doi: 10.1111/iji.
12228
Association study of interleukin-1 family and interleukin-6 gene single
nucleotide polymorphisms in recurrent aphthous stomatitis
S. Najafi*,†, H. Yousefi‡,§, M. Mohammadzadeh†, A. Z. Bidoki¶, I. Firouze Moqadam**,
E. Farhadi††, A. A. Amirzargar¶ & N. Rezaei‡,¶,‡‡
Summary
Recurrent aphthous stomatitis (RAS) is a common
painful, ulcerative oral inflammatory disorder with
unknown aetiology. Immune system and aberrant
cytokine cascade deemed to be critical in outbreaks of
RAS ulcers. Interleukin-1 (IL-1) and IL-6 are the most
potent pro-inflammatory cytokines. Single nucleotide
polymorphisms (SNPs) of IL-1 and IL-6 genes can
affect the secretion of these cytokines. The aim of this
study was to investigate the association between RAS
and IL-6 and IL-1 in Iranian subjects with minor RAS.
Genomic DNA was obtained from 64 Iranian patients
with RAS. IL-1a C 889 T, IL-1b C 511 T, IL-1b C
+3962 T, IL-1R C pst-I 1970 T, IL-1Ra C Mspa-
I11100 T, IL-6 C 174 G and IL-6 A nt +565 G poly-
morphisms were determined using polymerase chain
reaction with sequence-specific primers (PCR-SSP).
The frequency of C 174 C genotype in the patients
group was significantly different from the healthy con-
trol. No other significant differences were found in
genotype and alleles frequencies between the two
groups. These results indicate that certain SNPs of
IL-6 gene at position 174 which located in promoter
have association with predisposition of individuals to
RAS.
*Dental Research Center, School of Dentistry, Tehran University of
Medical Sciences, Tehran, Iran, †Department of Oral Medicine,
School of Dentistry, Tehran University of Medical Sciences, Tehran,
Iran, ‡Research Center for Immunodeficiencies, Children’s Medical
Center, Tehran University of Medical Sciences, Tehran, Iran,
§Department of Oral Medicine, Dental Branch, Tehran Azad Univer-
sity, Tehran, Iran, ¶Molecular Immunology Research Center; and
Department of Immunology, School of Medicine, Tehran University
of Medical Sciences, Tehran, Iran, **Molecular Medicine Research
Center, Hormozgan University of Medical Sciences, Bandar Abbas,
Iran, ††Hematology Department, School of Allied Medical Science,
Iran University of Medical Sciences, Tehran, Iran and ‡‡Universal
Scientific Education and Research Network (USERN), Tehran, Iran
Received 6 May 2015; revised 11 July 2015; accepted 19 July 2015
Correspondence: Nima Rezaei, Children’s Medical Center Hospital,
Gharib Street, Keshavarz Blvd, Tehran 14194, Iran. Tel: +9821
66929234; Fax: +9821 66929235; E-mail: rezaei_nima@tums.ac.ir
428
Introduction
Recurrent aphthous stomatitis (RAS), also known as
canker sores, is the most common oral chronic inflam-
matory ulcerative condition (Shulman, 2004). RAS has
been associated with three of clinical manifestations
including minor, major and herpetiform ulcers. A core
problem with RAS has been a lack of understanding
with regard to the aetiology of the disease, which is
the main cause of current palliative treatments. Evi-
dences from number of studies support the bewildering
association of genetic, immunologic, physiologic and
nutritional factors and microbial agents with develop-
ment of RAS (Akintoye & Greenberg, 2005; Chavan
et al., 2012).
Irregular counterbalancing CD4+ T cells and CD8+
T cells have been designated in RAS patients. Initially,
at the pre-ulcerative stage likewise at the healing stage,
a plethora of CD4+ T cells has been reported, while
CD8+ T cells dominant at the ulcerative stage (Chavan
et al., 2012). As with virtually, CD8+ T cells are cul-
prit in severity and progression of RAS. Compelling
evidences demonstrated that an aberrant cytokine cas-
cade eliciting cell-mediated immune response against
the oral mucosa is pivotal to development of RAS
(Buno et al., 1998). Of particular interest are those
cytokines deemed to be critical in engendering cyto-
kine release, chief among them are pro-inflammatory
cytokines including interleukin (IL)-1 and IL-6.
IL-1 family members including IL-1a and IL-1b have
been cytokines of interest owing to their pivotal role in
inducing local and systemic inflammatory immune
responses (Buno et al., 1998; Dinarello et al., 2012).
The secretion of IL-1a and IL-1b is ensued from recruit-
ing of the IL-1 receptor (IL-1R) on target cells. Avid
recognition of IL-1R by its antagonist IL-1Ra, which
binds to IL-1R with higher affinity compared with
cytokines, leads to ablation of cytokine secretion. Mice
models of IL-1Ra deficiency have developed various
inflammatory diseases (Ozkurede & Franchi, 2012).
IL-6 is produced in both infectious and noninfec-
tious inflammations (Matzinger, 2002). IL-6 makes
significant direct contributions to pathology of variety
of autoimmune diseases. Following a trauma, damaged
© 2015 John Wiley & Sons Ltd
International Journal of Immunogenetics, 2015, 42, 428–431
 IL-1a, IL-1b, IL-1R, IL-1Ra and IL-6 SNPs in aphthous stomatitis
cells produce IL-6, and such an acute production of
IL-6 stimulates various cell populations and induces
acute-phase proteins (Tanaka & Kishimoto, 2012).
Given the multifaceted role of gene polymorphism
in cytokine release, a great understanding of their
function will strengthen future therapeutics. This is the
first study among Iranian population, and no data are
available from Iranian patients with RAS in this
regard. This study was conducted to analyse the geno-
type frequencies of IL-1a, IL-1b, IL-1R, IL-1Ra and
IL-6 in a group of Iranian subjects with minor RAS.
Materials and methods
Subjects
Sixty-four Iranian patients with minor RAS, comprised
of 24 male and 40 female with mean age of
36.6 years, fulfilling the defined criteria and with epi-
sodes of aphthous for at least 2 ulcers per month,
more than 2 times per year, were enrolled in this
study. The diagnosis of RAS was made by oral medi-
cine specialist based on accepted clinical criteria (Ship
et al., 2000). The control group consisted of 140
ethnicity-, age- and sex-matched healthy individuals
without history of RAS or any other diseases from the
same geographic area (Amirzargar et al., 2008). Both
control and case groups were selected randomly. Preg-
nancy, smoking, and history of any other diseases such
as HIV, radiation therapy, Behcet’s syndrome, peri-
odontal diseases and any drug consumption, major
RAS and herpetic form ulcers considered as exclusion
criteria for both control and case groups. This study
protocol was approved by the Ethics Committee of
Tehran University of Medical Sciences. Written
informed consent was obtained from all subjects.
Genotyping
Genomic DNA was extracted from whole blood samples
with EDTA as anticoagulant, using a phenol–chloro-
form method. Single nucleotide polymorphisms (SNPs)
of the IL-1a, IL-1b, IL-1R, IL-1Ra and IL-6 genotyping
were performed by polymerase chain reaction with
sequence-specific primers (PCR-SSP) assay (PCR-SSP
kit; Heidelberg University, Heidelberg, Germany). The
technique was previously explained (Amirzargar et al.,
2008). Briefly, the PCR amplification was performed,
using a thermal cycler Techne Flexigene apparatus, and
the PCR products were viewed by agarose gel elec-
trophoresis. The allele and genotype frequencies of IL-
1a (C/T 889), IL-1b (C/T 511, C/T +3962), IL-1R
(C/T Pst-I1970), IL-1Ra (C/T Mspa-I 11100) and IL-6
(C/G 174, A/G nt565) were determined.
Statistics
All data were analysed using SPSS statistical software
package, version 15.0 (SPSS Inc., Chicago, IL, USA).
© 2015 John Wiley & Sons Ltd
International Journal of Immunogenetics, 2015, 42, 428–431
429
The frequency of gene polymorphisms was compared
between patients and control groups by chi-square
test. Allele frequencies were estimated by direct gene
counting. The odds ratio (OR) and 95% confidence
intervals (CI) were calculated at each SNP. P-value (P)
of <0.05 was considered statistically significant.
Results
IL-1 and IL-6 allelic and genotype frequencies in RAS
patients and healthy subjects are shown in the
Table 1.
IL-6 C 174 C genotype was significantly overrep-
resented in the RAS patients compared with control
group (10% in patients vs 2.9% in controls,
P = 0.044).
No significant differences were detected between
patients and controls regarding IL-1a T/C gene poly-
morphism at position 889. The frequency of IL-1b
C/T at position 511 was not significantly different
between two groups; however, C allele was the most
frequent allele among patients (51.7%) and controls
(55.4%) and T 511 C genotype was the most fre-
quent genotype in patients (50%) and controls (59%)
at this position.
IL-1b C allele at position +3962 was more frequent
among patients (74.2%) and controls (70.7%) com-
pared with T +3962 allele. And TT genotype was the
least frequent genotype among two groups (6.7% in
patients and 8.6% in controls), while the results at this
position did not reach significance.
No significant differences were detected between
two groups regarding IL-1Ra MspaI 11100. While C
allele at this position was less frequent than T allele
among both patients and controls, and CC genotype
was the least frequent genotype among patients
(1.6%) and controls (2.9%) at the same position.
Comparison of IL-1R pst-I 1970 allele and geno-
type frequencies between the patients and the controls
showed no significant differences between two
groups.
Discussion
Evidences showed association of IL-1 and IL-6 SNP
with several diseases (Amirzargar et al., 2005, 2006,
2008). In this study, the possible role of IL-1 and IL-6
gene polymorphisms in influencing the individual sus-
ceptibility to develop RAS was investigated.
The IL-6 174 promoter polymorphism plays a
functional role in IL-6 secretion. Although in a study
by Bazrafshani et al. (2002), a significant higher fre-
quency was detected for the G 174 allele in case
group, and it was reported that homozygosity in G
174 G was associated with greater risk of RAS in
our investigation, the GG genotype was more preva-
lent in the control group (30.2%) than in RAS patients
(28.3%); however, it was not significant. The results
of the present study showed significantly higher
430 S. Najafi et al.

Table 1. Comparison of alleles, genotypes and haplotypes frequencies of IL-1a, IL-1b, IL-1R, IL-1RA, IL-6 between patients with recurrent
aphthous stomatitis (RAS) and the control group

Alleles/Genotypes/ Odds ratio (95%

Haplotypes RAS (n = 60), n (%) Controls (n), n (%) P-value confidence interval)

IL-1a C 79 (65.8) 186 (68.4) 0.703 0.89 (0.55–1.44)

889 T 41 (34.2) 86 (31.6) 0.703 1.12 (0.69–1.81)
CC 24 (40) 62 (45.6) 0.568 0.80 (0.41–1.54)
TC 31 (51.7) 62 (45.6) 0.528 1.28 (0.66–2.45)
TT 5 (8.3) 12 (8.8) 0.870 0.94 (0.27–3.06)
IL-1b C 62 (51.7) 154 (55.4) 0.564 0.86 (0.55–1.35)
511 T 58 (48.3) 124 (44.6) 0.564 1.16 (0.74–1.83)
CC 16 (26.7) 36 (25.8) 0.949 1.04 (0.49–2.18)
TC 30 (50) 82 (59) 0.308 0.70 (0.36–1.34)
TT 14 (23.3) 21 (15.2) 0.231 1.71 (0.75–3.88)
IL-1b C 89 (74.2) 198 (70.7) 0.560 1.19 (0.71–1.98)
+3962 T 31 (25.8) 82 (29.3) 0.560 0.84 (0.50–1.40)
CC 33 (55) 70 (50) 0.621 1.22 (0.64–2.35)
TC 23 (38.3) 58 (41.4) 0.801 0.88 (0.45–1.71)
TT 4 (6.7) 12 (8.6) 0.445 0.76 (0.20–2.70)
IL-1R C 76 (63.9) 174 (62.1) 0.831 1.08 (0.67–1.72)
Pst-I 1970 T 43 (36.1) 106 (44.2) 0.831 0.93 (0.58–1.48)
CC 19 (32.2) 54 (38.6) 0.490 0.76 (0.38–1.51)
TC 37 (62.7) 66 (47.1) 0.064 1.89 (0.97–3.69)
TT 3 (5.1) 20 (14.3) 0.107 0.32 (0.07–1.21)
IL-1Ra C 24 (20) 64 (22.9) 0.616 0.84 (0.48–1.47)
Mspa-I 11100 T 96 (80) 216 (77.1) 0.616 1.19 (0.68–2.08)
CC 1 (1.6) 4 (2.9) 0.527 0.58 (0.02–5.65)
CT 22 (36.7) 56 (40) 0.775 0.87 (0.44–1.70)
TT 37 (61.7) 80 (57.1) 0.661 1.21 (0.62–2.35)
IL-6 C 49 (40.8) 101 (36.3) 0.460 1.21 (0.76–1.92)
174 G 71 (59.2) 177 (63.7) 0.460 0.83 (0.52–1.31)
CC 6 (10) 4 (2.9) 0.0441* 3.75 (0.89–16.59)
CG 37 (61.7) 93 (66.9) 0.582 0.80 (0.40–1.57)
GG 17 (28.3) 42 (30.2) 0.922 0.91 (0.44–1.87)
IL-6 A 21 (17.5) 50 (18) 0.978 0.97 (0.53–1.75)
Nt 565 G 99 (82.5) 228 (82) 0.978 1.03 (0.57–1.89)
AA 3 (5) 4 (2.9) 0.355 1.78 (0.30–9.79)
GA 15 (25) 42 (30.2) 0.564 0.77 (0.37–1.61)
GG 42 (70) 93 (66.9) 0.792 1.15 (0.57–2.34)

*The frequency of IL-6 C-174C genotype in the patients group was significantly different from the healthy control. No other significant differ-
ences were found in genotype and allele frequencies between the two groups.

frequency in C 174 C genotype in the patients group
(P = 0.044), and no significant differences were found
in G 174 G genotype. It has been indicated that inher-
itance of 174 G allele in comparison with C allele
encodes higher production of IL-6 (Fishman et al.,
1998), while G allele is more frequent among controls
in our investigation which is in consistent with the
result of Guimaraes et al. (2007) investigation; how-
ever, this different did not reach significance. Such con-
flicting results indicate that more studies in greater
population in different ethnic groups are needed.
IL-1 plays a critical role in initiation of an inflam-
matory process and a shift from acute to chronic
inflammation state (Rafiq et al., 2007). IL-1a and IL-
1b are two members of IL-1 family, coded by separate
genes. In the present study, no significant allele and
genotype differences for IL-1a at position 889 were
found, which is in line with the previous studies
(Bazrafshani et al., 2002). Earlier studies reported the
significant role of G 511 allele in IL-1b secretion in
RAS patients (Bazrafshani et al., 2002); hence, we
investigated the C 511 allele, and no significant dif-
ferences were found between the patients group and
the control group. This indicates the importance of G
511 allele but not C -511 allele in IL-1b secretion. It
is notable that a potential protective role from ulcera-
tive colitis for IL-1b 511 CC genotype has been indi-
cated in previous investigations (Muro & Morwiec,
2015), and in the current research, IL-1b 511 CC
genotype is more frequent among patients (26.7%)
compared with controls (25.8%), although the differ-
ence is not significant. The association between IL-1b
+3962 C allele with higher serum level of the cytokine
in patients with Crohn’s disease has been designated
(Muro & Morwiec, 2015), and in our study, C allele
at the same position is more prevalent among patients
(74.2%) in comparison with controls (70.7%); how-
ever, the difference did not reach significance. No sig-

© 2015 John Wiley & Sons Ltd

International Journal of Immunogenetics, 2015, 42, 428–431
 IL-1a, IL-1b, IL-1R, IL-1Ra and IL-6 SNPs in aphthous stomatitis
nificant differences were found in the distribution of
polymorphisms of C +3962 T for IL-1b, C pst-I 1970
T for IL-1R, C Mpsa-I 11100 T for IL-1Ra and A
nt565 G for IL-6 in our study.
Acknowledgements
This research has been supported by a grant from
Tehran University of Medical Sciences.
Conflict of interest
The authors declare that they have no conflict of interest.
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