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The aetiology of chronic obstructive pulmonary disease (COPD) is complex. While cigarette smoking is a well-established cause of COPD, a myriad of assessed genetic factors has given conflicting data. Since gene-environment interactions are thought to be implicated in aetiopathogenesis of COPD, we aimed to examine the matrix metalloproteinase (MMP) 9 C-1562T (rs3918242) functional variant and cigarette smoke in the pathogenesis of this disease. The distribution of the MMP9 C-1562T variant was analyzed in COPD patients and controls with normal pulmonary function from Serbia. Interaction between the C-1562T genetic variant and cigarette smoking was assessed using a case-control model. The response of the C-1562T promoter variant to cigarette smoke condensate (CSC) exposure was examined using a dual luciferase reporter assay. The frequency of T allele carriers was higher in the COPD group than in smoker controls (38.4% vs. 20%; OR = 2.7, P = 0.027). Interaction between the T allele and cigarette smoking was identified in COPD occurrence (OR = 4.38, P = 0.005) and severity (P = 0.001). A functional analysis of the C-1562T variant demonstrated a dose-dependent and allele-specific response (P < 0.01) to CSC. Significantly higher MMP9 promoter activity following CSC exposure was found for the promoter harboring the T allele compared to the promoter harboring the C allele (P < 0.05). Our study is the first to reveal an interaction between the MMP9-1562T allele and cigarette smoke in COPD, emphasising gene-environment interactions as a possible cause of lung damage in the pathogenesis of COPD. Environ. Mol. Mutagen. 57:447-454, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Aim: Alpha-1-antitrypsin (A1AT) is the main inhibitor of neutrophil elastase, and severe alpha-1-antitrypsin deficiency (A1ATD) is a genetic risk factor for early-onset emphysema. Despite the relatively high prevalence of A1ATD, this condition is frequently underdiagnosed. Our aim was to determine the distribution of the A1ATD phenotypes/alleles in patients with lung diseases as well as in the Serbian population., Methods: The study included the adults with chronic obstructive pulmonary disease (COPD) (n=348), asthma (n=71), and bronchiectasis (n=35); the control was 1435 healthy blood donors. The A1ATD variants were identified by isoelectric focusing or polymerase chain reaction-mediated site-directed mutagenesis., Results: PiMZ heterozygotes, PiZZ homozygotes, and Z allele carriers are associated with significantly higher risk of developing COPD than healthy individuals (odds ratios 3.43, 42.42, and 5.49 respectively). The calculated prevalence of PiZZ, PiMZ, and PiSZ was higher in patients with COPD (1:202, 1:8, and 1:1243) than in the Serbian population (1:5519, 1:38, and 1:5519)., Conclusion: The high prevalence of A1ATD phenotypes/allele in our population has confirmed the necessity of screening for A1ATD in patients with COPD. On the other hand, on the basis of the estimated number of those with A1ATD among the COPD patients, it is possible to assess the diagnostic efficiency of A1ATD in the Serbian population.

Imbalance between neutrophil elastase and alpha-1-antitrypsin (AAT) leads to emphysema in smokers as well as in patients with inherited alpha-1-antitrypsin deficiency. AAT as a proven inhibitor of apoptosis may play role in lung cancer (LC) progression. The aim was to analyse AAT protein variants and polymorphism in promoter region of the neutrophil elastase gene (ELA2) in patients with primary lung cancer. AAT phenotypisation by isoelectric focusing method and ELA2 gene promoter characterization by DNA sequencing were performed in 66 patients with primary lung cancer. Results showed that the frequency of M1 allele and PiM1 homozygotes in LC patients was significantly higher when compared to the healthy subjects (f  = 0.6360 and 0.7424 respectively). The most frequent ELA2 promoter region genotypes in LC patients were -903TT and -741GG. There were significantly more patients with intermediate and high ELA2 genotype activity, compared to those with low activity (91% vs. 9%, respectively). In conclusion, we found that PiM1 homozygosity could be associated with the lung cancer, probably due to increased synthesis of this antiapoptotic protein. Non-MM variants of AAT and ELA2 genotypes with predicted intermediate or high activity could also represent a risk factor for aggressive form of lung cancer associated with extrathoracic metastases.

The alpha-1-antitrypsin (A1AT) gene is highly polymorphic, with more than 100 genetic variants identified of which some can affect A1AT protein concentration and/or function and lead to pulmonary and/or liver disease. This study reports on the characterization of a p.G320R variant found in two patients, one with emphysema and the other with lung cancer. This variant results from a single base-pair substitution in exon 4 of the A1AT gene, and has been characterized as P by isoelectric focusing. Functional evaluation of the A1AT p.G320R variant was through comparing specific trypsin inhibitory activity in two patients with pulmonary disorders, carriers of the p.G320R variant, and 19 healthy individuals, carriers of normal A1AT M variants. Results showed that specific trypsin inhibitory activity was lower in both emphysema (2.45 mU/g) and lung cancer (2.07 mU/g) patients than in carriers of the normal variants (range 2.51-3.71 mU/g). This rare A1AT variant is associated with reduced functional activity of A1AT protein. Considering that it was found in patients with severe pulmonary disorders, this variant could be of clinical significance.

Background: The excess of matrix metalloproteinases (MMPs) might be associated with the airways destruction or dilatation in bronchiectasis. The functional promoter polymorphisms of MMP1 and MMP9 genes, involved in the extracellular matrix remodeling, might increase the expression of MMPs leading to the development of bronchiectasis., Methods: Detection of MMP1 G-1607GG and MMP9 C-1562T gene variants was performed on 37 patients with idiopathic disseminated bronchiectasis and 102 control subjects. We also described a novel method for simple and rapid detection of MMP1 G-1607GG polymorphism., Results: The frequency of -1607GG allele was significantly higher in the group of patients than in control subjects (P = 0.014). The heterozygote genotype showed association with bronchiectasis (odds ratio, 5.3; 95% confidence intervals, 1.4-20.0). The association was even stronger in homozygotes for -1607GG allele (odds ration, 8.7; 95% confidence intervals, 1.9-41.0). The allelic and genotype frequencies of MMP9 C-1562T variant did not show significant differences between the groups., Conclusions: This is the first report concerning a role of MMP1 G-1607GG and MMP9 C-1562T variants in pathogenesis of idiopathic disseminated bronchiectasis. The results of our study revealed the association of -1607GG allele and the lack of association of MMP9 C-1562T variant with the disease.

Chronic obstructive pulmonary disease (COPD) and lung cancer (LC) are a major cause of morbidity and mortality worldwide. In both diseases airways inflammation plays an important role. Functional promoter polymorphism, at the position -308, of tumor necrosis factor (TNF)-alpha represents attractive potential susceptibilty marker for both diseases. In order to investigate the role of this polymorphism in COPD and LC, a case-control study was performed. The patient groups consisted of 97 subjects with COPD and 70 subjects with LC, while the control group encompassed 102 subjects. Results of our study showed significant decrease of heterozygote for TNF-alpha-308 1/2 gene variant in COPD group in comparison to controls (p=0.043). According to our results heterozygous carriers of TNF-alpha-308 1/2 polymorphism had a2.3-fold decreased risk for COPD development (OR=0.44, 95%CI=0.20-0.97). In patients with lung cancer we also observed a trend of decreased distribution of TNF-alpha-308 1/2 heterozygotes, but statistical significance was not achieved. To our knowledge, this is the first study implicating decreased frequency of TNF-alpha-308 1/2 gene variant in patients with COPD and LC. Although these results need to be confirmed on larger cohort, they represent anew and interesting finding, not reported in other populations tested so far.

Chronic obstructive pulmonary disease (COPD) is a complex disease influenced by genetic and environmental factors. Cystic fibrosis transmembrane conductance regulator (CFTR) protein is an important component of the lung tissue homeostasis, involved in the regulation of the rate of mucociliary clearance. As it is known that certain CFTR variants have consequences on the function of CFTR protein, the aim of this study was to examine the possible role of F508del, M470V, Tn locus, and R75Q variants in COPD development and modulation. Total number of 86 COPD patients and 102 control subjects were included in the study. Possible association between COPD susceptibility, severity, and onset of the disease and allele or genotype of four analyzed CFTR variants was examined. No associations were detected between COPD development, onset of the disease and tested CFTR alleles and genotypes. However, VV470 genotype was associated with mild/moderate COPD stages in comparison to severe/very severe ones (OR = 0.29, 95%CI = 0.11-0.80, p = 0.016). Our study showed that patients with VV470 genotype had a 3.4-fold decreased risk for the appearance of severe/very severe COPD symptoms, and the obtained results indicate that this genotype may have a protective role. These results also suggest the importance of studying CFTR gene as a modifier of this disease.

Laboratory diagnosis of alpha-1-antitrypsin (AAT) deficiency is routinely performed by phenotyping methods, which include measurement of serum alpha-1-antitrypsin concentration and isoelectric focusing (IEF). Several DNA-based methods are also used for AAT deficiency testing, but they still have not become part of routine diagnostics. The aim of the study was to identify AAT variants using 2 different methods, isoelectric focusing and denaturing gradient gel electrophoresis (DGGE), and to compare obtained results as well as practical application of these 2 methods. The study has encompassed 27 emphysema patients. In all patients, AAT phenotypization was conducted using IEF, whereas genotypization was performed by DGGE. Variations detected by DGGE were characterized by DNA sequencing. Mutations in the AAT gene were detected in 6 patients. Three patients were homozygous for the Z allele, whereas 1 patient was heterozygous. In 2 patients, novel AAT variants, G320R and V321F, were detected. When results obtained by IEF and DGGE were compared, it was observed that IEF results were inconclusive or misinterpreted in 5 cases (18.5%). Both methods proved to be reliable for detection of the Z alleles, whereas discrepancy existed for M4 allele and rare variants. Therefore, the optimal strategy for diagnostics of AAT deficiency should encompass detection of the most common AAT variants by IEF and screening for the less common variants by DGGE in combination with sequencing.

We performed the complete screening of the CFTR gene in a group of 31 patients with COPD in order to investigate the impact of mutations and polymorphisms in the CFTR gene. The cumulative frequency of CFTR mutations (17.74%) was significantly higher than in our general population (P < 0.0001). The R75Q was significantly overrepresented in COPD patients (8.06%; P = 0.002). In all patients carrying the R75Q chronic bronchitis was a dominant symptom of COPD, and all were homozygous for the V470 allele. These findings suggest that R75Q mutation could be characteristic CFTR variant for COPD patients.