LACK OF ASSOCIATION OF TUMOR NECROSIS FACTOR-α G–308A AND TRANSFORMING GROWTH FACTOR-β1 C–509T POLYMORPHISMS IN PATIENTS WITH DEEP NECK SPACE INFECTIONS
Jevtović-Stoimenov T1, Despotović M1,*, Pešić Z2, Ćosić A2
*Corresponding Author: Milena Despotović, M.D., Department of Biochemistry, Faculty of Medicine, University of Niš, Bulevar dr Zorana Đinđića 81, 18000 Niš, Serbia; Tel.: +381-62-606-036; Fax: +381-18-423-8770; E-mail: milena.despotovic@ymail.com
page: 59

MATERIALS AND METHODS

Patients and Controls. Blood samples were collected from 41 patients admitted at the Department of Maxillo-facial Surgery, Dental Clinic, Niš, Serbia. The patients with deep neck infections were classified into three groups: abscess, phlegmon and others (e.g., cellulitis, suppurative parotitis, etc.), where the particular diagnoses that were included in “others” did not have a sufficient number of patients to perform statistical tests. Forty-four randomly selected healthy individuals, without known acute or chronic disease, were included in the study as a control group. An informed consent was obtained from all participants and the study was approved by the Ethical Committee of the Medical Faculty, University of Niš, Niš, Serbia. At the moment of admission to the hospital, venous blood samples were obtained from the median cubital vein and collected into EDTA vaccutainer tubes. Two-hundred microliters of blood were used for DNA isolation and the rest for biochemical analysis. An automatic hematology analyzer (MEK 6318K; Nihon Kohden, Tokyo, Japan) was used for WBC count determination. The CRP levels were measured using nephelometric immunoassay (Dade Behring, Marburg, Germany). DNA Isolation and Polymerase Chain Reaction Amplification. Genomic DNA was isolated from whole blood samples using QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany). The biallelic polymorphisms within TNF-a and TGF-b1 genes were determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Polymerase chain reaction was performed in a final volume of 25 mL containing 20 ng of DNA, 12.5 mL KAPA2G Fast HotStart ReadyMix (Kapa Biosystems Inc, Boston, MA, USA) and 20 pmol of each primer. Primer sequences used in this study are summarized in the Table 1. The PCR conditions were as follows: initial denaturation at 95°C for 2 min., followed by 35 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds and extension at 72°C for 15 seconds, ending with a final extension at 72°C for 1 min. The PCR products were electrophoresed on a 2.0% agarose gel, stained with ethidium bromide and visualized under UV light. The amplification products were digested using NcoI and HpyF3I restriction enzymes (Fermentas GmbH, St. Leon-Rot, Germany). Restriction enzyme digestion was carried out at 37°C overnight and analyzed by 8.0% polyacrylamide gel electrophoresis (PAGE). The gel was stained with ethidium bromide and visualized under UV light (Figures 1 and 2). The interpretation of the obtained results was performed according to Table 1. Genotype analyses were performed by two independent researchers. After the polymorphic alleles were established to be homozygous, the PCR-RFLP was repeated in order to confirm the obtained results. Statistical Analyses. The allele and genotype frequencies were determined in patients and controls. They were compared with the values predicted by the Hardy-Weinberg equilibrium using the c2 test. The two-tailed Fisher’s test was used when the number of expected cases was small. Genetic risk magnitudes (effect size) were estimated by calculating odds ratios (ORs) with 95% confidence intervals (95% CI). C-reactive protein levels and WBC counts were expressed by a median (25th to 75th percentiles). The correlation of TNF-a -308 and TGF-b1 -509 genotypes with CRP and WBC count values were determined using the Mann-Whitney U test. One-way analysis of variance (ANOVA) was used to compare the mean CRP levels and WBC counts between the groups. Probability values less than 0.05 (p <0.05) were considered statistically significant. Statistical analyses were performed using the SPSS version 13.0 statistical software package (SPSS Inc, Chicago, IL, USA).



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