Keywords
genetic polymorphisms - interferon-gamma - periodontitis
Introduction
Periodontitis is a multifactorial disease initiated by dental plaque. The bacterial
agents in the dental plaque induce inflammatory reactions of the host immune system,
leading to gingival inflammation that can further escalate to loss of dental attachment
and finally loss of mandibular bone in untreated patients. Periodontitis is common,
with a prevalence of 74.1% in Indonesia[1] and 40.0 to 77.5% worldwide in the adult population.[2]
[3]
[4]
[5]
The genetic status of the host has an important effect on the pathogenesis of periodontitis.
Genetic factors such as polymorphisms can stimulate or retard the production of specific
cytokines.[6] Previous studies have indicated pro- or anti-inflammatory polymorphisms in cytokine
genes such as tumor necrosis factor-α and interleukin 1β (IL-1β),[7] IL-6,[8] transforming growth factor-β,[9] IL-10,[10]
[11] and interferon-gamma (IFNg),[12]
[13] also concerning periodontitis.
IFNg is one of the key cytokines regulating immune reactions. IFNg is secreted by
CD4+ Th1 cells, CD8 cytotoxic cells, activated natural killer cells, and macrophages.[13] The level of IFNg is elevated in diseased periodontal tissue and related to the
severity of periodontitis.[14]
[15]
The IFNg gene is located at 12q15 and contains 4 exons.[16] Single nucleotide polymorphism at +874A/T (rs2430561) at the end of 5′-CA is repeated
at first intron related to changes of IFNg expression.[17] The allele of +874T is linked to the 12 CA repeats, and the A allele is connected
to the non-12 CA repeats.[18] The T allele-specific sequence binding to the transcription factor of nuclear factor
kappa B (NFkB). NFkB induces IFNg expression, so the +874 T allele is associated with
high IFNg expression.[19] A study in mice with the disruption of IFNg presented migration inflammatory cell
and receptor activator of nuclear factor K β-ligand levels in periodontium significantly
decreased in bone resorption compared with wild-type mice.[20] Another study has found that the production of IFNg in human peripheral blood CD4+
T cells produces IFNg as a response to IL-12 and IL-18, but also in the absence of
any antigenic stimulation.[21]
This study aims to investigate the IFNg +874A/T polymorphic genotypes in Indonesian
subjects, comparing patients with severe and mild periodontitis and the level of IFNg.
Materials and Methods
Patient Selection
All subjects with or without periodontitis were collected from three subdistricts
in central Jakarta (n = 100) from June 2018 to March 2019. The study was approved by the Research Ethics
Committee of the Faculty of Dentistry Universitas Indonesia, protocol number 070390418.
The inclusion criteria were male and female ≥18 years old, at least with 14 remaining
teeth, in good general health. Exclusion criteria were current pregnancy and lactation,
previous periodontal treatment within 6 months, use of antibiotics or immunosuppressant
medication within the last 3 months, and inability or unwillingness to provide informed
consent. All subjects provided written informed consent.
Clinical Examination
The diagnosis of periodontitis was based on an intraoral examination, bleeding on
probing, probing depth, assessments of clinical attachment loss using University of
North Carolina-15 probe (UNC-15 periodontal probe) Hu-Friedy (Chicago, IL, United
States), tooth mobility, number of remaining teeth in all fully erupted teeth except
third molars. The clinical parameters were examined by calibrated periodontists. Probing
depth was measured from the gingival margin to the bottom of the pocket, and clinical
attachment loss measured from the cementoenamel junction to the bottom of periodontal
pocket assessed in six sites (distobuccal, buccal, mesiobuccal, mesiopalatal/lingual,
palatal/lingual, distopalatal/lingual). The diagnostic criteria were based on the
severity of periodontitis by the 2017 World Workshop on the Classification of Periodontal
and Peri-implant Diseases and Conditions staging.[22] The case and control selection were based on clinical attachment loss ≥ 5 mm with
probing depth ≥ 6 mm as severe periodontitis (periodontitis stage 3 and stage 4) and
mild periodontitis when clinical attachment loss < 5 mm with probing depth < 6 mm
(periodontitis stage 1 and stage 2).
Subgingival Sample Collection
IFN-g was retrieved from the gingival crevicular fluid by sterile paper point (International
Organization for Standardization [ISO] 30, Roeko, Langenau, Germany). Each subject
used four paper points in four regions in the deepest pocket and randomly in the healthy
periodontal sulcus. The supragingival plaque was gently cleaned before sampling with
sterile cotton pellets and isolated with cotton rolls. The paper point was inserted
gently to the bottom of the selected pocket for 30 seconds All four paper points were
pooled together in one tube with 1 mL phosphate buffer saline and transferred to the
laboratory. In the laboratory, the samples were centrifuged for ~20 minutes at 1,000 g
(or 3000 rpm) ~30 minutes after collection. The supernatant was collected carefully,
and samples were stored immediately at–20°C.
Blood Sample Collection and DNA Extraction
Peripheral blood sample collected with vacutainer with ethylenediaminetetraacetic
acid ~3 mL by phlebotomists at the time of periodontal examination. The blood was
stored in −20°C until deoxyribonucleic acid (DNA) extraction. The genomic DNA was
extracted using standard proteinase K digestion and salt purification method.[23]
Interferon-Gamma Detection
IFNg was detected by enzyme-linked immunosorbent assay kit (MyBioSource, San Diego,
California, United States) according to the manufacturer's instructions. Briefly,
50 μL of standard reagent was added to each standard well and 50 μL of the sample
to each sample well. Adding sample diluent 50 μL to each black/control well, 20% of
the samples were duplicated. The Horseradish peroxidase-conjugate reagent was added
100 μL to each well, then covered with a closure plate membrane and incubated for
60 minutes at 37°C. The plate was then washed four times. Chromogen solution was added
to each well, then protected from light and incubated for 15 minutes at 37°C. After
adding stop solution of 50 μL to each well, the color in the wells changed from blue
to yellow. Finally, the optical density was read using Universal Microplate Reader
(Sunrise, Tecan, Austria) at 450 nm within 5 to 15 minutes after adding the stop solution.
The IFNg level was determined by comparing it with the standard curve. The detection
range was from 31.2to 1,000 pg/mL, but the lowest standard diluted twice so the final
detection range was from 7.8 to 1,000 pg/mL. The detected samples of more than 1,000
pg/mL or out of range were diluted and re-examined. The final measurement was multiplied
by the number of dilutions.
Polymorphism Detection
The genomic DNA was extracted from peripheral blood leucocytes. IFNg +874A/T polymorphism
was evaluated by restriction fragment length polymorphism-polymerase chain reaction
(RFLP-PCR) methods, described previously by Zambon et al.[24] The IFNgA/T was amplified using forward primer: 5′-GATTTTATTCTTACAACACAAAATCAAGAC-3′
and reverse primer: 5′-GCAAAGCCACCCCACTATAA-3′. PCR products (176 bp) were digested
with the HinfI enzyme at 37°C for 1 hour and 65°C for 20 minutes. The restriction
fragment separation (176 bp for allele A, 148 bp, and 28 bp for allele T) was by electrophoresis
at 70V, 400mA for 40 minutes on 1.5% agarose gel and staining with ethidium bromide.
Statistical Analysis
IFNg allele frequencies were tested with Hardy–Weinberg equilibrium for both groups
(patients and controls) using the chi-squared test. The significant association between
periodontitis and IFNg genotypes was made using the chi-squared test and Fisher's
exact test. The relative risk of periodontitis associated with a particular genotype
was estimated by the odds ratio (OR). The clinical parameters of periodontitis, comparison
between IFNg level and periodontitis, comparison between IFNg level and IFNg genotypes
were analyzed by Mann–Whitney U or Independent t-test. Kruskal–Wallis test was used to assess a correlation between polymorphs of
IFNg +874A/T and the levels of IFNg. All statistical tests were performed using the
SPSS software (IBM version 23, SPSS, Armonk, New York, United States). The two different
groups were tested for significance by two-tailed tests. A p-value p < 0.05 was considered statistically significant.
Results
A total 100 subjects were included in the study with demographic data presented in
[Table 1]. The subjects with severe periodontitis have a higher median age than the subjects
with mild periodontitis (47 [17] and 25 [15], respectively). The clinical parameters
of periodontitis are shown in [Table 2]. All parameters except the oral hygiene index showed statistically significant differences
between patients with mild and severe periodontitis (p < 0.05).
Table 1
Demographic data and periodontal status of the subjects
|
Variables
|
Mild periodontitis
(n = 43) n (%)
|
Severe periodontitis (n = 57) n (%)
|
p-Value
|
|
Age, median (IQR)
|
25 (15)
|
47 (17)
|
0.000
|
|
Sex (P)
|
|
Male
|
39 (57.4)
|
29 (42.6)
|
0.000
|
|
Female
|
4 (12.5)
|
28 (87.5)
|
|
|
BMI (mean ± SD)
|
24.04 ± 4.87
|
25.80 ± 4.04
|
0.051
|
|
Education
|
0.055
|
|
Above high school
|
9 (64.3)
|
5 (35.7)
|
|
|
High school
|
29 (44.6)
|
36 (55.4)
|
|
|
Below high school
|
5 (23.8)
|
16 (76.2)
|
|
|
Occupation
|
0.113
|
|
Working
|
33 (49.3)
|
34 (50.7)
|
|
|
Not working
|
10 (30.3)
|
23 (69.7)
|
|
|
Smoking status
|
0.050
|
|
Nonsmoker
|
21 (34.4)
|
40 (65.6)
|
|
|
Smoker
|
22 (56.4)
|
17 (43.6)
|
|
Abbreviations: BMI, body mass index; IQR, interquartile range; SD, standard deviation.
Note: Chi-square test.
Table 2
Clinical parameters of periodontitis
|
Mild periodontitis (n = 43)
|
Severe periodontitis (n = 57)
|
p-Value
|
|
Number of teeth[a]
|
26.30 ± 2.18
|
23.98 ± 3.568
|
0.001
|
|
Plaque index[a]
|
1.12 ± 0.439
|
1.32 ± 0.390
|
0.018
|
|
Papilla bleeding index[a]
|
1.06 ± 0.608
|
1.51 ± 0.751
|
0.001
|
|
Oral hygiene index[a]
|
2.15 ± 0.830
|
2.47 ± 0.824
|
0.061
|
|
Pocket depth[a]
|
1.59 ± 0.292
|
2.04 ± 0.515
|
0.000
|
|
Clinical attachment loss[a]
|
1.70 ± 0.348
|
2.80 ± 0.774
|
0.000
|
|
Tooth mobility[a]
|
0.00 ± 0.000
|
1.45 ± 2.80
|
0.000
|
a Mann–Whitney U test.
b Independent t-test.
The agarose gel electrophoresis results of the RFLP analysis for IFNg +874A/T polymorphism
are shown in [Fig. 1]. The expected fragment sizes are for genotype AA 176 bp; for TT 148 bp and 28 bp;
and for AT 176 bp, 148 bp, and 28 bp.
Fig. 1 Agarose gel electrophoresis of the restriction fragment length polymorphism analysis
for interferon-gamma874 polymorphism: lane 1 shows a 50 bp ladder, lanes 2–5 are samples.
The restriction fragment length was 176 bp for allele A, 148 and 28 bp for allele
T.
The chi-squared test showed that genotype frequencies of IFNg polymorphism were in
Hardy–Weinberg equilibrium (p > 0.05).
The distribution of the IFNg genotypes and allele frequencies for patient and control
groups is shown in [Table 3]. There were no significant differences between genotypes and alleles of the IFNg
+874A/T polymorphism between patients with mild and severe periodontitis (p > 0.05).
Table 3
Distribution of genotypic and allelic frequencies IFNg (+874A/T) in periodontitis
|
Mild periodontitis, n = 43 (%)
|
Severe periodontitis, n = 57 (%)
|
p-Value
|
OR (95% CI)
|
|
Genotype
|
|
AA
|
19 (44.2)
|
20 (35.1)
|
|
1
|
|
AT
|
20 (46.5)
|
31 (54.4)
|
0.37
|
1.47 (0.63–3.42)
|
|
TT
|
4 (9.3)
|
6 (10.5)
|
0.62
|
1.43 (0.35–5.85)
|
|
Allele
|
|
A
|
58 (67.44)
|
71 (62.28)
|
|
1
|
|
T
|
28 (32.56)
|
43 (37.72)
|
0.45
|
1.26 (0.69–2.26)
|
|
Dominant
|
|
AA
|
19 (44.2)
|
20 (35.1)
|
|
1
|
|
AT + TT
|
24 (55.8)
|
37 (64.9)
|
0.36
|
0.68 (0.30 - 1.54)
|
Abbreviations: CI, confidence interval; IFNg, interferon-gamma; OR, odds ratio.
Note: Chi-squared test.
The comparison level of IFNg in mild and severe periodontitis is presented in [Table 4]. [Fig. 2] illustrates the association between genotype IFNg +874A/T and IFNg level, p = 0.155 or no statistically different p > 0.05. Categoric dominant genotype (AA vs. AT + TT) also not showed an association
with the level of IFNg (p > 0.05) as seen from [Table 5].
Table 4
Level of IFNg in mild and severe periodontitis as measured by ELISA
|
Interferon-gamma
|
Mild periodontitis pg/mL (n = 43)
|
Severe periodontitis pg/mL (n = 57)
|
Detection range pg/mL
|
p-Value[a]
|
|
Mean ± SD
|
229.76 ± 189.15
|
322.92 ± 289.02
|
15.6–1,000
|
0.255
|
|
Median (IQR)
|
158.46 (250.40)
|
215.50 (541.64)
|
|
|
Abbreviations: ELISA, enzyme-linked immunosorbent assay; IQR, interquartile range;
SD, standard deviation.
a Mann–Whitney U test.
Table 5
Relationship between dominant genotypes of IFNg +874A/T and IFNg level
|
Dominant genotype
|
Median (min–max) (pg/mL)
|
p-Value[a]
|
|
Dominant
|
|
0.623
|
|
AA (n = 39)
|
166.8 (27.6–952.3)
|
|
|
AT and TT (n = 61)
|
190.3 (29.9–950.0)
|
|
|
Recessive
|
|
0.054
|
|
TT (n = 10)
|
471.2 (33.1–885.8)
|
|
|
AA and AT (n = 90)
|
165.9 (27.6–952.3)
|
|
Abbreviation: IFNg, interferon-gamma.
a Mann–Whitney U test; detection range 15.6–1000 pg/mL.
Fig. 2 Association between genotypes of interferon-gamma (IFNg) and IFNg level (pg/mL),
p > 0.05.
Discussion
Periodontitis is a complex multifactorial disease that typically has a relatively
mild phenotype, slowly progressing and chronic in nature. Periodontitis contributed
to the global burden of disease with a prevalence of 25.4% and manifest in many systemic
diseases.[25]
[26] Genetic and environmental factors affect individual phenotypes in complex diseases.
The genetic polymorphism in some situations may cause a change in protein that leads
to a change in innate and adaptive immunity.[27] Some single nucleotide polymorphisms were associated with severe chronic periodontitis.[11] Meta-analysis study about periodontitis and IFNg +874A/T showed the inconsistency
of the results of +874A/T polymorphism and periodontitis risk that may be attributed
to several factors such as race, type of periodontitis, study design, and environmental
factors. The limited study about +874A/T polymorphism makes the research in the Asian
population important to map the polymorphism accurately.[28]
In this study, there was no association between IFNg level and +874A/T polymorphism
which in line with previous studies found no significant correlation for alleles and
genotypes between periodontitis and controls.[6]
[8]
[9]
[13]
[29] On the contrary, Heidari et al showed a significant association for IFNg +874A/T
polymorphism between patients with chronic periodontitis and controls.[12] The IFNg +874A/T polymorphism showed different distribution in Asia. Allele A showed
low frequency in periodontitis patients,[6] while in this study we found allele T in low frequency.
IFNg has an essential role as a proinflammatory agent, which influences the initiation
and progression of periodontal disease. The T-allele of the IFNg +874A/T polymorphism
found increased production of this cytokine. This finding was confirmed in a previous
study where the AA group was showing significantly lower production of IFNg than the
AT group (p < 0.05).[30]
Nonsignificant associations may arise because genetic variation may be a risk factor
for a disease in one population but not in some other populations. The genotype and
allele frequencies are varying between ethnically and geographically distinct populations.[31]
Conclusions
IFNg +874A/T polymorphism was not significantly associated with the IFNg level of
the gingival crevicular fluid of the tested periodontitis patients. There was also
no significant association between the polymorphic genotypes and the severity of periodontitis
when categorized as mild or severe.
