|Year : 2015 | Volume
| Issue : 3 | Page : 220-224
A disintegrin and metalloprotease 33 polymorphism association with COPD in long-term tobacco smokers of the ethnic Kashmiri population of India
Sonaullah Shah1, Amir Rashid1, Zaffar A Shah2, Rafi Ahmad Jan1, Umar Hafiz Khan1, Imtiyaz A Bhat2, Suhail Mantoo1, Tajamul H Shah1, Parvaiz A Koul1
1 Department of Internal and Pulmonary Medicine, Sher-i-Kashmir Institute of Medical Sciences, Srinagar, Jammu and Kashmir, India
2 Department of Immunology, Sher-i-Kashmir Institute of Medical Sciences, Srinagar, Jammu and Kashmir, India
|Date of Web Publication||5-May-2015|
Dr. Sonaullah Shah
Department of Internal and Pulmonary Medicine, Sher-i-Kashmir Institute of Medical Sciences, Srinagar - 190 011, Jammu and Kashmir
Source of Support: None of the authors have any declaration of interest to disclose. All the authors have equally contributed in writing up of this manuscript., Conflict of Interest: None
| Abstract|| |
Background: Chronic obstructive pulmonary disease (COPD) is characterized by an interaction of various environmental influences especially cigarette smoking and genetic determinants. The prevalence of this disease is ever increasing and characterization of the genetic determinants of the disease has been undertaken globally. The 'A disintegrin and metalloprotease 33' (ADAM 33) gene is one candidate gene that has been studied. Objective: Our objective was to investigate whether single nucleotide polymorphisms in ADAM33 gene are associated with COPD in long-term tobacco smokers in the ethnic Kashmiri population of northern India. Materials and Methods: This was a randomized case-control study, which included 78 stable COPD (GOLD stage11-IV) patients, who were compared with 77 age- and sex-matched long-term tobacco smokers (>20 pack years) without any evidence of COPD. Polymorphic analysis for three single nucleotide polymorphisms (SNPs), (T1, T2, and Q1) of the ADAM33 gene was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by sequencing. The data were analyzed by descriptive statistics and comparative evaluation was done by parametric/non-parametric tests. Results: The analysis of the T1, T2, and Q1 SNPs, revealed that the frequencies of the T2GG, T1GG, and the Q1AG genotypes were significantly higher in patients with COPD in comparison with the controls (P < 0.001). Similarly, the T1G and T2G allele frequency was higher in the patients than in the controls (p = 0.177 and 0.43, respectively). Conclusion: Three SNPs of the ADAM33 gene were significantly associated with COPD in the Kashmiri population of India. This study establishes the possible role of ADAM33 SNPS in the causation of COPD. Further studies across different geographical areas in the country will unravel the contribution of this gene in the causation of COPD in India.
Keywords: A disintegrin and metalloprotease 33 polymorphism, chronic obstructive pulmonary disease, smoking
|How to cite this article:|
Shah S, Rashid A, Shah ZA, Jan RA, Khan UH, Bhat IA, Mantoo S, Shah TH, Koul PA. A disintegrin and metalloprotease 33 polymorphism association with COPD in long-term tobacco smokers of the ethnic Kashmiri population of India. Lung India 2015;32:220-4
|How to cite this URL:|
Shah S, Rashid A, Shah ZA, Jan RA, Khan UH, Bhat IA, Mantoo S, Shah TH, Koul PA. A disintegrin and metalloprotease 33 polymorphism association with COPD in long-term tobacco smokers of the ethnic Kashmiri population of India. Lung India [serial online] 2015 [cited 2019 Nov 13];32:220-4. Available from: http://www.lungindia.com/text.asp?2015/32/3/220/156222
| Introduction|| |
Chronic obstructive pulmonary disease (COPD) is a common disease, which is characterized by persistent airflow obstruction that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and lungs to noxious particles and gases.  The global burden of disease estimates suggest that COPD will rise from the sixth to the third-most common cause of death by 2020.  COPD is recognized to be a major non-communicable disease in India with huge socioeconomic implications.  In a recent survey the prevalence of chronic bronchitis, considered a surrogate for COPD, in India was estimated to be 3.49%.  However, in a spirometry-based study in Kashmir we showed that the prevalence of COPD was found to be as high as 19% among participants >40 years of age.  Though cigarette smoking has been implicated as a risk factor in upto 80% of the patients,  the occurrence of COPD in non-smoking individuals point towards other factors leading to its genesis. In a recent Burden of Obstructive Lung Disease (BOLD) study in Kashmir,  non-smoking COPD contributed significantly to the total prevalence of COPD among the participants, and even as biomass fuels are considered an important contributor to this population,  the presence of the disorder in non-smoking males (less exposed to biomass fuels) points to other factors that could be responsible for the etiopathogenesis of the disorder in our population. A genetic basis for the development of COPD has been suggested based on its more than chance occurrence in first-degree relatives, who are smokers. ,, Many candidate genes have been suggested to play a role in the pathogenesis of COPD, secondary to their hypothesized roles in triggering proteolytic and inflammatory pathways, because of inhalation of cigarette smoke or other noxious particles. The candidate genes investigated in case-control genetic association studies have included genes involved in protease-antiprotease pathways, oxidant-antioxidant pathways, and inflammatory response to cigarette smoke. ,,, Even as many studies are available that point towards the familial aggregation of COPD, severe alpha-1 antitrypsin (AAT) deficiency remains the only proven genetic risk factor for COPD. 
Ever since the identification of the A-disintegrin and metalloprotease 33 (ADAM 33) gene, by Van Erdgeweigh and co-workers, as a susceptibility gene for asthma and bronchial hyper-responsiveness,  it has been identified as an asthma susceptibility gene in ethnically diverse populations. ,, ADAM 33 is a transmembrane metalloproteinase belonging to a subgroup of a zinc-dependent metalloproteinase super-family comprising of over 30 members, which are structurally very complex.  The gene is expressed by lung fibroblasts and bronchial smooth muscle cells and is involved in cell adhesion, cell fusion, cell signaling, and proteolysis, by releasing various factors,  suggesting its potential role in pulmonary defenses and tissue remodeling. Some studies have linked polymorphisms in ADAM33 to airway hyper-responsiveness and airway inflammation in COPD, and to accelerated lung function decline. , As low lung function is associated with high mortality risk, particularly on account of COPD, it is important to study the genetic aspects, which increase the susceptibility to COPD and lung function decline. 
The present study was undertaken with this background, to ascertain the relationship between ADAM 33 gene polymorphism and COPD in the ethnic Kashmiri population of India, which had a high prevalence of this disease.  This study might give an insight into the genetic basis of this disease and help in exploring newer treatment options in future.
| Materials and Methods|| |
A prospective randomized case-control study was undertaken in the department of Pulmonary Medicine in Sher-i-Kashmir Institute of Medical Sciences, a tertiary care cum referral University Hospital, in Srinagar, Kashmir. The study subjects included 78 randomly selected, consenting diagnosed cases of COPD, who attended the hospital for routine follow-up, over a period of two years. Pulmonary function tests (PFT) were performed according to American Thoracic Society guidelines  and COPD was defined on the basis of a post bronchodilator FEV1/FVC ratio of less than 0.70 and FEV1 <80%. The patients were staged based on the FEV1/FVC ratio according to the GOLD stage.  Patients with age >40 years with GOLD stages II-IV, who had 20 or more pack years of smoking were included in the study.
The control group consisted of 77 healthy age- and sex-matched volunteers, documented to have normal PFTs. Healthy controls with a family history of COPD were excluded from the study. Patients with an expected survival of less than one year secondary to cancer or other chronic disease and patients who did not consent to be the part of the study were excluded. The demographic data, smoking history, examination findings, and basic laboratory data, including complete blood count, kidney function test, liver function tests, blood glucose; electrocardiogram, and chest skiagram were recorded in all participants.
A written informed consent was obtained from all the participants and the study was approved by the Ethical Committee of the Institute.
Genetic analysis was done for three SNPS (T1, T2, and Q1) of the ADAM 33 gene chosen [Figure 1], [Figure 2] and [Figure 3] from the published data showing an association with excessive lung function decline and/or presence of COPD. , DNA extraction was performed according to the manufacturer's protocol for Qiagen DNA extraction kits (Qiagen, Hilden, NRW, Germany). The DNA content was quantified by spectrophotometric absorption. Polymerase chain reaction (PCR) was performed using an iCycler Thermal Cycler (Bio-Rad, Hercules, CA, USA). Amplifications were carried out and the PCR product was amplified using the specific forward and reverse primers. Primers were designed and selected using Primer 3, version 0.4.0 software. The primer sequence and annealing temperatures for the amplification for different SNPs are given in [Table 1]. Genotyping of AD-T1 A > G (2280091), AD-T2 A > G (2280090), and AD-Q1 A > G (612709) genotypes were determined by using the PCR-Restriction Fragment Length Polymorphism (RFLP) method. The enzymes and the digested products are given in [Table 1] and [Table 2].
|Figure 1: RFLP picture of ADAM 33 T1 SNP A/G after restriction digestion with NcoI (3%) agarose gel electrophoresis. Lane 1-100 bp DNA Marker. Lane 2-Blank. Lanes 5, 9 heterozygous AG (400 and 260 bp, 140 bp), Lanes 3, 4, 6, 7, 8 homozygous wild AA (260 bp, 140 bp), Lanes 10 homozygous GG|
Click here to view
|Figure 2: RFLP picture of ADAM 33 T2 SNP A/G after restriction digestion with HpyCH4III (3%) agarose gel electrophoresis. Lane 1: 100 bp DNA Marker. Lanes 6, 7 homozygous GG (312 bp). Lanes 2, 4, 5, 8, 9 heterozygous AG (310 and 198 bp, 112 bp), Lanes 3 homozygous AA (198 bp, 112 bp)|
Click here to view
|Figure 3: RFLP picture of ADAM 33 Q-1 SNP A/G after restriction digestion with BtsCI (20% PAGE) agarose gel electrophoresis. Lane 6: 50 bp DNA Marker. Lanes 3, 5, 7 heterozygous AG (158 and 138 bp, 20 bp), Lane 4 homozygous AA (138 bp, 20 bp), Lanes 1, 2, 8 homozygous GG (158 bp)|
Click here to view
The data were analyzed by using the appropriate statistical tests, that is, descriptive statistics and comparative evaluation by parametric/non-parametric tests. The data was described as mean ± SD. Inter- and intra-group comparisons were done by using the students t-test. A p value of less than 0.05 was considered significant. Data analysis was performed using SPSS version 11.0 version.
| Results|| |
The study consisted of 78 COPD patients who were matched for age and sex with 77 healthy controls. The mean age of the patients was 57.7 ± 8.8 years (range 40-80 years) and that of the controls was: 56.5 ± 9.3 (range 40-78 years). Males constituted 70% in the cases and 71.4% in the controls. There was a predominance of rural patients, who were matched for controls, (71.8% in cases and 76.6% in controls). The duration of smoking (pack years) was also similar in both cases and controls (32.6 ± 8.5 and 32.3 ± 8.0, respectively).
Comparing the genotype and allele frequencies in the two groups, the T2GG genotype, the T1GG genotype, and the Q-1AG genotype were significantly more frequent in the cases than in the controls (P < 0.001). However, T2 AA, T1AA, Q1AA, and Q1GG genotypes were found to be expressed more in the controls [Table 3].
The frequencies of alleles, T1G and T2G were higher in the patient group than in the control group. The frequency of alleles T1A and T2A was significantly higher in controls than in cases [Table 4].
| Discussion|| |
The present case-control study is the first demonstration of an association between ADAM 33 polymorphisms and COPD in the ethnic Kashmiri population of India. In this association study, we genotyped 78 well-characterized COPD cases and 77 healthy controls, who were long-term tobacco smokers for the three SNPs (T1, T2 and Q-1) of the ADAM33 gene. The analysis revealed that the SNPs were significantly associated with COPD patients.
In 2002, Van Erdgeweigh and co-workers mapped the ADAM 33 (A distintegrin and metalloprotease 33)  gene polymorphism, as a suspected gene for asthma/bronchial hyper-responsiveness on chromosome 20p13, and since then, a number of studies have replicated the association of the ADAM 33 polymorphism with asthma in different populations. ,,
In addition to the association between asthma and the ADAM33 polymorphism, different alleles have been characterized, which are associated with an excessive decline in lung function.
In a cohort of 200 asthmatic patients followed over 20 years Jongepier et al. found that rare alleles of S2, T1, and T2 SNPs of ADAM33, were associated with an excessive decline in FEV1.  Simpson et al. also found an association of SNPs, F+1, M+1, T1, and T2 of ADAM 33 and impaired early life lung function in a population-based birth cohort of European children.  Similarly, Van-Dieman in a cohort of 1390 subjects in the general population, found that four SNPs (F+1, S1, S2, T2) were significantly associated with accelerated lung function decline.  Various other studies have also shown that various SNPs in ADAM33 were associated with reduced lung function and COPD included T1, ST+5, T2, Q1, S1, S2, V1, and V4. ,
There are only few studies that have shown a relationship between ADAM 33 gene variation and COPD. In the Dutch general population, Van Diemen and colleagues genotyped SNPs in ADAM 33 and found that the subjects homozygous for the minor alleles of SNPs, S2 and Q1, and heterozygous for SNP S1, have an excessive annual decline in FEV1 compared to the wild-type. They also saw a significantly greater frequency of minor alleles of SNP F+1, S1, S2, and T2 in COPD subjects compared to the entire general population. This was the first study of its kind to show an association between ADAM33 and COPD.  Later, Gosman found an association between SNPs (ST+5, T1, T2, and S2) with airway hyper-responsiveness, higher number of sputum inflammatory cells, and CD8 cells in bronchial biopsies, indicating that the gene was associated with the pathophysiology of COPD.  Sadeghnejad, in his case-control study, also demonstrated the association of SNPs (Q-1, S1, S2, V1, V4) with COPD and lung function decline in long-term smokers, in the Caucasian population.  Similar observations were also made by Wang et al. in the Chinese Han population for four SNPs (T2G, T1G, S2C, and Q-1G alleles (P < 0.001).  In our study, we observed that compared to the wild allele, heterozygous AG and homozygous GG genotypes of T1 SNP, homozygous GG genotype of T2 SNP, and heterozygous AG genotype of the Q-1 SNP were distributed significantly in higher (P < 0.05) frequency among cases when compared to controls; suggesting the increased susceptibility among carriers of these genotypes to develop COPD.
In a recent study, Jun-lung Xiao et al. found an association of four SNPs (V4, T2, T1, and S1) in long-term smoking COPD patients in the Tibetan population of China,  which was again comparable to our study. We also observed that the homozygous AA genotype of the T1 and T2 SNPs and the GG genotype of Q-1 were significantly higher in the controls pointing toward a possible protective role in the development of COPD. We were able to replicate the study by Wang et al. for the association of SNPs with COPD, but the other studies differed in terms of a positive association with all the SNPs. ,, Some studies showed no association of the T1 and T2 SNPs, as was also shown in our study, but they demonstrated this association in other SNPs.  An association of the q-1 SNP of ADAM33 and COPD was shown in these studies. ,,
T1 and T2 SNPs are located in exon 19 (which included an SH3 domain and a phosphorylation site) of the cytoplasmic tail, which could affect signaling. The SNP Q-1 located in the intron immediately before exon 16, containing an epidermal growth factor (EGF) domain,  which is important in lung morphogenesis.
These inconsistencies in replication could be on account of population heterogeneity, as observed by some studies. , Thus, differences in the haplotype structure or even in the occurrence of SNPs may exist between ethnicities, which have not yet been investigated sufficiently for ADAM33, but which are known to exist for a number of other genes.
Differences in the study populations in terms of genes, by unknown environment interactions may also explain some of the observed discrepancies in replication, as has been suggested to be the case with other genes being inconsistently replicated.  In addition, ethnicity-specific differences with environmental or genetic risk factors may account for the observed differences. 
| Conclusion|| |
There are many studies , that show a relationship of asthma with ADAM 33 SNPs, but to date only few studies , have shown an association of COPD with ADAM33. In these studies, some common SNPs are associated with both the diseases, suggesting that they contribute to accelerated airway dysfunction in various disease progressions. However, some specific SNPs are only associated with one disease. This phenomenon is consistent with the pathogenetic mechanisms of the two diseases. At present, the mechanistic roles of the disease-associated SNPs have yet to be elucidated, especially in the context of the pathophysiology of asthma and COPD.
There are some potential limitations of the study, as the number of patients studied was small and we could include only few SNPs in our study. Genome-wide association studies are needed to fully elucidate the potential candidate genes implicated in the genesis of COPD in our population.
| References|| |
Lopez AD, Shibuya K, Rao C, Mathers CD, Hansell AL, Held LS, et al
.Chronic obstructive pulmonary disease: Current burden and future projections. EurRespir J 2006;27:397-412.
Bhome AB. COPD in India: Lceberg or volcano. J Thorac Dis 2012;4:298-309.
Jindal SK, Aggarwal AN, Gupta D, Agarwal R, Kumar R, Kaur T, et al
. Indian study on epidemiology of asthma, respiratory symptoms and chronic bronchitis in adults (INSEARCH). Int J Tuberc Lung Dis 2012;16:1270-7.
Burney P, Jithoo A, Kato B, Janson C, Mannino D, Nizankowska-Mogilnicka E, et al
.;Burden of Obstructive Lung Disease (BOLD) Study. Chronic obstructive pulmonary disease mortality and prevalence: The associations with smoking and poverty--a BOLD analysis. Thorax 2014;69:465-73.
Eisner MD, Anthonisen N, Coultas D, Kuenzli N, Perez-Padilla R, Postma D, et al
.; Committee on Nonsmoking COPD, Environmental and Occupational Health Assembly. An official American Thoracic Society public policy statement: Novel risk factors and the global burden of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2010;182:693-718.
Salvi SS, Barnes PJ. Chronic obstructive pulmonary disease in non-smokers. Lancet 2009;374;733-43.
Silverman EK, Chapman HA, Drazen JM, Weiss ST, Rosner B, Campbell EJ, et al
. Genetic epidemiology of severe, early-onset chronic obstructive pulmonary disease. Risk to relatives for airflow obstruction and chronic bronchitis. Am J Respir Crit Care Med 1998;157:1770-8.
McCloskey SC, Patel BD, Hinchliffe SJ, Reid ED, Wareham NJ, Lomas DA. Siblings of patients with severe chronic obstructive pulmonary disease have a significant risk of airflow obstruction. Am J RespirCrit Care Med 2001;164:1419-24.
Dahl M, Nordestgaard BG, Lange P, Vestbo J, Tybjærg-Hansen A. Molecular diagnosis of intermediate and severe alpha (1)-antitrypsin deficiency: MZ individuals with chronic obstructive pulmonary disease may have lower lung function than MM individuals. ClinChem 2001;47:56-62.
Ohnishi K, Takagi M, Kurokawa Y, Satomi S, Konttiken YT. Matrix metalloproteinase-mediated extracellular matrix protein degradation in human pulmonary emphysema. Lab Invest 1998;78:1077-87.
Yoshikawa M, Hiyama K, Ishioka S, Maeda H, Maeda A, Yamakido M. Microsomal epoxide hydrolase genotypes and chronic obstructive pulmonary disease in Japanese. Int J Mol Med 2000;5:49-53.
Smith CA, Harrison DJ. Association between polymorphism in gene for microsomal epoxide hydrolase and susceptibility to emphysema. Lancet 1997;350:630-3.
Sakao S, Tatsumi K, Igari H, Shino Y, Shirasawa H, Kuriyama T. Association of tumor necrosis factor alpha gene promoter polymorphism with the presence of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001;163:420-2.
Kueppers F, Miller RD, Gordon H, Hepper NG, Offord K. Familial prevalence of chronic obstructive pulmonary disease in a matched pair study. Am J Med 1977;63:336-42.
Van Eerdewegh P, Little RD, Dupuis J, Del Mastro RG, Falls K, Simon J, et al
. Association of the ADAM33 gene with asthma and bronchial hyper responsiveness. Nature 2002;418:426-30.
Hirota T, Hasegawa K, Obara K, Matsuda A, Akahoshi M, Nakashima K, et al
. Association between ADAM33 polymorphisms and adult asthma in the Japanese population. Clin Exp Allergy 2006;36:884-91.
Bijanzadeh M, Ramachandra NB, Mahesh PA, Mysore RS, Kumar P, Manjunath BS, et al
. Association of IL-4 and ADAM33 gene polymorphisms with asthma in an Indian population. Lung 2010;188:415-22.
Howard TD, Postma DS, Jongepier H, Moore WC, Koppelman GH, Zheng SL, et al
. Association of ADisintegrin and Metalloprotease 33 (ADAM33) gene with asthma in ethnically diverse populations. J Allergy ClinImmunol 2003;112:717-22.
Edwards DR, Handsley MM, Pennington CJ. The ADAM metalloproteinases. Mol Aspects Med 2008;29:258-89.
Holloway JW, Laxton RC, Rose-Zerilli MJ, Holloway JA, Andrews AL, Riaz Z, et al
. ADAM33 expression in atherosclerotic lesions and relationship of ADAM33 gene variation with atherosclerosis. Atherosclerosis 2010;211:224-30.
Gosman MM, Boezen HM, van Diemen CC, Snoeck-Stroband JB, Lapperre TS, Hiemstra PS, et al
. A disintegrin and metalloprotease 33 and chronic obstructive pulmonary disease pathophysiology. Thorax 2007;62:242-7.
van Diemen CC, Postma DS, Vonk JM, Bruinenberg M, Schouten JP, Boezen HM et al
. A disintegrin and metalloprotease 33 polymorphisms and lung function decline in the general population. Am J Respir Crit Care Med 2005;172:329-33.
Standardization of spirometry, 1994 update. American Thoracic Society. Am J Respir Crit Care Med 1995;152:1107-36.
Global initiative for Chronic Obstructive Lung Disease (GOLD):Global strategy for Diagnosis, Management and Prevention of COPD, Updated 2008. Available from: http://www.goldcopd.org
. [Last accessed on 2014 Nov 11].
Jongepier H, Boezen HM, Dijkstra A, Howard TD, Vonk JM, Koppelman GH, et al
. Polymorphisms of ADAM 33 gene are associated with accelerated lung function decline in asthma. Clin Exp Allergy 2004,34:757-60.
Lee JH, Park HS, Park SW, Jang AS, Uh ST, Rhim T, et al
. ADAM33 polymorphism: Association with bronchial hyper-responsiveness in Korean Asthmatics. Clin Exp Allergy 2004;34:860-5.
Simpson A, Maniatis N, Jury F, Cakebread JA, Lowe LA, Holgate ST, et al
. Polymorphisms in adisintegrin and metalloprotease 33 (ADAM33) predict impaired early-life lung function.Am J RespirCrit Care Med 2005;172:55-60.
Sadeghnejad A, Ohar JA, Zheng SL, Sterling DA, Hawkins GA, Meyers DA, et al
. ADAM33 polymorphisms are associated with COPD and lung function in long-term tobacco smokers. Respir Res 2009;10:21.
Wang X, Li L, Xiao J, Jin C, Huang K, Kang X, et al
. Association of ADAM33 gene polymorphisms with COPD in a northeastern Chinese population. BMC Med Genet 2009;10:132.
Xiao J, Han J, Wang X, Hua D, Su D, Bao Y, et al
. Association of ADAM33 gene with susceptibility to COPD in Tibetan population of China. MolBiol Rep 2011;38:4941-5.
Yoshinaka T, Nishii K, Yamada K, Sawada H, Nishiwaki E, Smith K, et al
. Identification and characterization of novel mouse and human ADAM33s with potential metalloprotease activity. Gene 2002;282:227-36.
Tan J, Liu AP, Sun C, Bai YF, Lv F.Association of ADAM33 gene polymorphisms with COPD in the Mongolian population of China. Ann Hum Biol 2014;41:9-14.
Vercelli D. Genetics, epigenetics, and the environment: Switching, buffering, releasing.J Allergy ClinImmunol 2004;113:381-87.
Burchard EG, Ziv E, Coyle N, Gomez SL, Tang H, Karter AJ, et al
. The importance of race and ethnic background in biomedical research and clinical practice. N Engl J Med 2003;348:1170-5.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]