International Journal of Pediatric Otorhinolaryngology 79 (2015) 557–560
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Chronic tonsillitis is not associated with beta defensin 1 gene polymorphisms in Turkish population Fatih Arslan a, Seda Turkoglu Babakurban a,*, Selim S. Erbek a, Feride I. Sahin b, Yunus Kasım Terzi b a b
Department of Otolaryngology, Baskent University, Faculty of Medicine, M. Fevzi Cakmak Caddesi, 5. Sokak, No: 48, 06490 Ankara, Turkey Department of Medical Genetics, Baskent University, Faculty of Medicine, Kubilay Sokak, No: 36 Maltepe, 06570 Ankara, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 October 2014 Received in revised form 26 December 2014 Accepted 22 January 2015 Available online 30 January 2015
Background: Defensins are antimicrobial peptides expressed on mucosal surfaces. They function as part of the innate immune system. Palatine tonsils play important roles in innate immune system. However, our knowledge on the pathophysiology of chronic tonsils is limited. Objective: The aim of this study was to investigate the association between beta defensin 1 gene single nucleotide polymorphisms and chronic tonsillitis. Study design: Prospective, non-randomized, controlled clinical study. Setting: Tertiary referral center. Subjects and methods: Eighty six patients with chronic tonsillitis and eighty controls without history of chronic tonsillitis were enrolled in this study. Genotypes were determined by restriction fragment length polymorphism analyses after polymerase chain reaction. Results: Genotype and allele frequencies of the -20G/A (rs11362), -44C/G (rs1800972) and -52G/A (rs1799946) single nucleotide polymorphisms were not statistically different between patients and control groups (p > 0.05). Conclusion: In this study, we found that DEFB1 gene -20G/A, -44C/G and -52G/A single nucleotide polymorphisms were not associated with chronic tonsillitis. Studies, which analyse other polymorphism of the beta defensin 1 gene in large case series, should be conducted to understand the role of DEFB1 gene on chronic tonsillitis. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: DEFB1 gene Chronic tonsillitis Genotype Polymorphism
1. Introduction The lymphatic tissue of the oral cavity, wherein the innate and acquired immune system establishes contact with one another, is in the direct contact with environment which is overloaded with bacteria, virus and fungi. These various antigens enter the body especially through the mouth. Palatine tonsils are located in the oropharynx so that this critical location gives them a significant role in initiating immune responses [1]. Palatine tonsils have roles related with innate and acquired immune reactions. Innate immune response represents the first line of host defense and it enables the palatine tonsils to recognize
* Corresponding author. Tel.: +90 3122238534/+90 5057806296; fax: +90 3122237597. E-mail addresses: [email protected] (F. Arslan), [email protected], [email protected] (S.T. Babakurban), [email protected] (F.I. Sahin), [email protected] (Y.K. Terzi). http://dx.doi.org/10.1016/j.ijporl.2015.01.028 0165-5876/ß 2015 Elsevier Ireland Ltd. All rights reserved.
and response to pathogenic organisms. Besides, it instructs the adaptive immune system [2]. Defensins play an important role in the innate immune system [3]. Defensins can also induce adaptive answer by their signaling function [4]. The defensins are of 3–5 kDa, cationic, cysteine-rich proteins [5]. Human defensins are divided into alpha and beta subgroups [1]. Genes encoding defensins are located on chromosome 8 (8p 22–23) [6]. There are 28 human beta defensins (hBDs) but expression characteristics were demonstrated only in four of them [7]. In general, hBDs are endogenous antibiotics against gram-negative bacteria, gram-positive bacteria, fungi, viruses and protozoa [8]. This protein was first isolated from hemofiltrate [9]. Expression of hBD1 was showed in epithelial cells of the urinary tract, respiratory tract, and keratinocytes [10–14]. Human beta defensin 1 expression is constitutive [13], but it was showed that microbial pathogens and cytokines stimulate the expression of beta defensins in oral epithelial cells during inflammation [15]. Thereafter, palatine tonsils are capable of producing proinflammatory molecules shaping the immune response [16].
558
F. Arslan et al. / International Journal of Pediatric Otorhinolaryngology 79 (2015) 557–560
Chronic infectious or inflammatory diseases of upper aerodigestive tract are commonly encountered disease in the general population. One of these inflammatory diseases is chronic tonsillitis [17]. There are a lot of unknowns about processes underlying these inflammations. Determination of the innate immune system components in the palatine tonsils will lead to provide better information about the role of the palatine tonsils in immune response [16]. Beta defensins are a component of innate immune response. It was shown that the expression of hBD1 varies between individuals [18]. Therefore, our hypothesis was that individuals may exhibit increased susceptibility to chronic tonsillitis upon the differences in genotypes of beta defensin 1 (DEFB1) gene. However, as far as we know, there is no study investigating the association of DEFB1 genotypes with chronic tonsillitis. The aim of this study was to determine whether three coding single nucleotide polymorphisms (SNPs) (-20G > A -rs11362, -44C > G-rs1800972 and -52G > Ars1799946) located in 5’-untranslated region (UTR) of DEFB1 gene were related to susceptibility to chronic tonsillitis.
2. Materials and methods A prospective and controlled clinical study was conducted. Our study group was comprised of 86 consecutive patients who underwent tonsillectomy with the diagnosis of chronic tonsillitis. Chronic tonsillitis was considered in patients who received at least three or more courses of antibiotic therapy for the treatment of tonsillitis in the last 12 months and/or in patients who had sore throat with or without halitosis that did not respond to antibiotic therapy for at least 3 months [19]. Paradise criteria [20] were used for tonsillectomy decision. The control group consisted of 80 healthy volunteers without chronic tonsillitis or history of tonsillectomy. There was a big discrepancy between the ages of patients and controls because of ethical problems with children related with taking blood in the absence of any medical problem. Furthermore because of decreasing the incidence of chronic tonsillitis in the advanced ages, it was an advantage that ages of volunteers were more than patients. Patients with known immunodeficiency and malignancy were excluded from the study. Baskent University Institutional Review Board and Ethics Committee approved this study, and all participants provided informed consent (Project number: KA 11/244). 2.1. Genotyping Five milliliters of venous blood was collected from each subject. Genomic DNA was extracted using the QIAamp DNA Blood Mini kit (QIAGEN GmbH, Hilden, Germany). The polymerase chain reaction (PCR)—restriction fragment length polymorphism method was performed for genotyping. Two regions of the DEFB1 gene, each of which contained a SNP site, were amplified with forward (50 CACCAGGGGTTAGCGATTAG-30 ) and reverse (50 -CAACTGCAGGCCACTCAAC-30 ) primers [21]. The resulting 400 bp amplicon was digested with restriction enzymes BstNI, BsmAI and NlaIV to detect 20G > A (rs11362), -44C > G (rs1800972) and -52G > A (rs1799946) SNPs, respectively. Following the restriction enzyme digestion, samples were run on agarose gel, and genotypes were determined. 2.2. Statistical evaluation SPSS software (Statistical Package for the Social Sciences, version 15.0, SSPS Inc, Chicago, IL) was used for statistical assessments. DEFB1 genotypes in patients with chronic tonsillitis and in controls were analyzed by the chi-square and the Pearson correlation analyze test.
3. Results Our patient group was consisted of 86 consecutive patients with chronic tonsillitis; 44 were men and 42 were women (mean age 10.49 9.64 years). The control group was consisted of 80 healthy volunteers; 51 were men and 29 were women (mean age 27.11 16.03 years). There was a statistically significant difference in terms of age, between the study and control groups (p < 0.001) (Table 1). When gender was considered, there was no statistically significant difference between the two groups (p = 0.572) (Table 1). The ethnicity of the patients and volunteers were the same, Turkish. DEFB1 genotype distributions and allele frequencies of the patients and controls are given in Table 2. The distribution of the -20G/A, -44C/G and -52G/A genotypes and the frequencies of the alleles did not differ between the patient and control groups (p > 0.05). 4. Discussion Our study is the first one to investigate the association between DEFB1 SNPs and chronic tonsillitis. We found no association between DEFB1 variants and chronic tonsillitis. Therefore we suggested that DEFB1 -20G/A, -44C/G and -52G/A SNPs are not associated with susceptibility of chronic tonsillitis in our population. The polymorphisms are different substitutions causing relatively conservative amino acid changes in the proteins. -20G/A, 44C/G and -52G/A SNPs are situated in the 50 untranslated region of the DEFB1 gene. They are suggested as important for regulating gene expression [22]. Different findings in the pathogenesis of chronic inflammatory diseases reveal the presence of defensins [23,24]. Braida et al. [22] reported an association between -44C/G SNP in the DEFB1 gene and HIV-1 infection in children born to seropositive mothers. They hypothesized that hBD1 is important for protecting skin and mucosa of newborns. The (-44) C allele was highly conserved in primate species. It was shown to correlate with the risk of Candida spp. carriage [8]. Beside this, Milanese et al. [23] demonstrated significant correlation between SNPs in the 50 UTR of the DEFB1 gene and the risk of being infected with HIV-1 in Brazilian children. Therefore, this polymorphism is thought to be important in the modulation of DEFB1 expression. Levy et al. [25] suggested that DEFB1 interacts with infectious and other environmental exposures in a time dependent manner to influence the disease process. They found an association among SNPs -1816 T > C, +692 G > A and asthma. The authors considered that their findings support the role for DEFB1 in airway inflammation. The other findings supporting this study that epithelial cells constitutively express hBD1 in the airway [26]. Thus Beside, DEFB1 can contribute to airway inflammation by its direct antibiotic activity and chemoattractant activity [27]. As in airway epithelium, Chae et al. [28] showed that hBD1 expressed in the surface epithelia of palatine tonsil. Therefore they thought that hBD-1 of the palatine tonsil may also play an important role in innate defense against microorganisms. On the other hand,
Table 1 Demographic data.
Age (mean SD) Gender
Patients with chronic tonsillitis
Controls
p Value
10.49 9.64 years 44 Were men 42 Were women
27.11 16.03 years 51 Were men 29 Were women
A) Genotype GG Genotype GA Genotype AA Allele G Allele A
26 39 21 91 81
(%30.23) (%45.34) (%22.87) (%52.9) (%47.09)
36 27 17 99 61
(%45) (%33.75) (%21.25) (%61.87) (%38.13)
0.13
(-44C > G) Genotype CC Genotype CG Genotype GG Allele C Allele G
63 19 4 145 27
(%73.25) (%22.09) (%4.65) (%84.3) (%15.69)
62 11 7 135 25
(%77.5) (%13.75) (%8.75) (%84.37) (%15.63)
0.25
(-20 G > A) Genotype GG Genotype GA Genotype AA Allele G Allele A
33 40 13 106 66
(%38.37) (%46.51) (%15.11) (%61.62) (%38.37)
23 35 22 81 79
(%28.75) (%43.75) (%27.5) (%50.62) (%49.38)
0.22
0.57 0.42
0.84 0.15
0.56 0.43
Schwaab et al. [17] found that hBD1 to 3 were expressed at same concentration as in acute tonsillitis, hypertrophic tonsil and peritonsillar abscess. The authors attributed this condition to changed external circumstances as reduced number of macrophages, antigen presenting cells, dendritic cells and colonies in the biofilm. In accordance with this study, Wang et al. [29] showed that hBD1 was similar in the patients and the normal group, but hBD2 expressed significantly higher levels in chronic tonsillitis group. In another study, Claeys et al. [4] demonstrated that there was strong hBD2 expression in tonsillar tissue, in with no (regardless of) significant difference between hypertrophic tonsillar disease and recurrent tonsillitis, compared with nasalparanasal mucosa and adenoids. Therefore, the authors concluded that defensin induction needs more intensive and prolonged microorganical triggering. In another chronic inflammation, Loo et al. [30] demonstrated that the DEFB1 SNP at genomic position -1654 was present more frequently in chronic periodontitis than healthy group. They proposed that this SNP could be a potential marker for the assessment of risk for periodontal disease. However, an association between DEFB1 gene polymorphism and periodontitis could not be established in previous studies as in our study [31]. Our study suggests that DEFB1 SNPs at genomic positions 20G/A, -44C/G and -52G/A are not associated with chronic tonsillitis. Despite this disconnection, some limitations of our study might affect the results. First, this is a population-based study with a limited number of subjects. Although we only investigated three SNPs in the 50 UTR of DEFB1, additional polymorphisms in different localizations have been described in the literature for DEFB1. In this regard, it could be planned to compare the expression level of DEFB1 in tonsil tissues of patient with chronic tonsillitis and in those of control subjects and to characterize the cellular source of their mRNA expression. Beside this, further studies with larger groups are required to establish the role of the DEFB1 in chronic tonsillitis and its clinical consequences. 5. Conclusion There are a lot of unknowns about processes underlying chronic tonsillitis. Therefore, determination of the innate immune system components in the palatine tonsils will lead to be better informed
559
about the role of the palatine tonsils in immune response. Beta defensins are a component of the innate immune response and we investigated SNPs of DEFB1 gene. This is the first report on the association of DEFB1 polymorphisms with chronic tonsillitis. We did not find any association, but changes in DEFB1 structure or function may lead to a modification of antimicrobial defense in the oral mucosa. So that, DEFB1 may still play a role in the pathophysiology of chronic tonsillitis. References [1] H. Nave, A. Gebert, R. Pabst, Morphology and immunology of the human palatine tonsil, Anat. Embryol. 204 (2001) 367–376. [2] M.J. Lange, J.C. Lasiter, M.L. Misfeldt, Toll-like receptors in tonsillar epithelial cells, Int. J. Pediatr. Otorhinolaryngol. 73 (2009) 613–621. [3] J.E. Meyer, U.H. Beier, T. Go¨ro¨gh, S. Schreıber, C. Beck, S. Maune, Defensin and chemokine expression patterns in the palatine tonsil: a model of their local interaction, Eur. Arch. Otorhinolaryngol. 263 (2006) 319–326. [4] S. Claeys, T. de Belder, G. Holtappels, P. Gevaert, B. Verhasselt, P. van Cauwenberge, et al., Human beta-defensins and toll-like receptors in the upper airway, Allergy 58 (2003) 748–753. [5] M. Pazgier, A. Prahl, D.M. Hoover, J. Lubkowski, Studies of the biological properties of human beta-defensin 1, J. Biol. Chem. 282 (2007) 1819–1829. [6] L. Liu, C. Zhao, H.H.Q. Heng, T. Ganz, The human beta defensin-1 and alphadefensins are encoded by adjacent genes: two families with differing disulfide topology share a common ancestry, Genomics 43 (1997) 316–320. [7] B.C. Schutte, J.P. Mitros, J.A. Bartlett, J.D. Walters, H.P. Jia, M.J. Welsh, et al., Discovery of five con- served beta-defensin gene clusters using a computational search strategy, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 2129–2133. [8] M. Zasloff, Antimicrobial peptides of multicellular organisms, Nature 415 (2002) 389–395. [9] K.W. Bensch, M. Raida, H.J. Ma¨gert, P. Schulz-Knappe, W.G. Forssmann, hBD-1: a novel beta-defensin from human plasma, FEBS Lett. 368 (1995) 331–335. [10] P.B. McCray Jr., L. Bentley, Human airway epithelia express a beta-defensin, Am. J. Respir. Cell Mol. Biol. 16 (1997) 343–349. [11] M.J. Goldman, G.M. Anderson, E.D. Stolzenberg, U.P. Kari, M. Zasloff, J.M. Wilson, Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis, Cell 88 (1997) 553–560. [12] E.V. Valore, C.H. Park, A.J. Quayle, K.R. Wiles, P.B. McCray Jr., T. Ganz, Human betadefensin-1: an antimicrobial peptide of urogenital tissues, J. Clin. Invest. 101 (1998) 1633–1642. [13] C. Zhao, I. Wang, R.I. Lehrer, Widespread expression of beta-defensin hBD-1 in human secretory glands and epithelial cells, FEBS Lett. 396 (1996) 319–322. [14] O.E. Sørensen, J.B. Cowland, K. Theilgaard-Mo¨nch, L. Liu, T. Ganz, N. Borregaard, Wound healing and expression ofantimicrobial peptides/polypeptides in humankeratinocytes, a consequence of common growth factors, J. Immunol. 170 (2003) 5583–5589. [15] G. Diamond, L. Ryan, Beta-defensins: what are they really doing in the oral cavity? Oral Dis. 17 (2011) 628–635. [16] M.J. Lesmeister, M.R. Bothwell, M.L. Misfeldt, Toll-like receptor expression in the human nasopharyngeal tonsil (adenoid) and palantine tonsils: a preliminary report, Int. J. Pediatr. Otorhinolaryngol. 70 (2006) 987–992. [17] M. Schwaab, A. Gurr, S. Hansen, A.M. Minovi, J.P. Thomas, H. Sudhoff, et al., Human beta-defensins in different states of diseases of the tonsilla palatine, Eur. Arch. Otorhinolaryngol. 267 (2010) 821–830. [18] S. Krisanaprakornkit, A. Weinberg, C.N. Perez, B.A. Dale, Expression of the peptide antibiotic human beta-defensin 1 in cultured gingival epithelial cells and gingival tissue, Infect. Immun. 66 (1998) 4222–4228. [19] D.L. Witsell, L.J. Orvidas, M.G. Stewart, M.T. Hannley, E.M. Weaver, B. Yueh, et al., Quality of life after tonsillectomy in adults with recurrent or chronic tonsillitis, Otolaryngol. Head Neck Surg. 138 (2008) 1–8. [20] R.F. Baugh, S.M. Archer, R.B. Mitchell, R.M. Rosenfeld, R. Amin, J.J. Burns, et al., Clinical practice guideline: tonsillectomy in children, Otolaryngol. Head Neck Surg. 144 (2011) 1–30. [21] R.J. Jurevic, M. Bai, R.B. Chadwick, T.C. White, B.A. Dale, Single nucleotide polymorphisms (SNPs) in humanb-defensin 1: high-throughput SNP assays and association with Candida carriage in type I diabetics and nondiabetic controls, J. Clin. Microbiol. 41 (2003) 90–96. [22] L. Braida, M. Boniotto, A. Pontillo, P.A. Tovo, A. Amoroso, S. Crovella, A singlenucleotide polymorphism in the human beta-defensin 1 gene is associated with HIV-1 infection in Italian children, AIDS 18 (2004) 1598–1600. [23] T.F. Leung, C.Y. Li, E.K. Liu, N.L. Tang, I.H. Chan, E. Yung, et al., Asthma and atopy are associated with DEFB1 polymorphisms in Chinese children, Genes Immun. 7 (2006) 59–64. [24] M. Milanese, L. Segat, A. Pontillo, L.C. Arraes, J.L. de Lima Filho, S. Crovella, DEFB1 gene polymorphisms and increased risk of HIV-1 infection in Brazilian children, AIDS 20 (2006) 1673–1675. [25] H. Levy, B.A. Raby, S. Lake, K.G. Tantisira, D. Kwiatkowski, R. Lazarus, et al., Association of defensin beta-1 gene polymorphisms with asthma, J. Allergy Clin. Immunol. 115 (2005) 252–258. [26] P.K. Singh, H.P. Jia, K. Wiles, J. Hesselberth, L. Liu, B.A. Conway, et al., Production of beta-defensins by human airwy epithelia, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 14961–14966.
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[27] D. Yang, O. Chertov, S.N. Bykovskaia, Q. Chen, M.J. Buffo, J. Shogan, et al., Betadefensins: linking innate and adaptive immunity through dendritic and T cell CCR6, Science 286 (1999) 525–528. [28] S.W. Chae, S.H. Lee, J.H. Cho, H.M. Lee, G. Choi, S.J. Hwang, Expression of human betadefensin 1 mRNA in human palatine tonsil, Acta Otolaryngol. 121 (2001) 414–418. [29] C. Wang, Z. Dong, Z. Yang, Expression of human beta-defensin in palatine tonsil, Lin Chuang Er Bi Yan Hou Ke Za Zhi. 18 (2004) 129–131.
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Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Chronic tonsillitis is not associated with beta defensin 1 gene polymorphisms in Turkish population Fatih Arslan a, Seda Turkoglu Babakurban a,*, Selim S. Erbek a, Feride I. Sahin b, Yunus Kasım Terzi b a b
Department of Otolaryngology, Baskent University, Faculty of Medicine, M. Fevzi Cakmak Caddesi, 5. Sokak, No: 48, 06490 Ankara, Turkey Department of Medical Genetics, Baskent University, Faculty of Medicine, Kubilay Sokak, No: 36 Maltepe, 06570 Ankara, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 October 2014 Received in revised form 26 December 2014 Accepted 22 January 2015 Available online 30 January 2015
Background: Defensins are antimicrobial peptides expressed on mucosal surfaces. They function as part of the innate immune system. Palatine tonsils play important roles in innate immune system. However, our knowledge on the pathophysiology of chronic tonsils is limited. Objective: The aim of this study was to investigate the association between beta defensin 1 gene single nucleotide polymorphisms and chronic tonsillitis. Study design: Prospective, non-randomized, controlled clinical study. Setting: Tertiary referral center. Subjects and methods: Eighty six patients with chronic tonsillitis and eighty controls without history of chronic tonsillitis were enrolled in this study. Genotypes were determined by restriction fragment length polymorphism analyses after polymerase chain reaction. Results: Genotype and allele frequencies of the -20G/A (rs11362), -44C/G (rs1800972) and -52G/A (rs1799946) single nucleotide polymorphisms were not statistically different between patients and control groups (p > 0.05). Conclusion: In this study, we found that DEFB1 gene -20G/A, -44C/G and -52G/A single nucleotide polymorphisms were not associated with chronic tonsillitis. Studies, which analyse other polymorphism of the beta defensin 1 gene in large case series, should be conducted to understand the role of DEFB1 gene on chronic tonsillitis. ß 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: DEFB1 gene Chronic tonsillitis Genotype Polymorphism
1. Introduction The lymphatic tissue of the oral cavity, wherein the innate and acquired immune system establishes contact with one another, is in the direct contact with environment which is overloaded with bacteria, virus and fungi. These various antigens enter the body especially through the mouth. Palatine tonsils are located in the oropharynx so that this critical location gives them a significant role in initiating immune responses [1]. Palatine tonsils have roles related with innate and acquired immune reactions. Innate immune response represents the first line of host defense and it enables the palatine tonsils to recognize
* Corresponding author. Tel.: +90 3122238534/+90 5057806296; fax: +90 3122237597. E-mail addresses: [email protected] (F. Arslan), [email protected], [email protected] (S.T. Babakurban), [email protected] (F.I. Sahin), [email protected] (Y.K. Terzi). http://dx.doi.org/10.1016/j.ijporl.2015.01.028 0165-5876/ß 2015 Elsevier Ireland Ltd. All rights reserved.
and response to pathogenic organisms. Besides, it instructs the adaptive immune system [2]. Defensins play an important role in the innate immune system [3]. Defensins can also induce adaptive answer by their signaling function [4]. The defensins are of 3–5 kDa, cationic, cysteine-rich proteins [5]. Human defensins are divided into alpha and beta subgroups [1]. Genes encoding defensins are located on chromosome 8 (8p 22–23) [6]. There are 28 human beta defensins (hBDs) but expression characteristics were demonstrated only in four of them [7]. In general, hBDs are endogenous antibiotics against gram-negative bacteria, gram-positive bacteria, fungi, viruses and protozoa [8]. This protein was first isolated from hemofiltrate [9]. Expression of hBD1 was showed in epithelial cells of the urinary tract, respiratory tract, and keratinocytes [10–14]. Human beta defensin 1 expression is constitutive [13], but it was showed that microbial pathogens and cytokines stimulate the expression of beta defensins in oral epithelial cells during inflammation [15]. Thereafter, palatine tonsils are capable of producing proinflammatory molecules shaping the immune response [16].
558
F. Arslan et al. / International Journal of Pediatric Otorhinolaryngology 79 (2015) 557–560
Chronic infectious or inflammatory diseases of upper aerodigestive tract are commonly encountered disease in the general population. One of these inflammatory diseases is chronic tonsillitis [17]. There are a lot of unknowns about processes underlying these inflammations. Determination of the innate immune system components in the palatine tonsils will lead to provide better information about the role of the palatine tonsils in immune response [16]. Beta defensins are a component of innate immune response. It was shown that the expression of hBD1 varies between individuals [18]. Therefore, our hypothesis was that individuals may exhibit increased susceptibility to chronic tonsillitis upon the differences in genotypes of beta defensin 1 (DEFB1) gene. However, as far as we know, there is no study investigating the association of DEFB1 genotypes with chronic tonsillitis. The aim of this study was to determine whether three coding single nucleotide polymorphisms (SNPs) (-20G > A -rs11362, -44C > G-rs1800972 and -52G > Ars1799946) located in 5’-untranslated region (UTR) of DEFB1 gene were related to susceptibility to chronic tonsillitis.
2. Materials and methods A prospective and controlled clinical study was conducted. Our study group was comprised of 86 consecutive patients who underwent tonsillectomy with the diagnosis of chronic tonsillitis. Chronic tonsillitis was considered in patients who received at least three or more courses of antibiotic therapy for the treatment of tonsillitis in the last 12 months and/or in patients who had sore throat with or without halitosis that did not respond to antibiotic therapy for at least 3 months [19]. Paradise criteria [20] were used for tonsillectomy decision. The control group consisted of 80 healthy volunteers without chronic tonsillitis or history of tonsillectomy. There was a big discrepancy between the ages of patients and controls because of ethical problems with children related with taking blood in the absence of any medical problem. Furthermore because of decreasing the incidence of chronic tonsillitis in the advanced ages, it was an advantage that ages of volunteers were more than patients. Patients with known immunodeficiency and malignancy were excluded from the study. Baskent University Institutional Review Board and Ethics Committee approved this study, and all participants provided informed consent (Project number: KA 11/244). 2.1. Genotyping Five milliliters of venous blood was collected from each subject. Genomic DNA was extracted using the QIAamp DNA Blood Mini kit (QIAGEN GmbH, Hilden, Germany). The polymerase chain reaction (PCR)—restriction fragment length polymorphism method was performed for genotyping. Two regions of the DEFB1 gene, each of which contained a SNP site, were amplified with forward (50 CACCAGGGGTTAGCGATTAG-30 ) and reverse (50 -CAACTGCAGGCCACTCAAC-30 ) primers [21]. The resulting 400 bp amplicon was digested with restriction enzymes BstNI, BsmAI and NlaIV to detect 20G > A (rs11362), -44C > G (rs1800972) and -52G > A (rs1799946) SNPs, respectively. Following the restriction enzyme digestion, samples were run on agarose gel, and genotypes were determined. 2.2. Statistical evaluation SPSS software (Statistical Package for the Social Sciences, version 15.0, SSPS Inc, Chicago, IL) was used for statistical assessments. DEFB1 genotypes in patients with chronic tonsillitis and in controls were analyzed by the chi-square and the Pearson correlation analyze test.
3. Results Our patient group was consisted of 86 consecutive patients with chronic tonsillitis; 44 were men and 42 were women (mean age 10.49 9.64 years). The control group was consisted of 80 healthy volunteers; 51 were men and 29 were women (mean age 27.11 16.03 years). There was a statistically significant difference in terms of age, between the study and control groups (p < 0.001) (Table 1). When gender was considered, there was no statistically significant difference between the two groups (p = 0.572) (Table 1). The ethnicity of the patients and volunteers were the same, Turkish. DEFB1 genotype distributions and allele frequencies of the patients and controls are given in Table 2. The distribution of the -20G/A, -44C/G and -52G/A genotypes and the frequencies of the alleles did not differ between the patient and control groups (p > 0.05). 4. Discussion Our study is the first one to investigate the association between DEFB1 SNPs and chronic tonsillitis. We found no association between DEFB1 variants and chronic tonsillitis. Therefore we suggested that DEFB1 -20G/A, -44C/G and -52G/A SNPs are not associated with susceptibility of chronic tonsillitis in our population. The polymorphisms are different substitutions causing relatively conservative amino acid changes in the proteins. -20G/A, 44C/G and -52G/A SNPs are situated in the 50 untranslated region of the DEFB1 gene. They are suggested as important for regulating gene expression [22]. Different findings in the pathogenesis of chronic inflammatory diseases reveal the presence of defensins [23,24]. Braida et al. [22] reported an association between -44C/G SNP in the DEFB1 gene and HIV-1 infection in children born to seropositive mothers. They hypothesized that hBD1 is important for protecting skin and mucosa of newborns. The (-44) C allele was highly conserved in primate species. It was shown to correlate with the risk of Candida spp. carriage [8]. Beside this, Milanese et al. [23] demonstrated significant correlation between SNPs in the 50 UTR of the DEFB1 gene and the risk of being infected with HIV-1 in Brazilian children. Therefore, this polymorphism is thought to be important in the modulation of DEFB1 expression. Levy et al. [25] suggested that DEFB1 interacts with infectious and other environmental exposures in a time dependent manner to influence the disease process. They found an association among SNPs -1816 T > C, +692 G > A and asthma. The authors considered that their findings support the role for DEFB1 in airway inflammation. The other findings supporting this study that epithelial cells constitutively express hBD1 in the airway [26]. Thus Beside, DEFB1 can contribute to airway inflammation by its direct antibiotic activity and chemoattractant activity [27]. As in airway epithelium, Chae et al. [28] showed that hBD1 expressed in the surface epithelia of palatine tonsil. Therefore they thought that hBD-1 of the palatine tonsil may also play an important role in innate defense against microorganisms. On the other hand,
Table 1 Demographic data.
Age (mean SD) Gender
Patients with chronic tonsillitis
Controls
p Value
10.49 9.64 years 44 Were men 42 Were women
27.11 16.03 years 51 Were men 29 Were women
A) Genotype GG Genotype GA Genotype AA Allele G Allele A
26 39 21 91 81
(%30.23) (%45.34) (%22.87) (%52.9) (%47.09)
36 27 17 99 61
(%45) (%33.75) (%21.25) (%61.87) (%38.13)
0.13
(-44C > G) Genotype CC Genotype CG Genotype GG Allele C Allele G
63 19 4 145 27
(%73.25) (%22.09) (%4.65) (%84.3) (%15.69)
62 11 7 135 25
(%77.5) (%13.75) (%8.75) (%84.37) (%15.63)
0.25
(-20 G > A) Genotype GG Genotype GA Genotype AA Allele G Allele A
33 40 13 106 66
(%38.37) (%46.51) (%15.11) (%61.62) (%38.37)
23 35 22 81 79
(%28.75) (%43.75) (%27.5) (%50.62) (%49.38)
0.22
0.57 0.42
0.84 0.15
0.56 0.43
Schwaab et al. [17] found that hBD1 to 3 were expressed at same concentration as in acute tonsillitis, hypertrophic tonsil and peritonsillar abscess. The authors attributed this condition to changed external circumstances as reduced number of macrophages, antigen presenting cells, dendritic cells and colonies in the biofilm. In accordance with this study, Wang et al. [29] showed that hBD1 was similar in the patients and the normal group, but hBD2 expressed significantly higher levels in chronic tonsillitis group. In another study, Claeys et al. [4] demonstrated that there was strong hBD2 expression in tonsillar tissue, in with no (regardless of) significant difference between hypertrophic tonsillar disease and recurrent tonsillitis, compared with nasalparanasal mucosa and adenoids. Therefore, the authors concluded that defensin induction needs more intensive and prolonged microorganical triggering. In another chronic inflammation, Loo et al. [30] demonstrated that the DEFB1 SNP at genomic position -1654 was present more frequently in chronic periodontitis than healthy group. They proposed that this SNP could be a potential marker for the assessment of risk for periodontal disease. However, an association between DEFB1 gene polymorphism and periodontitis could not be established in previous studies as in our study [31]. Our study suggests that DEFB1 SNPs at genomic positions 20G/A, -44C/G and -52G/A are not associated with chronic tonsillitis. Despite this disconnection, some limitations of our study might affect the results. First, this is a population-based study with a limited number of subjects. Although we only investigated three SNPs in the 50 UTR of DEFB1, additional polymorphisms in different localizations have been described in the literature for DEFB1. In this regard, it could be planned to compare the expression level of DEFB1 in tonsil tissues of patient with chronic tonsillitis and in those of control subjects and to characterize the cellular source of their mRNA expression. Beside this, further studies with larger groups are required to establish the role of the DEFB1 in chronic tonsillitis and its clinical consequences. 5. Conclusion There are a lot of unknowns about processes underlying chronic tonsillitis. Therefore, determination of the innate immune system components in the palatine tonsils will lead to be better informed
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about the role of the palatine tonsils in immune response. Beta defensins are a component of the innate immune response and we investigated SNPs of DEFB1 gene. This is the first report on the association of DEFB1 polymorphisms with chronic tonsillitis. We did not find any association, but changes in DEFB1 structure or function may lead to a modification of antimicrobial defense in the oral mucosa. So that, DEFB1 may still play a role in the pathophysiology of chronic tonsillitis. References [1] H. Nave, A. Gebert, R. Pabst, Morphology and immunology of the human palatine tonsil, Anat. Embryol. 204 (2001) 367–376. [2] M.J. Lange, J.C. Lasiter, M.L. Misfeldt, Toll-like receptors in tonsillar epithelial cells, Int. J. Pediatr. Otorhinolaryngol. 73 (2009) 613–621. [3] J.E. Meyer, U.H. Beier, T. Go¨ro¨gh, S. Schreıber, C. Beck, S. Maune, Defensin and chemokine expression patterns in the palatine tonsil: a model of their local interaction, Eur. Arch. Otorhinolaryngol. 263 (2006) 319–326. 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