ASSOCIATION OF THE MMP7 –181A>G PROMOTER POLYMORPHISM WITH EARLY ONSET OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE
Tacheva T, Dimov D, Anastasov A, Zhelyazkova Y, Kurzawski M, Gulubova M, Drozdzik M, Vlaykova T
*Corresponding Author: Assistant Professor Tanya Tacheva, Department of Chemistry and Biochemistry, Medical Faculty, Trakia University, 11 Armeiska Str., Stara Zagora, Bulgaria. Tel: +359878334176. E-mail: tanya.ta4eva@abv.bg
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MATERIAL AND METHODS

Patients and Controls. We have genotyped 191 patients with COPD and 215 healthy volunteers or individuals unaffected by lung or cancer diseases. The inclusion criteria for COPD were as follows: age higher than 40 years; forced expiratory volume in one second (FEV1) of <80.0%; forced expiratory volume in one second (FEV1)/ forced vital capacity (FVC) ratio of ≤70.0%; FEV1 reversibility after inhalation of 400 μg Salbutamol of <12.0%. In both groups, the age of inclusion in the study and smoking status were noted; in the patients’ group: age of diagnosis, the spirometric indexes, duration and the stages of the disease (GOLD stages) were also reported. The available demographic and clinical data are presented in Table 1. Informed consent was obtained from patients and controls before the beginning of the study. DNA Isolation and Genotyping. Genomic DNA was isolated from 0.2 mL of whole blood using a commercial kit for isolation of genomic DNA from blood (GenElute™ Mammalian Genomic DNA Miniprep Kit, Sigma-Aldrich, St. Louis, MO, USA). The genotyping for the MMP7 –181A>G (rs11568818) was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based method. The final volume of each reaction was 15 μL, containing 0.5 U Dream Taq Polymerase (Fermentas, Waltham, MA, USA), 1.5 μL 10 × PCR buffer (with 1.5 mM MgCl2), 0.6 μL dNTPs (Sigma-Aldrich) in a final concentration of 200 μM for each of the four dNTPs, 0.3 μL of each primer in concentration of 20 pmol/μL (MMP7F: 5’-TGG TAC CAT AAT GTC CTG AAT G-3’; MMP7R: 5’-TCG TTA TTG GCA GGA AGC ACA CAA TGA ATT- 3’) and distilled water to the end volume. The temperature profile of the PCR reactions included primary denaturing of the template DNA for 3 min. at 94°C, followed by 30 cycles of denaturation for 30 seconds at 94°C, annealing for 30 seconds at 53.6°C and poly-merization for 30 seconds at 72°C. The PCR reaction was terminated by a final extension for 5 min. at 72°C. The restriction reaction was performed with 5U EcoRI in final volume of 5 μL for 16 hours at 37°C. The obtained restriction products were analyzed by 4.0% agarose gel stained with ethidium bromide. The results were documented by the Gel documentation system (Syngene; Synoptics Ltd., Cambridge, Cambridgeshire, UK). Statistical Analyses. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS), version 16.0 for Windows (SPSS Inc., Chicago, IL, USA). Continuous variables were analyzed for normality of the distribution using the Kolmogorov-Smirnov test (One-Sample Kolmogorov-Smirnov D-Test in SPSS, version 16; SPSS Inc.). When the level of signif-icance in this test was lower than 0.05 (p <0.05), the hypothesis for normal distribution was rejected. The continuous variables with normal distribution were com-pared between two or more independent groups by the Student t-test or one-way analysis of variance (ANOVA) test with least significant difference (LSD) post hoc ana-lysis, while those with an abnormal distribution were analyzed with the Mann-Whitney U or Kruskal-Wallis tests. The frequencies of distribution in the contingency tables were analyzed using χ2 test, or Fisher’s exact test, when needed. The odds ratio (OR) and 95% confidence interval (95% CI) were calculated by binary logistic regression with age and sex as covariates. The Hardy-Weinberg equilibrium (HWE) was calculated by an inter-active calculation tool for χ2 tests of goodness of fit and independence [24]. Factors with a p value of <0.05 were considered to be statistically significant.



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