Open Journal of Stomatology, 2011, 1, 25-28 OJST
doi:10.4236/ojst 2011.12005 Published Online June 2011 (
Published Online June 2011 in SciRes.
Human leukocyte antigen associations and c-reactive protein
levels in lebanese patients with aggressive periodontitis
HLA and CRP in aggressive periodontitis
Marita Chakhtoura, Nada M. Souccar, Nayla S. Al-Akl, Alexander M. Abdelnoor
Department of Microbiology & Immunology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon.
Received 6 May 2011; revised 16 June 2011, accepted 25 June 2011.
Background: Periodontal disease, which affects tooth-
supporting structures, results from disequilibrium
between the oral micro flora and host defense mecha-
nisms. It has been classified into chronic (CP) or ag-
gressive (AP) periodontitis according to disease onset,
localization and progression. Because of their invol-
vement in generating immune responses, Human
Leukocyte Antigen (HLA) alleles are considered can-
didate genetic risk markers for periodontitis. Addi-
tionally, periodontitis appears to contribute to the
severity of some systemic conditions such as cardio-
vascular disease and adverse pregnancy outcome as
indicated by elevated levels of C-reactive protein
(CRP). Aim: The aims of this study were to deter-
mine if there is an HLA-AP association in Lebanese
patients, and to determine CRP levels in patients and
compare them to those in healthy controls. Materials
and methods: The study groups included 26 patients
with AP and 39 healthy controls. HLA profiles were
determined by DNA typing and CRP levels by ELI-
SA. Results: HLA-A*30 (P-value = 0.010), HLA-
B*41 (P1 = 0.012 and P2 = 0.014), HLA-DRB1*13 (P1
= 0.031 and P2 = 0.063) alleles seemed to be associ-
ated with protection agains t AP in Lebanese pa tients.
No linkage disequilibrium existed between alleles
associated with AP. Ten of 26 AP patients (38.5%)
and 10 of 39 (25.7%) controls had elevated CRP lev-
els. Conclusion: In conclusion, protective, but no sus-
ceptible HLA alleles were detected in AP. CRP levels
were not elevated in the entire AP group, and were
not significantly different from controls. No linkage
disequilibrium existed between alleles.
Keywords: HLA; CRP; Periodontitis; Associations
Periodontitis, a disease affecting teeth and tooth-su-
pporting structures, results from a disequilibrium be-
tween normal microbial flora and host defense mecha-
nisms [1]. It is an inflammatory condition that is closely
related to the presence of bacteria found in dental plaque,
mainly Gram-negative anaerobic rods [2,3]. The disease
could ultimately lead to the formation of periodontal
pockets upon bone loss and apical migration of the junc-
tional epithelium [3,4]. It is classified into aggressive
periodontitis (AP) and chronic periodontitis (CP) [5,6].
Subjects with the aggressive form have rapid attach-
ment loss, bone loss, familial aggregation and are usu-
ally younger than 30 years of age. Those with the chro-
nic form have at least four sites with pocket depths > 4
mm and at least four sites with attachment level mea-
surements < 4 mm, as well as radiographic evidence of
alveolar bone loss [7].
Genetic factors for periodontitis have been reviewed
by Stabholz et al. [8]. The prevalence of periodontitis
within families is used as a major diagnostic criterion for
the aggressive form. A number of studies have indicated
an association between a single nucleotide polymor-
phism and periodontitis. These included single nucleo-
tide polymorphisms in the interleukin-1 gene, FcγR gene
and the interleukin-10 gene. Genetic factors such as the
Human Leukocyte Antigen (HLA) alleles, have been re-
ported to correlate with pre-disposition to or protection
against several diseases including periodontitis [9-11]. In
particular, HLA-A9 and HLA-B15 seem consistently
associated with aggressive periodontitis in Caucasians
[10,12,13], while HLA-A2, HLA-B5 and HLA-A28 are
potential protective factors against periodontitis among
Caucasians [10,14]. However, studies involving HLA-
periodontitis association in the Lebanese ethnic group
are lacking.
Periodontitis seems to contribute to the severity of
several systemic conditions such as cardiovascular dis-
ease [15] and adverse pregnancy outcome [16,17], through
elevated C-reactive protein (CRP) levels. One hypothe-
M. Chakhtoura et al. / Open Journal of Stomatology 1 (2011) 25-28
Copyright © 2011 SciRes. OJST
sis suggests that this is likely achieved when inflamma-
tory effects from periodontal disease cause oral bacterial
byproducts to enter blood and trigger the production of
acute phase reactants such as CRP, subsequently leading
to artery inflammation or preterm birth [18]. CRP is an
acute phase reactant found in plasma. Its physiological
function is to enhance phagocytosis of infectious agents
by macrophages. Elevated level of CRP is considered an
indication of infection and inflammation. It is produced
in the liver and elevated levels are thought to be due to
an increase in the production of interleukin-6 by macro-
phages and adiptocytes [19].
In an earlier study we reported on the detection of
Porphyromonas gingivalis (Pg) in specimens obtained
from dental plaques of pregnant women with periodonti-
tis [20]. The aims of this study were to see if HLA-AP
and CRP-AP associations existed in Lebanese patients.
2.1. Subjects and Specimens
A total of 65 subjects aged between 21 and 56 were re-
cruited for this study. Twenty six patients (11 males and
15 females) with aggressive periodontitis (AP) were
matched according to age, gender and smoking habits to
26 controls. Half of the controls and patients were
smokers. Additional 13 controls were available and were
included in the study; they were divided into 7 males and
6 females; 7 of them were smokers. Blood from all sub-
jects was collected in citrated and plain tubes. The for-
mer was used to separate the mononuclear cells and the
latter was used to obtain serum for CRP determinations.
A clinical exam that involved a full mouth periodontal
probing was done on each patient and probing depth on
each tooth (6 sites per tooth), bleeding on probing (BOP)
[21], recessions, attachment loss (AL) [7] and Plaque
index [22] were recorded. Periodontal disease was diag-
nosed clinically when bleeding on probing (BOP) ex-
isted in 35% or more of all tooth sites, and /or patients
had at least 1 site on 4 different teeth with pocket depth
4 mm and clinical attachment loss 2 mm. In addition,
a radiologic examination using a panoramic or periapical
radiographs to visualize disease pattern and severity, as
well identify other dental pathologies was performed.
Aggressive periodontitis patients were identified using
the recorded measurements (including rapid attachment
loss, bone loss, familial aggregation).
2.2. Informed Consent
Prior review and approval of this project was done by
the Institutional Review Board (IRB). A written consent
was obtained from each subject included in the project.
2.3. DNA Extraction
Peripheral Mononuclear cells (PMNC) were separated
from whole blood using Ficoll-Isopaque gradient cen-
trifugation (GE Healthcare, Pis). DNA was extracted
from PMNC using the QIAamp DNA mini kit (Qiagen,
Germany), according to the manufacturer’s protocol.
2.4. Determination of HLA Profiles
HLA profiles of all patients and controls were deter-
mined by DNA typing using the polymerase chain reac-
tion-sequence specific primer (micro-SSP) (One Lambda,
Canoga Park, CA) according to the manufacturer’s in-
2.5. Determination of CRP Levels
CRP levels in the sera of all participants were deter-
mined by ELISA using the high sensitivity C-reactive
protein enzyme immunoassay test kit (BioCheck, Inc.,
Foster City, CA) according to the manufacturer’s instru-
ctions. Specimens were run in duplicates and absorbance
values were read at 450 nm. CRP concentrations were
calculated and expressed in mg/l. According to the ma-
nufacturers guidelines normal adult CRP values were be-
tween 0.068 mg/l and 8.2 mg/l.
2.6. Statistical Analysis
Relative Risks (RR) and P-values were determined for
each HLA allele in the AP-control groups, using Statcalc
(Epi-Info, version 6.1). Linkage disequilibrium was cal-
culated for any two associated HLA alleles using SPSS
17.0 for Windows.
Relative Risk and 95% confidence interval (CI) were
determined to see if there was a significant association
between periodontitis and elevated CRP levels using the
method of Hutchon, DJR [23]. An RR greater than one
and a CI that does not include the number “1” indicated
a significant association.
3.1. Dental Analysis
The dental parameters were all statistically significant
difference between patient and control groups (P 0.05).
The average values of tested indices for the patient group
indicated a moderate periodontitis (Table 1).
3.2. HLA Allele-Aggressive Periodontitis (AP)
HLA-A*30 (P-value = 0.010), HLA-B*41 (P1 = 0.012
and P2 = 0.014) and HLA-DRB1*13 (P1 = 0.031 and P2
= 0.063) seemed to associate with protection against AP
in Lebanese (Table 2). None of these alleles were in
linkage disequilibrium.
3.3. C-Reactive Protein Levels
Ten of 26 AP patients (38.5%) had elevated CRP levels
M. Chakhtoura et al. / Open Journal of Stomatology 1 (2011) 25-28
Copyright © 2011 SciRes. OJST
(values > 8.2 mg/l) (Table 2).
Ten of 39 individuals in the control group (25.7%)
had elevated CRP levels (values > 8.2 mg/l) (Table 3).
Although the number of patients with elevated CRP lev-
els was more than that in controls, and the Relative Risk
was greater than 1 (1.406), the 95% confidence interval
indicated that the association between AP and elevated
CRP levels was not significant.
In determining HLA-disease associations the frequency
of an allele in the diseased and normal populations are
used to calculate a Relative Risk, and both populations
must belong to the same ethnic group. A Relative Risk
greater than 1 with p-value of less than 0.05 indicates an
association. In normal populations some HLA allele
frequencies vary among different ethnic groups and it
follows that an established association in one ethnic
group does not necessarily apply to another ethnic group
[24, 25]. This appears to be the case in the present study.
Whereas those alleles associated with protection against
AP in Caucasians were reported to be HLA-A2, HLA-
B5 and HLA-A28 [10,14], in Lebanese they were HLA-
A*30, HLA-B*41 and HLA-DRB1*13. Moreover, no
Table 1. Baseline dental characteristics of all participants.
Parameter Study Group Control Group
Probing depth (mm) 5.3 ± 1.9 3.2 ± 1.5
Clinical attachment loss (mm) 4.2 ± 2.3 1.2 ± 0.8
Bleeding on probing 182/240 sites
74/240 sites
Study group: periodontal disease, control group: periodontally healthy; P
0.05 for all parameters.
Table 2. HLA alleles associated with protection against ag-
gressive periodontitis; AP = Aggressive periodontitis.
HLA Allele Relative Risk and P-value
HLA-A*30 0.27, P = 0.010
Ignored by Statcalc
HLA-B*41 P1 = 0.012, P2 = 0.014
Ignored by Statcalc
HLA-DRB1*13 P1 = 0.031, P2 = 0.063
Table 3. Serum C-Reactive Protein (CRP) levels in Aggressive
Periodontitis (AP) and Controls (C).
Number of subjects
with elevated CRP levels with normal CRP levels
Patients 10 16
Controls 10 29
Relative Risk = 1.406; 95% Confidence Interval = 0.78 - 2.53; No signifi-
cant association between elevated CRP and AP.
linkage disequilibrium seemed to exist between asso-
ciated alleles. The positive association of HLA-A9 and
HLA-B15 with AP in Caucasians [10,12,13] but not in
Lebanese is also probably accounted for by the ethnic
differences between the two populations. In addition,
HLADRB1*13 seemed to have a more frequent occur-
rence in patients with AP that are Germans of Caucasian
descent [26] and HLA-B*15 was also found to be asso-
ciated with AP in Indian population [27].
It would be expected that all patients with AP had ele-
vated CRP levels, but this was not the case. In the pre-
sent study we did not find a significant association be-
tween elevated CRP levels and AP. Not all of the pa-
tients had elevated CRP levels and a number of perio-
dontitis-free, apparently healthy controls had elevated
CRP levels. The causes of elevated CRP levels are non-
specific and this may account for elevated levels in ap-
parently normal controls. In as much as the patients with
normal CRP levels are concerned, probably inflamma-
tion is initially confined to the oral cavity and the in-
flammatory mediators had not yet found their way to the
liver to induce the production of CRP.
A number of reports indicated that periodontitis and
elevated CRP levels were related to premature labor [15,
18]. Earlier we reported a lack of a relationship between
periodontitis, elevated CRP levels and premature labor
in Lebanese women [20]. It is worth noting that during
the later phase of pregnancy in periodontitis-free females,
CRP levels are elevated [28,29].
In conclusion, protective, but no susceptible HLA al-
leles were detected in AP. The association between AP
and elevated CRP was statistically non-significant in
We have no conflicting interests.
This study was supported by funds from the Medical
Practice Plan (MPP), Faculty of Medicine, American
University of Beirut.
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