ARTICLE
Auteur(s) : Nívea Maria
de Freitas1, Ana Vitória Imbronito1,
Adriana Costa Neves2, Fábio Daumas Nunes3,
Francisco Emilio Pustiglioni1, Roberto Fraga Moreira
Lotufo1
1Department of Periodontology, Faculty of Odontology,
University of São Paulo, Brazil
2Butantan Institute, São Paulo, Brazil
3Department of Oral Pathology, Faculty of Odontology,
University of São Paulo, Brazil
accepté le 27 Juillet 2007
Aggressive periodontitis (AP) comprises a group of rare periodontal
diseases characterized by frequently severe clinical manifestations
that affect young individuals, which progress rapidly and can
involve multiple family members. The diagnosis of AP requires the
exclusion of the presence of systemic diseases that may severely
compromise host defenses and cause premature tooth loss.Although
the presence of bacteria is essential for the onset of the disease,
the number and type of these microorganisms are not sufficient to
explain differences in the inflammatory and immune responses and,
consequently, in the severity of the periodontal disease [1]. It
has currently become evident that in the case of most chronic
diseases other factors exist that do not cause disease but that
modify the course of the disease, rendering it more severe. Among
these factors are genetic alterations, called polymorphisms, which
are commonly found in the population [2]. Gene polymorphisms are
locations within the genome that vary in sequence between
individuals and are very prevalent, affecting at least 1% of the
population [3]. Many genes responsible for cytokine production
exhibit polymorphisms [4] that can modify the production of
proinflammatory cytokines such as interleukin-1 (IL-1) and tumor
necrosis factor-α (TNF-α) [5, 6]. In all cases, it has been
suggested that the host response to bacterial etiological agents
may be exaggerated or deficient, resulting in periodontal breakdown
at an early age. Polymorphisms in various cytokine genes can
influence the level of secretion of these substances and explain
the variations in individual immune-inflammatory responses to a
bacterial aggression [2]. Recent studies have indicated that the
IL-1 gene polymorphism might be associated with a greater severity
of the disease in patients with chronic periodontitis and AP
[7-9].IL-1 is the cytokine most frequently found in inflammatory
processes and is involved in the onset and progression of
connective tissue and bone destruction. Bacterial products such as
lipopolysaccharide increase the synthesis of IL-1 [10]. IL-1
mediates the recruitment of inflammatory cells to the sites of
infection, promotes bone resorption, stimulates the production of
fibroblasts, induces the synthesis of prostaglandin E2 by
macrophages and fibroblasts, stimulates the production of
metalloproteinases that degrade extracellular matrix proteins, and
participates in the host immune response [11]. The role of the
IL-1α and IL-1β gene polymorphisms was evaluated in patients with
generalized or localized, early-onset periodontitis (EOP) by Diehl
and coworkers [7]. In the case of generalized juvenile
periodontitis (GJP), allele 1 at the IL-1 locus was transmitted
more frequently than allele 2. The results of linkage
disequilibrium analysis suggested that the IL-1β polymorphism may
be more important for the association with localized juvenile
periodontitis (LJP) [7]. On the other hand, Walker et al. (2000)
[12] ruled out the importance of allele 1 of gene IL-1β in the
African-American population and Hodge et al. 2001 [13] concluded
that there was no association between the IL-1 polymorphism and GJP
of European origin.TNF-α is a proinflammatory cytokine produced
mainly by macrophages, with a biological role similar to that of
IL-1. It induces the secretion of collagenase, prostaglandin E2 and
IL-6 by human fibroblasts and in human bone cell culture, and
possesses important immunologic activities. TNF-α also stimulates
bone resorption by osteoclasts, but is 500 times less potent than
IL-1 [5]. TNF-α has also been detected in gingival fluid from
patients with gingivitis and periodontitis [14]. Several studies
have reported an association between the TNFA gene polymorphism
(allele 2) and infectious and autoimmune diseases such as cerebral
malaria, rheumatoid arthritis, lupus erythematosus and diabetes
[15]. Few studies are available in the literature regarding the
association between the TNFA gene polymorphism and periodontal
disease. Kinane et al. [16], studying the association between EOP
and TNFA (-308) and IL-10 polymorphisms, did not observe any
association between these polymorphisms and GJP. In addition, no
association was observed between the TNFA (-308) gene polymorphism
and AP in patients with generalized or localized AP [17].The
association between the TNFA (-308) gene polymorphism and GJP was
evaluated in a Japanese population by Endo et al. [18]. The results
showed no difference in polymorphism frequency between patients
with GJP and healthy controls, and indicated that its frequency is
low in the Japanese population. It is possible that the prevalence
of these polymorphisms varies among different races as observed in
studies evaluating the IL-1A and IL-1B polymorphism in Chinese
subjects [19] and in individuals of Hispanic origin [20] without
periodontal disease.The objective of the present study was to
evaluate the association between the IL-1A (-889) and TNFA (-308)
gene polymorphisms and generalized AP in Caucasian Brazilian
patients.
Methods
Patients and controls
The study was approved by the Research Ethics Committee of the
Dental School, University of São Paulo. All patients received
detailed information about the objective of the study and signed a
free, informed consent form to participate.
This case-control study involved 100 individuals from the city
of São Paulo in the southeastern region of Brazil. Patients with
aggressive periodontitis and the control individuals were recruited
from the Dental School at University of São Paulo. Patients and
healthy controls came from the same geographical region and had a
similar socio-economic status.
The AP group comprised 30 Caucasian patients (patients with
parents and grandparents of different European origins) (six males
and 24 females), with a mean age of 25.93 ± 3.27 (range 21 to 30
years). The patients presented attachment loss
> 4 mm in at least three permanent teeth, in
addition to molars and incisors. The control group consisted of 70
white, age-matched subjects (seven males and 63 females), mean age
of 26.4 + 3.16 years (range 21 to 30 years) without clinical
evidence of attachment loss in any tooth. These individuals had at
least 24 teeth.
A complete medical and dental history was obtained for the
patients and control subjects, with all individuals showing good
general health. Excluded from the study were patients with
orthodontic devices, insufficient restorations margins, patients
with a history of diabetes, pregnant or breast-feeding women,
patients chronically using anti-inflammatory drugs, smokers, and
subjects with a history of hepatitis and HIV infection.
The following clinical parameters were recorded: bleeding index
upon probing, probing pocket depth at six sites/teeth, clinical
attachment level at 6 sites/teeth, observation of clinical
mobility, bleeding upon probing and the presence of
plaque/calculus. All patients in the AP group had severe,
generalized aggressive periodontitis, based on the International
Workshop for a Classification of Periodontal Disease and Conditions
in 1999 [21]. The clinical examination was performed by only one
examiner (NMF) which was calibrated (kappa value = 0.82).
Fourteen periapical radiographs were taken from patients with
AP. The patients were submitted to periodontal treatment and
enrolled in a periodontal control and maintenance program.
Collection of saliva, enzymatic digestion and DNA
extraction
Non-stimulated saliva samples were collected into a sterile
universal container, transferred to 2 mL tubes and stored in a
freezer at -20°C until the time of DNA extraction. The patients
were instructed not to eat or brush their teeth for 30 min
before saliva collection.
A 500 μL aliquot of saliva was transferred to a 2 mL
tube and centrifuged at 1000 rpm for 5 min. The supernatant
was discarded and the pellet was washed twice with 1 mL 1X
PBS. The material was again centrifuged at 2000 rpm for 5 min
and the supernatant was discarded. The pellet was washed with
1 mL 1X PBS, centrifuged and the supernatant was again
discarded. The pellet was then submitted to enzymatic digestion
with proteinase K.
For digestion, 200 to 400 μL of sterile lysis buffer containing
1 M NaCl, 1 M Tris [tris(hydroxymethyl)aminomethane], pH 8, 0.5 M
EDTA (ethylenediaminetetraacetic acid), pH 8, 10% SDS (sodium
dodecyl sulfate) (Sigma Chemical Co., St. Louis, MO, USA) and
proteinase K (Invitrogen, Carlsbad, CA, USA) at a final
concentration of 500 μg/mL were added to the tube. The tubes were
shaken in a water bath at 55°C for 3 to 7 days until complete
dissolution of the pellet. Proteinase K solution (200-400 μg/mL)
was added at 24h intervals and the tubes were inverted at least
once a day. After complete dissolution of the material, the tubes
were incubated at 95°C for 10 min for proteinase K
inactivation.
DNA was extracted from the samples by the ammonium acetate and
isopropanol method standardized at the Laboratory of Molecular
Biology, Dental School, University of São Paulo [22]. Briefly, 200
μL 4 M ammonium acetate (Synth, BR) were added to the tube
containing the lysate for protein precipitation. The material was
homogenized for 20 s, incubated on ice for 5 min and
centrifuged at 13,000 x g for 3 min. Precipitated protein was
observed at the bottom of the tube and the supernatant containing
DNA was transferred to another tube. For precipitation of DNA, 600
μL 100% isopropanol was added and the mixture was homogenized and
centrifuged at 16,000 x g for 5 min. The supernatant was
discarded and the DNA pellet was washed with 600 μL 70% ethanol and
centrifuged at 16,000 x g for 2 min. The alcohol was removed
and the sample was evaporated to dryness at room temperature. The
DNA pellet was dissolved in 30 to 50 μL TE buffer (10 mM Tris-HCl,
pH 7.4 and 1 mM EDTA, pH 8) and stored at 4°C until quantification
in a spectrophotometer.
Genotyping of IL-1A (-889) and TNF-a (-308)
The extracted DNA was amplified by PCR using primers specific for
each gene whose sequences were obtained from the literature and
verified using the GenBank [accession number X03833.1 for IL-1A
(-889) and AF247608.1 for TNFA (-308)]. IL-1A (-889): 5′-AAG CTT
GTT CTA CCA CCT GAA CTA GGC- 3′ [23] and 5′-TTA CAT ATG AGC CTT CCA
TG-3′. TNFA (-308) 5′- AAG CAA TAG GTT TTG AGG GCC AT-3′ and 5′-
TCC TCC CTG CTC CGA TTC CG -3′ [24]. All reactions were carried out
in 0.5 mL PCR tubes containing 1% formamide (Invitrogen), 1X
PCR buffer (200 mM Tris-HCl, pH 8.4, 500 mM KCl) (Invitrogen), 0.3
mM dNTP mix (2’-deoxynucleotide 5’-triphosphates: dATP, dTTP, dCTP,
dGTP) (Invitrogen), MgCl2 (Invitrogen), sense and
antisense primers (Invitrogen), 2 U Taq DNA polymerase
(Invitrogen), 100 to 300 ng genomic DNA, and sterile water in final
volume of 25 μL. The MgCl2 concentration was 2 mM for
the IL-1A (-889) gene and 2.5 mM for the TNFA (-308) gene. The
primer concentration was 500 pM for the IL-1A (-889) gene and 250
pM for the TNFA (-308) gene.
The PCR conditions were optimized for the different primers
using as a basis one initial denaturation cycle at 94°C for
3 min, followed by 38 cycles of denaturation at 94°C for
1 min, annealing at 50°C for 50 s for the IL-1A (-889)
gene and at 61°C for 50 s for the TNFA (-308) gene and
extension at 72°C for 1 min, and a final extension step at
72°C for 7 min.
Restriction fragment length polymorphism (RFLP) analysis
RFLP analysis is based on the pattern of DNA fragments produced by
digestion of the PCR product with an endonuclease specific for the
determination of polymorphisms. The RFLP technique involves the
cleavage of DNA molecules with restriction enzymes, separation of
the generated fragments by gel electrophoresis and their
visualization in the form of bands.
IL-1A polymorphism in the position -889 has a cytosine (allele
1) substituted by thymine (allele 2), and the cytosine allele
completes a NcoI site. Allele 1 yielded products of 83bp and 16bp,
and allele 2 resulted in a product of 99bp. The three bands were
shown in heterozygous individuals.
TNFA (-308) polymorphism in the position -308 had a guanine
(allele 1) substituted by adenine (allele 2) and the guanine allele
completes a NcoI site. Allele 1 yielded products of 87bp and 20bp,
and allele 2 resulted in a product of 107bp. Heterozygous
individuals had the three bands.
For RFLP analysis of the IL-1A (-889) and TNFA (-308) genes, 5
μL of the PCR product was digested with 2 U of the NcoI restriction
enzyme in 1X specific buffer and sterile water to a final volume of
20 μL at 37°C for 5 h. The resulting fragments were separated
by size on 15% polyacrylamide gel containing 5% glycerol
(2.6 mL 10X TAE, 5.2 mL 50% glycerol, 25 mL 30%
acrylamide, 520 μL 10% APS, 52 μL TEMED, and Milli-Q water, q.s.p.
50 mL). The PCR product was mixed with running buffer (10 mM
EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol). Two
molecular weight standards were used for the determination of
fragment size: low DNA Mass Ladder (Invitrogen) and 10-bp DNA
Ladder (Invitrogen). The gels were run in 1X TAE buffer for
approximately 6 h at room temperature.
After the run, the gel was stained with silver and developed for
visualization of the DNA bands. First, the gel was fixed in 10%
ethanol and 0.75% acetic acid for 20 min, followed by staining
with 0.2% silver for 30 min, washing in Milli-Q water for
2 min and development with a solution of 3% NaOH and 0.03%
formaldehyde for 20 min. Finally, the gel was incubated with
10% acetic acid for 12 min and washed with Milli-Q water for
12 min. All steps were performed in the dark and under shaking
[25]. The gels were then scanned for subsequent analysis.
Statistical analysis
Statistical analysis was performed using the standard statistical
software (Stat View J-4.5 application program, SAS Institute Inc.,
NC, USA). The chi-square test was used to compare the sex
distribution between control and aggressive periodontitis group.
Hardy-Weinberg equilibrium in both groups was tested for genotyping
frequencies by a chi-square test with one degree of freedom (d.f.).
The associations of allele and distributions in aggressive
periodontitis patients and healthy controls were assessed in 2 X 2
contingency tables (d.f. = 1). The 2 X 2 tables were analyzed using
chi-square test or Fisher’s exact test. A p-value < 0.05 was
considered significant.
Results
The GAP group and control group were similar regarding age and
gender distribution (p > 0.05). GAP patients had a mean of 15.53
(± 4.42) teeth with a probing depth greater than 5 mm, a mean
probing depth of 5.35 ± 0.9 and clinical attachment loss of 6.33 ±
0.7; control patients had a mean probing depth of 2.11 ± 0.3 and
clinical attachment loss of 2.45 ± 0.25.
Examination of polyacrylamide gels showed fragments of 99 bp
(allele 2) and 83 bp and 16 bp (allele 1) for patients and controls
heterozygous for IL-1A (-889). For the TNFA (-308) polymorphism
fragments of 87 bp and 20 bp (allele 1) and 107 bp (allele 2) were
visible for heterozygotes.
The genotype and allele frequencies of the two genes are shown
in tables 1 and 2. With respect to the IL-1A (-889) gene, genotype
frequencies in the control group were 54.3% for genotype 1/1, 37.1%
for genotype 2/1 (heterozygous) and 8.6% for genotype 2/2.
Statistically similar genotype frequencies were obtained for the
study groups: 56.7% for genotype 1/1, 40% for genotype 2/1, and
3.30% for genotype 2/2. The frequency of allele 1 and allele 2 was
72.9% and 27.1% in control group and in the AP group the frequency
for allele was 76.7% 1 and for allele 2 was 23.3%. Both groups were
in Hardy-Weinberg equilibrium (aggressive periodontitis group,
χ2= 0.41; healthy group, χ2= 0.26, d.f. = 1,
p > 0.05).
The homozygous TNFA (-308) 1 allele was present in 68.6% of the
control group and in 80% of AP group, while the heterozygous TNFA
(-308) 2 allele was present in 27.1% of the control group and 20%
of AP patients. The homozygous TNFA (-308) 2 allele was present in
4.3% of the control group and it was not detected in the AP group.
The frequency of allele 1 and allele 2 was 82.15% and 17.85%
respectively in the control group, and in the AP group the
frequency for allele 1 was 90% and for allele 2 was 10%. Both
groups were in Hardy-Weinberg equilibrium (aggressive periodontitis
group, χ2= 0.37; healthy group, χ2= 0.39,
d.f. = 1, p > 0.05).
Comparison of the IL-1A (-889) and TNFA (-308) gene
polymorphisms between the AP and control groups revealed no
significant association with AP, i.e., the two groups did not
differ in terms of changes in the genes analyzed (p > 0.05). The
association with any allele or combination of alleles of the
polymorphisms and AP are presented in table
3.
Table 1 Frequency of IL-1A (-889) genotype and alleles
in generalized aggressive periodontitis patients and healthy
controls
|
|
Controls
|
GAP patients
|
|
|
N
|
(%)
|
N
|
(%)
|
|
Genotype
|
1/1
|
38
|
(54.3)
|
17
|
(56.7)
|
|
2/1
|
26
|
(37.1)
|
12
|
(40.0)
|
|
2/2
|
6
|
(8.6)
|
1
|
(3.3)
|
|
Total
|
70
|
(100)
|
30
|
(100)
|
|
ARRAY(0x22df44)
|
|
Alleles
|
Allele 1
|
102
|
(72.9)
|
46
|
(76.7)
|
|
Allele 2
|
38
|
(27.1)
|
14
|
(23.3)
|
|
Total
|
140
|
(100)
|
60
|
(100)
|
Table 2 Frequency of TNFA (-308) genotype and alleles
in generalized aggressive periodontitis patients and healthy
controls
|
|
Controls
|
GAP patients
|
|
|
N
|
(%)
|
N
|
(%)
|
|
Genotype
|
1/1
|
48
|
(68.6)
|
24
|
(80.0)
|
|
2/1
|
19
|
(27.1)
|
6
|
(20.0)
|
|
2/2
|
3
|
(4.3)
|
0
|
(0)
|
|
Total
|
70
|
(100)
|
30
|
(100)
|
|
ARRAY(0x236c74)
|
|
Alleles
|
Allele 1
|
115
|
(82.15)
|
54
|
(90.0)
|
|
Allele 2
|
25
|
(17.85)
|
6
|
(10.0)
|
|
Total
|
140
|
(100)
|
60
|
(100)
|
Table 3 Associations between IL-1A (-889) and TNFA
(-308) genes
|
IL-1A
|
|
1/1
|
2/1
|
2/2
|
|
TNFA
|
1/1
|
N
|
40
|
26
|
6
|
|
(%)
|
(72.7)
|
(68.4)
|
(85.7)
|
|
2/1
|
N
|
14
|
10
|
1
|
|
(%)
|
(25.5)
|
(26.3)
|
(14.3)
|
|
2/2
|
N
|
1
|
2
|
0
|
|
(%)
|
(1.8)
|
(5.3)
|
0
|
|
Total
|
N
|
55
|
138
|
7
|
|
(%)
|
(100)
|
(100)
|
(100)
|
Discussion
Our results demonstrated that there were no significant association
between GAP and the IL-1A (-889) and TNFA (-308) gene polymorphisms
in nonsmoking patients with GAP and nonsmoking subjects without
periodontal disease. As shown in table 1
and 2, the distribution of the IL-1A
(-889) and TNFA (-308) frequencies did not reveal any significant
differences between the groups.
The two groups were age-matched, and no smokers or former
smokers were included since smoking has been identified as the
major environmental risk factor associated with increased incidence
and severity of periodontitis [26-28]. In addition, patients with
the localized form of AP were also excluded in order to obtain a
homogenous study group.
The findings presented here disagree with the results provided
by Kornman et al. [8] for nonsmoking European patients with chronic
periodontitis, which pointed to strong evidence of association
between IL-1A (-889) gene polymorphism and the severity of
periodontal disease. In that study, patients carrying allele 2
presented a 19-fold higher risk of developing periodontal disease
than patients carrying allele 1.
Few studies have evaluated this association for various forms of
periodontitis in different countries. The present results agree
with those reported by other investigators studying patients with
AP. Diehl et al. [7] reported a more frequent transmission of
allele 1 of the IL-1A (-889) gene in Afro-American and Caucasian
patients with EOP compared to allele 2. Hodge et al. [13] and
Gonzalez et al. [29] also did not find any association between the
IL-1A (-889) gene polymorphism and juvenile periodontitis. Thus,
the different forms of the disease, as well as possible population
differences, should be taken into account.
With respect to the TNFA (-308) gene, the genotype frequencies
obtained in the present study were 68.6% for genotype 1/1, 27.1%
for genotype 2/1 and 4.3% for genotype 2/2 in the control group,
and 80% for genotype 1/1 and 20% for genotype 2/1 in the study
group. The frequency of allele 1 was 82.15% in the control group
and 90% in the study group. Allele 2 was observed in 17.85% of
control subjects and in 10% of patients with AP.
This result agrees with Kinane et al. [16] who also did not
observe any association between TNFA gene polymorphism and
generalized AP. Shapira et al. [17] observed a slight, but not
significant, increase in the prevalence of genotype 1/1 in patients
with AP, contrary to the results obtained in this study. Although
the detection of genotype 2/2 in the control group was not
statistically different from the absence of this genotype in the AP
patients, maybe a larger group of patients would provide more
convincing results. The frequencies of TNFA genotype 2/2 are
relatively low both for control and diseased patients. The
calculation of sample size to have 80% power to detect a difference
between AP patients and healthy controls at the = 0.05 level of
significance, shows that approximately 234 patients in each group
would need to be evaluated.
AP is a multifactorial disease resulting from the complex
interactions between the host, microbiota and environment. The
difficulty in associating a gene polymorphism with AP might be
explained by the lack of higher expression of a single gene in the
disease. Probably, there are other genes altering the expression of
genes and influencing the clinical expression of the disease.
Furthermore, multiple polymorphisms might be necessary for an
increase in the severity of the disease. In addition, specific
genes may vary among different populations and/or ethnic groups,
and true heterogeneity in the susceptibility to the disease might
be present.
In conclusion, the present study revealed no association between
the IL-1A (-889) and TNFA (-308) gene polymorphisms and generalized
AP.
Acknowledgements
This research was supported by Fundação de Amparo à Pesquisa do
Estado de São Paulo Grant 02/04015-8.
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111: 395.
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