do they indicate susceptibility to chronic obstructive pulmonary disease and non-small cell lung cancer. Genet Mol Biol 2004;27:1-10.
doi:10.1016/j.jcrc.2009.06.004
e7 [25] Tyssen R, Rovik JO, Vaglum P, et al. Helpseeking for mental health problems among young physicians: is it the most ill that seeks help?— A longitudinal and nationwide study. Soc Psychiatry Psychiatr Epidemiol 2004;39:989-93. [26] Chew-Graham CA, Rogers A, Yassin N. “I wouldn't want it on my CV or their records”: medical students' experiences of help-seeking for mental health problems. Med Educ 2003;37:873-80. [27] Hooper C, Meakin R, Jones M. Where students go when they are ill: how medical students access health care. Med Educ 2005;39:588-93. [28] Center C, Davis M, Detre T, et al. Confronting depression and suicide in physicians. JAMA 2003;289(23). [29] Patti E, Acosta J, Chavda A, et al. Prevalence of anxiety and depression among emergency department staff. N Y Med J 2007;2(2). [30] Tjia J, Givens JL, Shea JA. Factors associated with undertreatment of medical student depression. J Am Coll Health 2005;53(5):219-24. [31] Levine RE, Litwins SD, Frye AW. An evaluation of depressed mood in two classes of medical students. Acad Psychiatry 2006;30(3):235-7. [32] Mehmet Aktekin, Taha Karaman, Yesim Yigiter Senol, et al. Anxiety, depression and stressful life events among medical students: a prospective study in Antalya, Turkey. Med Educ 2001;35(1):12-7. [33] Inam SNB, Saqib A, Alam E. Prevalence of anxiety and depression among medical students of private university. J Pak Med Assoc 2003; 53(2):44-7. [34] Smith CK, Peterson DF, Degenhardt BF, et al. Depression, anxiety, and perceived hassles among entering medical students. Psychol Health Med 2007;12(1):31-9.
doi:10.1016/j.jcrc.2009.06.003
Genetic polymorphism of GSTT1 and GSTM1 and susceptibility to chronic obstructive pulmonary disease (COPD) Mona M. Faramawy a , Tasnim O. Mohammed a , Aysheh M. Hossaini b , Reem A. Kashem b , Rania M. Abu Rahma c a
Second-year Medical student of Dubai Medical College for Girls, Dubai, United Arab Emirates Third-year Medical student of Dubai Medical College for Girls, Dubai, United Arab Emirates c Fourth-year Medical student of Dubai Medical College for Girls, Dubai, United Arab Emirates b
Abstract Background: Chronic obstructive pulmonary disease (COPD) represents a major public health care problem worldwide due to its increasing prevalence, morbidity and mortality. Chronic obstructive pulmonary disease is known to be the fourth leading cause of death and the only cause of death, which is increasing. It is generally accepted that cigarette smoking is the most important risk factor for COPD. Nevertheless, only 10% to 20% of chronic smokers develop the severe impairment of pulmonary functions associated with COPD. This indicates the presence of genetic predisposing factors in its pathogenesis. Objective: To test the hypothesis that genetic polymorphism of glutathione S-transferase θ 1 (GSTT1) and/or glutathione S-transferase μ 1 (GSTM1) is associated with COPD in smokers. Materials and Methods: A case-control study was done on 34 patients with COPD and 34 matched controls. DNA was extracted from white blood cells by salting out method. GSTT1 and GSTM1 genotypes were amplified by polymerase chain reaction. The fragments were then analyzed by agarose gel electrophoresis. Statistical analysis was done using SPSS program.
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I. Ahmed et al.
Results: The frequency of carriers of null GSTT1 genotype was 50% among cases compared to 44.1% in the control group. Carriers of null GSTT1 were at minor risk of developing COPD when compared with carriers of the wild GSTT1 genotype (OR, 1.3; 95% CI, 0.5-3.3). In case of GSTM1, the frequency of carriers of null GSTM1 genotype was 52.9% among cases compared to 26.5% in controls. Carriers of null GSTM1 were at much higher risk of developing COPD (OR, 3.13; 95% CI, 1.1-8.6). Furthermore, the risk of developing COPD was increased among carrier of null GSTT1 & GSTM1 haplotype (OR, 3.6; 95% CI, 1.1-11.6). Conclusion: Carriers of null GSTM1 genotype were at high risk of developing COPD especially when they were null GSTT1 and GSTM1 haplotype.
1. Introduction
2. Materials and methods
Chronic obstructive pulmonary disease (COPD) represents a major public health care problem worldwide due to its increasing prevalence, morbidity, and mortality [1]. Although COPD is now the 12th largest burden in the world, it is estimated that it will rise to be the fifth largest burden by 2020 [2]. Chronic obstructive pulmonary disease is known to be the fourth leading cause of death and the only cause of death, which is increasing [3]. Chronic obstructive pulmonary disease is characterized by airflow limitation that is not fully reversible, which usually progresses, together with an abnormal inflammatory response to noxious particles or gases. Patients may have chronic bronchitis, emphysema, small airways diseases, or a combination of these, with or without systemic manifestations of the disease [4]. It is generally accepted that cigarette smoking is the most important risk factor for COPD. Nevertheless, only 10% to 20% of chronic smokers develop the severe impairment of pulmonary functions associated with COPD. This indicates the possible contribution of environmental cofactors to the development of COPD [5]. Another possible reason why only a small proportion of smokers develop COPD might be genetic variation in the enzymes that detoxify cigarette smoke products. These enzymes include mEPHX, GST, and CYP1A1, and this indicates the presence of genetic predisposing factors in its pathogenesis [3]. GSTs are a super family of enzymes involved in the conjugation of a wide range of electrophilic substances with glutathione, thereby, facilitating detoxification and further metabolism and excretion. Among the isoenzymes of GST, the homozygous GSTM1-null genotype has been reported to show some association with the pathogenesis of lung cancer and, especially, emphysema [5]. GSTT1 conjugates glutathione and various potential carcinogens, which are present in cigarette smoke, and its null type mutant has been suggested as a risk factor for many diseases. There is increasing evidence that several genes influence the development of COPD [3].
The study group consisted of 34 patients with 10 or more years of smoking-related COPD recruited from Ain Shams Hospital, Cairo, Egypt. Chronic obstructive pulmonary disease was diagnosed on the basis of medical history, chest radiographic findings, physical examination, and spirometric data, according to American Thoracic Society guidelines [6]. The control group included 34 asymptomatic smokers or ex-smokers with smoking history of 10 or more pack-years without clinical or laboratory evidence of COPD. All control subjects exhibited normal pulmonary function (FEV1/FVCN 70% and FEV1N 80% pred) [7]. Demographic data for cases and control groups are shown in Table 1. Ethical approval and informed consent were obtained.
1.1. Aim To test the hypothesis that genetic polymorphism of GSTT1 and/or GSTM1 is associated with COPD in smokers.
2.1. Blood collection Blood sample from cases and controls were collected by venipuncture, into an EDTA-containing tube.
2.2. DNA extraction DNA was extracted from white blood cells by a salting out method [8]. The DNA purity and concentration were determined by spectrophotometer measurement of absorbance at 260 and 280 nm.
2.3. Analysis of GSTT1 and GSTM1 genotype For genotype analysis, the GSTT1 and GSTM1 were amplified by using multiplex polymerase chain reaction (PCR) protocol as described in the literature[9-11]. Polymerase chain reaction was done on 96-well Amp PCR System 9700 Thermocycler (Applied Biosystems). Primer Table 1
Demographic data of cases and controls
Subject
Control (n = 34)
Case (n = 34)
Mean age ± SD Smoking pack/y FEV1 % pred FEV1/FVC
55.1 ± 7.8 y 35.0 ± 7.4 73.9 ± 2.2 83.5 ± 2.1
54.6 ± 7.7y 35.3 ± 7.0 67.4 ± 7.4 56.4 ± 14.0
ICCC 2009 Meeting Abstracts
e9
3. Results The frequency of carriers of null GSTT1 genotype was 50% among cases in the COPD group compared to 44.1% in the control group. Carriers of null GSTT1 were at minor risk of developing COPD when compared with carriers of the wild GSTT1 genotype (OR, 1.3; 95% CI, 0.5-3.2). In case of GSTM1, the frequency of carriers of null GSTM1 genotype was 52.9% among cases compared to 26.5% in controls. Carriers of null GSTM1 were at much higher risk of developing COPD (OR, 3.1; 95% CI, 1.1-8.6). Furthermore, the risk of developing COPD was increased among carrier of null GSTT1 and GSTM1 haplotype (OR, 3.6; 95% CI, 1.111.6). All results are shown in Table 2. Fig. 1 Patterns of multiplex PCR for GSTM1 and GSTT1 genes. CYP1A1 gene was used as an internal positive control. Lane 2 DNA marker. Lane 5 wild genotype for both. Lanes 1, 3, 6, 7, and 8 represent null genotype for GSTM1 gene and wild for GSTT1. Lane 4 null for both.
sequences and PCR conditions were as follows (oligonucleotides were synthesized by Sigma Aldrich, Germany). For detection of GSTM1 polymorphism, the forward primer was 5′-GAACTCCCTGAAAAGCTAAAGC-3′ and reverse primer was 5′-GTTGGGCTCAAATATACGGTGG-3′. For detection of GSTT1 polymorphism, the forward primer was 5′-TTCGTTACTGGTCCTCACATCTC-3′ and reverse primer was 5′-TCACGGGATCATGGCCAGCA-3′. To confirm that the PCR had worked in subjects homozygous for GSTM1 or GSTT1 gene deletion, one pair of primers was used as an internal control to amplify a 312 bp fragment of CYP1A1 gene: the forward primer was 5′GAACTGCCACTTCAGCTGTCT-3′, and the reverse primer was 5′CAGCTGCATTTGGAAGTGCTC-3′. Multiplex PCR mixture was carried out in a 50-μL reaction volume containing 100 ng of genomic DNA, 0.2 μmol/L of each primer, 0.8 mmol/L dNTPs, 2.0 mmol/L MgCl2 in10% PCR buffer, and 1.5 U of DNA polymerase (Promega, UK). PCR involved an initial 2-minute denaturation at 95°C, 30 cycles of denaturing at 94°C for 1 minute, annealing at 64°C for 1 minute, and extension at 72°C for 1 minute, with a final extension at 72°C for 5 minute. Detection of different genotypes were done by 1.5% agarose gel electrophoresis, the presence of band 480 bp indicates the presence of GSTT1 while the presence of band of 215 bp indicates the presence of GSTM1 (Fig. 1).
2.4. Statistical analysis Statistical analysis was done with SPSS software version 11.0 (SPSS Inc, Chicago, Ill). Difference in genotype prevalence and association between case and control group were assessed by the χ2 test. Odds ratio (OR) and 95% confidence interval (CI) were used to describe the strength of association.
4. Discussion Chronic obstructive pulmonary disease 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[12-14]. Cigarette smoking has been generally accepted as one of the most important environmental factors[12,13,15]. However, only a relatively small proportion of heavy smokers develop COPD, which indicates that there is genetic basis for COPD. Detoxification genes are potential candidates in the susceptibility of patients with COPD. Polymorphisms in these genes alter the metabolism of xenobiotics, such as polycyclic aromatic hydrocarbons present in cigarette smoke [16]. In the present work, we reported our preliminary results from a case-control study, which showed that the frequency of null GSTM1 was more in cases compared to control. And by statistical analysis, it was clear that carrier of null GSTM1 were at much higher risk of developing COPD (OR, 3.1; 95% CI, 1.1-8.6). Three studies were done in the same field,
Table 2
Frequency and odds among cases and control
Genotype
Cases
Control
n = 34
n = 34
OR
95%CI
χ2
GSTM1 Null Present
18 (52.9%) 9 (26.5%) 3.13 1.1-8.6 16 (47.1%) 25 (73.5%)
4.97⁎
GSTT1 Null Present
17 (50.0%) 15 (44.1%) 1.3 17 (50.0%) 19 (55.9%)
0.24
Null GSTT1 and GSTM1 Null both 13(38.2%) 5 (14.7%) 3.6 Other 21 (61.8%) 29 (85.3%) haplotypes ⁎ P b .05.
0.5-3.2
1.1-11.6 4.83⁎
e10 agree with our results [17-19]. They reported similar odds ratio (OR, 2.8 and 2.2 and 2.1, respectively). While 2 other studies did not show a significant result and with much less OR, that of He et al [20] and Gaspar et al [21] (with OR = 0.95 and 0.6, respectively) did. In our study, the analysis of the association of null GSTT1 with COPD revealed that carriers of null GSTT1 were at minor risk of developing COPD (OR, 1.3). These data are compared with other similar analyses in COPD patients in other studies as Cheng [18] et al, He [20] et al, and Gaspar et al [21] with insignificant result and even less OR (0.9, 1.23, and 0.9, respectively). The combined analysis of GSTM1 and GSTT1 polymorphism showed that individuals who were carrier of GSTM1 and GSTT1 haplotype were at much higher risk of developing COPD (OR, 3.6; 95% CI, 1.1-11.6). This agree with results of He and coworkers [20], who reported that carrier of null GSTM1, null GSTT1, and GSTP1 wild-type were at a risk of developing COPD (OR, 2.8; P b .05).
5. Conclusion Our data provide evidence that smokers with null genotype of GSTM1 were more susceptible for developing COPD. Furthermore, they are usually more susceptible for rapid decline in lung function. Novel genes would allow the assessment of new mechanisms and pathways in disease and provide new therapeutic opportunities. At-risk individuals could be identified by screening and strongly advised to abstain from smoking and avoid occupations where there are high loads of environmental dusts.
Acknowledgments This study was done under kind supervision of Dr Neveen Salah El Din (biochemistry department) and Dr Marwa (community medicine department). Thanks to Dr Sahar Elhannan and Mr Khan for their technical support.
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doi:10.1016/j.jcrc.2009.06.004