SINGLE NUCLEOTIDE POLYMORPHISMS IN IL-1A
RS1800587, IL-1B RS1143634 AND VITAMIN D RECEPTOR
RS731236 IN STAGE III GRADE B/C PERIODONTITIS
Özturk Özener H.1, Tacal Aslan B.2, Eken B.F.2, Agrali Ö.B.1,
Yildrim H.S.1, Altunok E.Ç.3, Ulucan K.2, Kuru L.1
*Corresponding Author: Assist. Prof. Dr. Hafize Öztürk Özener, Address: Marmara University, Faculty
of Dentistry, Department of Periodontology Basıbuyuk, Maltepe, Istanbul, Turkey. Fax number: +90 216
421 02 21; E-mail: hafize.ozturk@marmara.edu.tr
page: 10
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METHODS
Study Population
The protocol of this cross-sectional study was approved
by the Clinical Studies Ethical Committee of
Faculty of Dentistry, Marmara University on 23.10.2017
with the reference number 2017-143, and registered at
clinicaltrials.gov (identification number NCT04806971).
According to the 2013 revision of the 1975 Helsinki
Declaration, each participant signed an informed consent
form prior to the study. All individuals provided a written
informed consent. The present study recruited 200 individuals
from the Department of Periodontology, Faculty
of Dentistry, Marmara University between January 2018
and September 2019. The study population was made up
of 100 periodontitis patients and 100 periodontally healthy
subjects of Turkish origin.
Inclusion criteria for entry were as follows: previously
untreated periodontitis patients, systemically healthy
study population. Exclusion criteria included smoking
(current and past); presence of systemic disease such as
diabetes, rheumatic fever, kidney or liver diseases, neurologic
deficiencies, immunologic diseases; pregnancy;
received antibiotics within the last 6 months; use of any
medication that may influence periodontium (chronic use
of non-steroidal anti-inflammatory drugs, cyclosporine,
nifedipine, phenytoin, etc.).
Periodontitis group: Stage III, Grade B/C periodontitis
patients were diagnosed on the base of the new classification
criteria [2,24]. Stage III periodontitis patients
enrolled had ≥20 teeth, probing depth (PD) ≥6 mm, clinical
attachment loss ≥5 mm, and bone loss reaching to the
middle third of the root radiographically. Grade B was
assessed by indirect consideration of radiographic bone
loss throughout the most affected tooth in the dentition as
a function of age (% bone loss/age: 0.25-1.0), and Grade
C was evaluated based on the radiographic bone loss in
the most affected tooth in whole dentition as a function of
age (% bone loss/age: >1.0). Subjects in the periodontitis
group were categorized into two subgroups, 51 patients
Grade B and 49 patients Grade C.
Healthy group: The periodontally healthy subjects
with the absence of any sign of clinical inflammation, not having periodontitis history; absence of detectable bone
and/or attachment loss; <10% of sites with bleeding on
probing (BOP); PD ≤3 mm; presence of 28 permanent
teeth without extensive restorations or caries. In addition,
all subjects in the healthy group did not show any local or
systemic pathology.
Periodontal Examination
Periodontal examinations were carried out by the
same periodontist (HOO) using a periodontal probe (PCP
15 UNC, Hu-Friedy, Chicago, USA). Plaque index (PI)
[25] and gingival index (GI) [26] were evaluated at 4 sites,
PD, clinical attachment level (CAL) and BOP at 6 sites
per tooth, excluding third molars. A calibration session
was performed by using 10 subjects (not part of the study
groups) to assess the intra-examiner reliability and full
mouth PD and CAL scores were measured at two separate
sessions, 24 hours apart. The intra-examiner correlation
for PD and CAL measurements were calculated as 92.2%
and 93% reproducibility, respectively.
Genotyping
4–6 ml venous blood samples were collected from
the antecubital fossa of each individual and stored in ethylenediaminetetraacetic
acid (EDTA) vacationer. DNA
was extracted from the blood by using commercially available
kits (Invitrogen, USA), following manufacturer’s
guidelines. Sections of the IL-1A, IL-1B and VDR genes
that contain the SNPs of interest were amplified. All genotyping
assays were validated prior to processing the
study samples. After the validation step, negative controls
(water), positive controls of known genotypes and a
subset of study samples were genotyped in duplicate. All
samples were genotyped for rs1800587, rs1143634 and
rs731236 polymorphisms using Real-Time PCR (Thermofisher
Quantstudio 3, USA) by using a Taqman genotyping
assay (Catalog no: 4362691 Thermofisher, USA).
The 10 μl reaction volume comprised of 5 μl genotyping
master mix (Applied Biosystems, Foster City, CA), 0.5 μl
genotyping assay (Applied Biosystems), 3.5 μl nucleasefree
H2O (Thermofisher, USA), and 1 μl DNA. Reactions
were incubated in a 96-well optical plate for denaturation
at 92°C for 15 s, followed by annealing and extension at
60°C for 1 min (40 cycles).
Statistical Analysis
SPSS software version 25 was used for analyses. The
descriptive statistics were presented using mean and standard
deviation. Frequencies and percentages were used for
categorical data. The Kolmogorov Smirnov test was performed
to investigate to whether the variables are normally
distributed or not. For association analysis between SNPs
and periodontitis susceptibility, various genetic models
were used by SNPassoc. The Mann-Whitney U test was
done to compare the groups, since the variables were not
normally distributed. Chi-Square test and Fisher’s exact
test, where appropriate, was applied for comparison of the
proportions of the groups. The study population obeys the
rule of the Hardy-Weinberg equilibrium law, which was
carried out with a goodness-of-fit χ2. Odds ratios with a
95% confidence interval were calculated for risk factors.
A 5% type-I error level was used to interpret a statistical
significance.
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