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
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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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