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Cytokine Gene Polymorphism in Recurrent Acute Otitis Media
Veli-Pekka Joki-Erkkilä, MD;
Heikki Puhakka, MD;
Mikko Hurme, MD
Arch Otolaryngol Head Neck Surg. 2002;128:17-20.
ABSTRACT
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Background There is increasing evidence that a strong genetic component is involved
in the predisposition to recurrent acute otitis media (rAOM). Cytokines play
a key role in the pathogenesis of otitis media. Constitutional polymorphisms
in cytokine genes may lead to individual variations in cytokine secretion.
Objective To elucidate the role of cytokine gene polymorphisms in rAOM.
Setting University hospital.
Participants and Methods Blood samples for genetic analysis were obtained from 63 individuals
with rAOM from 20 different families and from 400 healthy blood donors. The
medical history of the rAOM group was based on medical records and interview
data. We studied the polymorphisms of tumor necrosis factor  , interleukin
(IL) 1, IL-1ß, and IL-1 receptor antagonist genes.
Results The distribution of cytokine alleles in the rAOM group did not differ
significantly from that of the control group. However, in patients with rAOM
without a history of allergic disorders, allele frequencies of IL-1-889
differed significantly from those of controls (P
= .03).
Conclusions There is no clear association between the polymorphism of studied cytokine
genes and rAOM. However, the IL-1 gene polymorphism may be associated
with recurrent middle ear infections in a subgroup of patients without allergic
disorders.
INTRODUCTION
ACUTE OTITIS media (AOM) is one of the most common childhood illnesses.
Although nearly all children experience at least one otitis episode, only
about 15% of children experience recurrent attacks.1
Recurrent AOM (rAOM) is a challenging clinical problem consuming many medical
resources. Moreover, frequent otitis episodes are also associated with a child's
developmental delay.2
Recurrent AOM has a multifactorial background that is still only partly
understood. Epidemiologic data have revealed several external factors predisposing
to otitis media, including day care outside the home, use of a pacifier, and
parental smoking.3 However, recent twin and
triplet studies4-5 have suggested
that there is a strong genetic component involved in middle ear infections.
This is supported by earlier findings6 of incidence
differences of AOM in different ethnic groups and epidemiologic analyses3 that have shown that a positive family history of
AOM is the most important risk factor for AOM. Genetically determined immunoglobulin
markers have also been studied in children with rAOM, and allotype G2m(23)
has been linked with otitis proneness.7-8
Moreover, an association between the HLA-A2 antigen and rAOM has been reported
but not between HLA-A2 and secretory otitis media.9-10
Cytokines are bioactive proteins that widely mediate host responses
to inflammatory stimuli. In the pathogenesis of an inflammation in the respiratory
tract, they regulate proliferation, chemotaxis, and the activation of inflammatory
cells.11 Constitutional polymorphisms in cytokine
genes may lead to individual variations in cytokine secretion.12-13
The outcomes of many infectious diseases, such as malaria,14
meningococcal meningitis,15 and Epstein-Barr
virus,16 have been associated with these polymorphisms.
Cytokines also play a key role in the pathogenesis of otitis media.17-19 The aim of the present
work was to study the polymorphisms of tumor necrosis factor (TNF) ,
interleukin (IL) 1, IL-1ß, and IL-1 receptor antagonist (IL-1Ra)
genes in patients with rAOM.
PARTICIPANTS AND METHODS
This study was conducted at Tampere University Hospital, Tampere, Finland,
and included 20 different families with a high occurrence of AOM. In each
family, at least 1 parent and all the children had a history of rAOM. The
criterion for rAOM was at least 6 AOM episodes within 12 months or at least
10 AOM episodes during a lifetime. The medical history was based on medical
records and interview data. A blood sample for genetic analysis was obtained
from 63 patients with rAOM.
Control blood samples were obtained from 400 unselected healthy blood
donors (The Finnish Red Cross Blood Transfusion Centre, Tampere) living in
the same area as the study group members. Donors were aged 18 to 60 years.
The history of AOM in the control population was not known.
The study was approved by the ethical committee of Tampere University
Hospital, and written informed consent was received from the parents in each
family with rAOM.
ANALYSIS OF CYTOKINE GENE POLYMORPHISMS
DNA specimens from citrated whole blood samples were prepared using
standard methods.
Tumor Necrosis Factor -308
A 107–base pair fragment of the TNF- gene promoter region
containing the G A substitution was amplified using polymerase chain
reaction (PCR) primers 5'-AGGCAATAGGTTTTGAGGGCCAT-3' and 5'-TCCTCCCTGCTCCGATTCCG-3'.
The amplified product was digested with NcoI and
analyzed using electrophoresis on 9% polyacrylamide gel.20
Interleukin 1-889
The base exchange at position -889 of the IL-1 gene was analyzed
as previously described.21 Oligonucleotides
5'-AAGCTTGTT CTACCACCTGAACTAGGC-3' and 5'-TTACATATGAGC CTTCCATG-3'
flanking the polymorphic site were used as primers in PCR. The products were
digested with NcoI, and the resultant products were
analyzed using 9% polyacrylamide gel electrophoresis.
Interleukin 1ß-511
The region that contains the AvaI polymorphic
site at position -511 of the IL-1-ß gene was amplified using PCR.22 The oligonucleotides 5'-TGGCATTGATCTGGTTCATC-3'
and 5'-GTTTAGGAATCTTCCCACTT-3' were used as primers. Fragments
were analyzed using electrophoresis on 9% polyacrylamide gel stained with
ethidium bromide.
Interleukin 1ß+3953
Position +3953 within exon 5 of the IL-1ß gene has a single base
pair polymorphism.23 The polymorphic region
containing the TaqI restriction site was amplified
using the following primers: 5'-GTTGTCATCAGACTTTGACC-3' and 5'-TTCAGTTCATATGGACCAGA-3'.
Fragments were analyzed using electrophoresis on 9% polyacrylamide gel stained
with ethidium bromide.
Interleukin 1Ra
The IL-1Ra exon 2 polymorphism was analyzed as described previously.24 Oligonucleotides 5'-CTCAGCAACACTCCTAT-3'
and 5'-TCCTGGTCTGCAGGTAA-3' were used as primers in PCR. The final
PCR product was analyzed using electrophoresis on a 2% agarose gel stained
with ethidium bromide.
STATISTICAL ANALYSIS
The distribution of the TNF--308, IL-1-889, IL-1ß-511,
IL-1ß+3953, and IL-1Ra allele frequencies in the rAOM families was compared
with that of healthy blood donors. Statistical analysis was performed using
the  2 test.
RESULTS
The families included in the study were extremely prone to otitis (Table 1), but none of the patients had
been known to have an immunologic deficiency. In every patient, the primary
condition was rAOM, but secretory otitis media later developed in 20 (32%).
Three patients also experienced subacute or chronic mastoiditis, and mastoidectomy
was performed in 2 patients. In the study group, 18 patients were adults and
45 were children; 57% were male (n = 36) and 43% were female (n = 27). Seventeen
patients from 7 different families had a history of allergic disorders (atopic
dermatitis, asthma, or allergic rhinitis): 10 in 2 families and 7 in 5 families.
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Table 1. Characteristics of 20 Families With rAOM*
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The distribution of proinflammatory cytokine alleles (TNF--308,
IL-1-889, IL-1ß-511, and IL-1ß+3953) (Table 2) and anti-inflammatory IL-1Ra alleles (Table 3) did not differ significantly in the rAOM group vs the control
group. When calculating the children's data separately, the result remained
basically the same. The greatest difference was in the distribution of IL-1-889
alleles, but this did not reach statistical significance (P = .07). However, in a subgroup of patients with rAOM without a history
of allergic disorders (atopy, asthma, or allergic rhinitis), allele frequencies
of IL-1-889 differed significantly from those in the control group
(allele 1: 0.78 vs 0.67; P = .03).
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Table 3. Polymorphism of IL-1Ra in 63 Patients With rAOM vs 400 Controls*
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COMMENT
Cytokines are of central importance in the pathogenesis of inflammation.
Three main proinflammatory cytokines—TNF-, IL-1, and IL-6—and
anti-inflammatory cytokines such as IL-1Ra and IL-10 down-regulate the inflammatory
process.12-13 The production of
cytokines is regulated by genetic elements. Polymorphisms associated with
these genes are thought to lead to individual variations in cytokine secretions
and are inherited in mendelian fashion.12-13
Cytokine gene polymorphism is associated with the outcomes of various infectious
diseases.14-16
In the past few years, several studies have been published regarding
cytokines and otitis media, and it has been possible to determine various
types of cytokines from middle ear effusions (IL-1ß, IL-2, TNF-,
interferon  ,19 IL-6,25
and IL-826) and from nasopharyngeal secretions
(IL-1ß, IL-6, and TNF-),18 indicating
that cytokines play a key role in the pathogenesis of AOM. High concentrations
of TNF- in middle ear effusions correlate with the persistence of secretory
otitis media.27 Furthermore, IL-1ß, IL-6,
and TNF- concentrations measured from the nasopharyngeal secretions
of children with rAOM are lower compared with healthy children.18
It has also been postulated that children with rAOM may have low IL-2 production,
leading to lower serum IgA levels in mucosal secretions.28
On the other hand, inflammatory cytokines can also increase bacterial adherence
to and invasion of cells29; hence, if a respiratory
tract virus infection, which usually precedes an AOM attack, induces too high
or prolonged cytokine production, this might theoretically increase the risk
of developing AOM.
According to results of twin and triplet studies,4-5
the weight of genetic factors in middle ear infections is 0.45 to 0.64 among
males and 0.74 to 0.79 among females. The genetic foundation of susceptibility
to rAOM is unclear. Prellner et al found that the genetically determined immunoglobulin
marker G2m(23) was present in 95% of children with rAOM but in only 64% of
children with no history of AOM (P = .01).7-8 Kalm et al9
revealed that the HLA-A2 antigen occurred significantly more often in the
rAOM group than in controls (80% vs 56%; P<.01)
and that the frequency of the HLA-A3 antigen was lower. The TNF- gene
is located on the short arm of chromosome 6 in the major histocompatibility
complex between HLA class I and III loci and can be coinherited with certain
HLA antigens.30 However, in the present study,
we could not find differences in allele frequencies of TNF--308 between
the study groups.
The IL-1 gene family includes IL-1, IL-1ß, and IL-1Ra, which
are situated on the short arm of chromosome 2.31
In the present study, the allele frequencies of the IL-1 gene did not differ
significantly in the rAOM group vs controls. The greatest difference was in
the allelic distribution of the IL-1ß-511 gene, but it did not reach
statistical significance (P = .08). However, in a
subgroup of patients with rAOM who did not have any allergic disorders, there
were significant differences in the IL-1-889 allele frequencies. Because
these differences were not so great and because the number of patients in
the subgroup analysis was small, the value of this finding should be considered
cautiously. On the other hand, it is possible that the pathogenesis of middle
ear inflammation in allergic patients differs from that in nonallergic patients.
In the present study we paid extra attention to selecting families who
were extremely prone to otitis; however, the control population was not ideal
because the occurrence of AOM in this population was not known. We have no
reason to assume that our control population would have had an unusually high
occurrence of AOM. Also, the frequency of TNF alleles reported20
in other populations did not differ from that in our control population.
Otitis media has a multifactorial background. The pathogenesis of AOM
is regulated by many external and internal factors, including normal flora,32 bacterial-viral interactions,33
and individual immunologic reactions. Recent studies have shown that a strong
genetic component is involved in middle ear infections,4-5
but the genetic foundation of susceptibility to rAOM is unclear. In the present
work, we studied the polymorphisms of TNF--308, IL-1-889, IL-1ß-511,
IL-1ß+3953, and IL-1Ra genes in patients with rAOM, but we did not find
any clear association between the studied cytokine genes and susceptibility
to recurrent middle ear infections, except in a subgroup of patients without
any allergic disorders. More data are needed to assess the value of this finding.
AUTHOR INFORMATION
Accepted for publication August 16, 2001.
This study was supported by a grant from The Medical Research Fund of
Tampere University Hospital.
Corresponding author and reprints: Veli-Pekka Joki-Erkkilä,
MD, Department of Otorhinolaryngology, Head and Neck Surgery, Tampere University
Hospital, PO Box 2000, FIN-33521 Tampere, Finland (e-mail: vp.jokierkkila{at}nokianlaakariasema.fi).
From the Departments of Otorhinolaryngology, Head and Neck Surgery
(Drs Joki-Erkkilä and Puhakka), and Clinical Microbiology (Dr Hurme),
Tampere University Hospital, and the Department of Microbiology and Immunology,
University of Tampere Medical School (Dr Hurme), Tampere, Finland.
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