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The FcγRIIa polymorphism in Turkish children with asthma bronchial and allergic rhinitis
Clinical Biochemistry 40 (2007) 392 – 396
The FcγRIIa polymorphism in Turkish children with asthma bronchial and allergic rhinitis Figen Gulen a,⁎, Remziye Tanac a , Serdar Altinoz a , Afig Berdeli b , Dost Zeyrek a , Huseyin Koksoy a , Esen Demir a a
Aegean University School of Medicine, Department of Paediatrics, Division of Allergy and Pulmonology, TR-35100, Bornova/azmir, Turkey b Aegean University School of Medicine, Molecular Research Laboratory, Turkey Received 14 September 2006; received in revised form 27 October 2006; accepted 8 November 2006 Available online 5 January 2007
Abstract Objective: The aim of the present study was to evaluate the FcγRIIa polymorphism in Turkish children with atopic asthma and allergic rhinitis. Design and methods: In this study, 372 atopic children (192 asthma bronchial, 180 allergic rhinitis) between ages of 5 and 16 years old (11.3 ± 2.9) who were followed at Aegean University Paediatric Allergy and Pulmonology Outpatient Clinics and 234 healthy subjects as the control group were included. The evaluation of subjects included routine biochemical blood analysis and allergic workup based on the following laboratory determinants. The FcγRIIa polymorphism was determined using the polymerase chain reaction method. Results: Distribution of R131R genotype was significantly different among patient groups compared to controls (for asthmatic children OR: 2.64 95%CI: 1.22–5.79, p = 0.006; for allergic rhinitis OR: 2.58 95%CI: 1.18–5.71, p = 0.009). Frequency of 131R allele was significantly different among patient groups compared to controls (for asthmatic children OR: 1.66 95%CI: 1.22–2.26, p = 0.0007; for allergic rhinitis OR: 1.93 95%CI: 1.42–2.63, p = 0.00001). Conclusion: This study shows that FcγRIIa gene 131R allele represents an important genetic risk factor for bronchial asthma and allergic rhinitis susceptibility. © 2007 Published by The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: FcγRIIa gene; Polymorphism; Allergic rhinitis; Asthma bronchial; Children
Introduction Allergic diseases are considered to be multifactorial inflammatory diseases involving complex interactions between genetic and environmental influences [1]. The impact of genetic factors on the pathogenesis of allergy is currently an area of intense investigation. Epidemiological studies find more and more genes that are likely to contribute to allergic inflammation [2]. Fc receptors (FcR) are glycoproteins found on the surface of hamatopoietic cells that bind the Fc portion of immunoglobulin and provide a link between the humoral and cellular immune systems. Interaction of FcR with immunoglobulin provides the host with protection against infection by means of phagocytosis, antibody-dependent cellular cytotoxicity, release of inflammatory ⁎ Corresponding author. Fax: +90 232 342 6990. E-mail address: [email protected] (F. Gulen).
mediators, and enhancement of antigen presentation [3]. The efficacy of IgG-induced FcγR function displays inter-individual heterogeneity due to genetic polymorphisms of three FcγR subclasses: FcγRIIa, FcγRIIIa, and FcγRIIIb. Three FcγR subclasses display functionally relevant genetically determined polymorphisms [4]. FcγRII is found on the surface of monocytes, macrophages, neutrophils, platelets, basophils, eosinophils, and other cells [5]. FcγRIIa receptors exhibit a genetically determined polymorphism (FcγRIIa-R131 and FcγRIIa-H131), resulting in differential ability to recognize and be activated by human and murine IgG isotypes. This polymorphism results from a single base substitution from guanidine to adenine at nucleotide 494 of the coding region in exon 4, which leads to an amino acid change from arginine to histidine at position 131 of the second extracellular domain [6]. The H131 allele (494A) codes for histidine at position 131 and has a high affinity for human IgG2 and a low affinity for
0009-9120/$ - see front matter © 2007 Published by The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2006.11.014
F. Gulen et al. / Clinical Biochemistry 40 (2007) 392–396 Table 1 Characteristics of the study and control groups
Diagnosis Asthma (%) Allergic rhinitis (%) Age, years ± SD (range) Gender Girls Boys IgE(kU/L), mean ± SD Eosinophils (per mm3)
393
Patients and methods
Study group, n: 372
Control group, n: 234
p
192 (51.6) 180 (48.4) 11.5 ± 3.1 (5–16)
11.7 ± 2.6 (6–16)
=0.11 =0.53
140 232 401 ± 119 362 ± 211.4
100 134 33.6 ± 17.4 138 ± 102.8
<0.001 =0.001
murine IgG1, while the R131 allele (494G) has arginine at position 131 and has little or no affinity for human IgG2 but has a high affinity for murine IgG1. To a lesser extent, the two genotypes also differ in their ability to ligate human IgG3, with the homozygous FcγRIIa-H131 genotype having the higher affinity for IgG3. Thus, this polymorphism may influence susceptibility to diseases, especially in situations where IgG2, and to a lesser extent IgG3, are the predominant antibody subclasses produced. The phagocytic capacity of leukocytes with R131R for IgG2 and IgG3 opsonized bacteria is lower than the ones with H131H [5,7,8]. The FcγRIIa genotype might influence the ability to clear encapsulated organisms [9]. The FcγRIIa genotype is also related to a risk of other immune-mediated diseases, including heparin-associated thrombocytopenia [10,11] and immune complex clearance in systemic lupus erythematosus [12]. Fcγ receptor II (FcγRIIa) polymorphism may be relevant to FcγRIIa function. This might be linked to variability in immune response and therefore related to the pathogenesis of atopic diseases. The important role of FcγR in anaphylaxis has been shown in mice, unfortunately there are no available data showing the relevance of FcγR receptors in humans anaphylaxis. The anaphylaxis reaction in mice has been considered to be a typical immediate hypersensitivity response determined by the activation of mast cells via antigen-induced aggregation of an IgE-sensitized high affinity receptor for IgE (FcεRI) causing the release of systemic mediators [13,14]. In addition to modulating IgG-triggered hypersensitivity responses, FcγRII and III on mast cells are potent regulators of IgE-mediated responses and reveal the existence of a regulatory pathway for IgE triggering of effector cells through IgG FcR that could contribute to the etiology of the atopic response [15,16]. The aim of the present study was to evaluate the FcγRIIa polymorphism in Turkish children with atopic asthma and allergic rhinitis.
The study consisted of 372 atopic children (192 asthma bronchial, 180 allergic rhinitis; 140 girls, 232 boys; age range 5– 16 years). The children were followed in the Aegean University Paediatric Allergy and Pulmonology Outpatient Clinics. The control group comprised 234 ethnically matched unrelated volunteers (100 girls, 134 boys; age range 6–16 years). All subjects were Caucasians. The inclusion criteria were as follows: –patients with allergic rhinitis or bronchial asthma –a positive spIgE against respiratory allergens –a positive SPT against respiratory allergens. Diagnosis of allergy Evaluation of clinical status The asthmatic patients were evaluated according to GINA criteria [17] and allergic rhinitis patients were evaluated according to symptom scoring system [18,19]. Evaluation of subjects included routine serum biochemical analysis and allergic workup based on the following laboratory determinants: eosinophilic granulocyte count in the peripheral blood, serum total IgE level, specific IgE levels against some inhaled allergens, and skin prick test. The eosinophilic granulocyte count was considered to be elevated when it exceeded 4% of total white blood cell count. Total IgE and specific IgE levels were estimated with enzyme immune assay method by using a 3M diagnostic System Kit. Total IgE levels were related to the age-specific limits provided by the manufacturer. Values that exceeded 100 kU/L were considered to be high. The presence of allergenspecific IgE antibodies was estimated with selected aeroallergens, i.e. house dust, ascarids (Dermatophagoides pteronyssinus and Dermatophagoides farinae), feathers, cat and dog hair, and pollens of Graminaceae, weeds, and trees. Pharmacia CAP system was used; 0.35 kU/L was regarded as positive. Skin prick test All patient's skin prick test was performed by a pediatric allergy specialist. Standard respiratory allergen panel included tree pollen, grass pollen, dust mite, fungus, animal dander, grains pollen, wild grass, flower pollen, and latex (Stallergenes S.A., France). Allergens were first dropped on the anterior side
Table 2 Genotype distribution of FcγRIIa-131 in asthmatic children and controls FcγRIIa genotypes HH HR RR a
Asthma group observed n
%
80 88 24
41.7 45.8 12.5
Hardy–Weinberg expectancy.
Asthma group expected, n a 78.6 88.4 24.8
Control group observed n
%
130 92 12
55.6 39.3 5.1
Control group expected, na
OR
95%CI
p
131.6 87.7 14.6
1 1.31 2.64
– 0.87–1.96 1.22–5.79
1 0.175 0.006
394
F. Gulen et al. / Clinical Biochemistry 40 (2007) 392–396
Table 3 Genotype distribution of FcγRIIa-131 in children with allergic rhinitis and controls FcγRIIa genotypes HH HR RR a
Allergic rhinitis group observed n
%
62 96 22
34.4 53.3 12.2
Allergic rhinitis group expected, n a 66.9 85.6 27.3
Control group observed n
%
130 92 12
55.5 39.3 5.1
Control group expected, na
OR
95%CI
p
131.6 87.7 14.6
1 1.76 2.5
– 1.17–2.67 81.18–5.71
1 0.004 0.009
Hardy–Weinberg expectancy.
of the forearm, and then pierced by stallerpoint. After 20 min, the test was evaluated. Histamine solution of 1 mg/mL was used as positive control and serum physiologic was used as negative control. Results were evaluated according to EAACI criteria [20]. The result of the control test was considered to be positive if the diameter of the blister was 3 mm larger than that of a negative control or if some reaction occurred that was at least half the size of that with histamine. Results were evaluated according to EAACI criteria [20]. The children were classified into three groups as follows: children with atopic bronchial asthma (192 subjects); children with allergic rhinitis (180 subjects); healthy children as control group (234 subjects). Exclusion criteria Children with recurrent respiratory tract infections, bronchitis obliterans, and chronic rhinopharyngitis were excluded from the study. Patients with clinical and laboratory findings of other chronic systemic diseases or immune deficiency were also excluded. DNA purification Genomic DNA from patients and healthy controls was extracted from peripheral blood leukocytes with QIAamp DNA Blood Mini Kits (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. FcγRIIa genotyping FcγRIIa genotyping was performed by using the method defined by Flesch BK et al. [21]. We used a 25-μL PCR mixture containing 2.5 μI of genomic DNA, 2.5 μI of 10× PCR buffer (Applied Biosystems, Foster City, CA, USA), 2 mM MgCl2, 200 μmol/L of each dNTP (Promega, Madison, USA), and 0.5 U AmpliTag DNA Polymerase (Applied Biosystems). In addition, 0.5 μmol/L of H131-specific sense primer (5′ATCC-CAGAAATTCTCCCA-3′) from the second extracellular domain or 0.5 μmol/L R 131-specific sense primer (5′-ATCCCAGAAATTCTCCCG-3′) was used. All primer oligonucleotides were synthesized by Qiagen Operon Co. and 0.5 μmol/I common anti-sense primer from an area downstream of the intron (5′-CAATTTTGCTGCTATGGGC-3′). The resulting fragment was 253 bp in length. As internal PCR control, we used 0.125 μmol/I human growth hormone (HGH)-1 forward primer (5′-CAGTGCCTTCCCAACCATTCCCTTA-3′) and
0.125 μmol/I HGH-II reverse primer (5′-ATCCACTCACGGATTTCTGTTGTGTTTC-3′), which resulted in a 439-bp fragment. A thermal cycler (GeneAmp 9700, Applied Biosystems) was used to perform a hot-start PCR as follows; 5 min at 95 °C, 10 cycles of 1 min at 95 °C, 2 min at 57 °C, and 1 min at 72 °C; thereafter, to enhance the sensitivity, we used 22 cycles of 1 min at 95 °C, 2 min at 54 °C, and 1 min at 72 °C and a final extension step for 5 min at 72 °C. The PCR amplification products were separated on a 2% agarose gel and visualized by ethidium bromide staining. To validate accuracy and reproducibility of the results obtained using the above described techniques, we randomly ran 20% of samples from both groups (study and control groups) for FcγRIIa genotypes by using directly DNA sequencing technique with ABI PRISM 310 Genetic Analyzer System (Applied Biosystems). The study was approved by the local ethics committee and informed consent was obtained from all parents of the children. Statistical analysis Statistical analysis of the data was performed with SPSS 11.0 computer program. Differences in demographic factors were tested with the chi-square test. Genotypic distribution and allelic frequencies were statistically compared by Fisher's exact test calculated on contingency tables using the GraphPad PRISM program (version 4.0 for Windows, GraphPad Software, San Diego, CA, USA). Odd ratios with 95% confidence intervals (95%CI) were calculated using the same software. Deviation from the Hardy–Weinberg expectancy was examined with the chi-square test. P < 0.05 is accepted as statistically significant. Results The characteristics of the study population are given in Table 1. Genotypic distributions of this case-control study did not Table 4 The frequency of FcγRIIa alleles in asthmatic children and controls FcγRIIa alleles
H R
Asthma
Control group
n
(%)
n
(%)
248 136
64.6 35.4
352 116
75.2 24.8
OR
95%CI
p
1.66
1.22–2.26
0.0007
F. Gulen et al. / Clinical Biochemistry 40 (2007) 392–396 Table 5 The frequency of FcγRIIa alleles in children with allergic rhinitis and controls FcγRIIa alleles
H R
Allergic rhinitis
Control group
n
(%)
n
(%)
220 140
61.1 38.9
352 116
75.2 4.8
OR
95%CI
p
1.93
1.42–2.63
0.00001
deviate significantly from the Hardy-Weinberg equilibrium expectations in each group (p > 0.05). Genotype distribution of FcγRIIa in the asthmatic children and control group is presented in Table 2. R131R genotype (12.5%) was significantly more frequent in asthmatic children compared to control group (5.1%) (OR: 2.64 95%CI: 1.22–5.79, p = 0.006). Genotype distribution of FcγRIIa in the allergic rhinitis group and the control group is presented in Table 3. R131R genotype was significantly more frequent in the allergic rhinitis group (12.2%) compared to the control group (5.1%) (OR: 2.58 95%CI: 1.18–5.71, p = 0.009). The frequencies of alleles were as follows: 131R allele 136 (35.4%), 131H allele 248 (64.6%) in asthmatic children, and 131R allele 140 (38.9%), 131H allele 220 (61.1%) in allergic rhinitis. 131R allele was more frequent among asthmatic children and children with allergic rhinitis, compared to the control group (OR: 1.66 95%CI: 1.22–2.26, p = 0.0007 and OR: 1.93 95%CI: 1.42–2.63, p = 0.00001, respectively) (Tables 4 and 5). Discussion Fcγ receptor (FcγR) induced leukocyte functions including antibody-dependent cellular cytotoxicity, phagocytosis, superoxide generation, degranulation, cytokine production, and regulation of antibody production are all essential for host defense. Three FcγR subclasses display functionally relevant genetically determined polymorphisms [4]. FcγR polymorphisms are now considered to be heritable risk factors for autoimmune and infectious diseases and support for a relevant role of these polymorphisms has been obtained in previous studies [22,23]. There is convincing evidence that FcγRIII and FcγRII alleles are risk factors for systemic lupus erythematosus [21,24]. In some other reports, it has been shown that patients carrying the FcγRIIa-R131R polymorphism suffer from different severe infections and infection complications [25,26,27]. Allergic diseases are considered to be multifactorial inflammatory diseases involving complex interactions between genetic and environmental influences [1]. The FcγR may also be involved in anaphylactic response. The ability of the immune system to respond appropriately to foreign antigen depends on a balance of activating and inhibitory signals. Recently, the role of cell surface inhibitory receptors in attenuating immune responses, thereby preventing pathologic conditions including autoimmunity and atopy, has been recognized [28]. The important role of FcγR in anaphylaxis has been shown in mice, unfortunately there are no available data showing the relevance of FcγR receptors in humans anaphylaxis [13,14].
395
In addition to modulating IgG-triggered hypersensitivity responses, FcγRII and III on mast cells are potent regulators of IgE-mediated responses and reveal the existence of a regulatory pathway for IgE triggering of effector cells through IgG FcR that could contribute to the etiology of the atopic response [15,16]. In small number of studies examining the relation between atopic diseases and FcγR, FcγRIIb−/− mice were found to be more sensitive to allergic rhinitis [29], but the study of Pawlik et al. found no association between the FcγRIIa genotypes and atopic diseases. The study included 140 atopic children, 77 with food allergy and 126 healthy subjects as the control group. The distribution of FcγRIIa genotypes in atopic children did not differ from that of healthy controls [30]. This study examined the hypothesis that FcγRIIa polymorphism may be a heritable factor influencing susceptibility to atopic asthma and allergic rhinitis in Turkish children. The study consisted of 372 atopic children (192 asthma bronchial, 180 allergic rhinitis) and 234 healthy subjects. Distribution of R131R genotype was significantly different among patient groups compared to controls (for asthmatic children OR: 2.64 95%CI: 1.22–5.79, p = 0.006; for allergic rhinitis OR: 2.58 95%CI: 1.18–5.71, p = 0.009). Frequency of 131R allele was significantly different among patient groups compared to controls (for asthmatic children OR: 1.66 95%CI: 1.22–2.26, p = 0.0007; for allergic rhinitis OR: 1.93 95%CI: 1.42–2.63, p = 0.00001). As a conclusion, this study shows that FcγRIIa gene 131R allele represents an important genetic risk factor for bronchial asthma and allergic rhinitis susceptibility. References [1] Holgate St. Genetic and environmental interaction in allergy and asthma. J Allergy Clin Immunol 1999;104:1139–46. [2] Vercelli D. Genetic polymorphism in allergy and asthma. Curr Opin Immunol 2003;15:609–13. [3] Van de Winkel JGJ, Capel PJA. Human IgG Fc receptor heterogenity: molecular aspects and clinical implications. Immunol Today 1993;14: 215–20. [4] van Sorge NM, van der Pol WL, van de Winkel JG. Fcgamma R polymorphisms: implications for function, disease susceptibility and immunotherapy. Tissue Antigens 2003;61:189–202. [5] Parren PWHI, Warmerdam PAM, Boeije LCM, et al. On the interaction of IgG subclasses with the low affinity FcγRIIa (CD32) on human monocytes, neutrophils, and platelets: analysis of a functional polymorphism to human IgG2. J Clin Invest 1992;90:1537–46. [6] Warmerdam PAM, Van de Winkel JGJ, Gosselin EJ, Capel PJA. Molecular basis for a polymorphism of human Fcγ receptor II (CD32). J Exp Med 1990;172:19–25. [7] Sanders LA, Feldman RG, Voorhorst-Ogink MM, et al. Human immunoglobulin G (IgG) Fc receptor IIa (CD32) polymorphism and IgG2-mediated bacterial phagocytosis by neutrophils. Infect Immun 1995;63:73–81. [8] Lehrnbecher T, Foster CB, Zhu S, et al. Variant genotypes of the lowaffinity Fcgamma receptors in two control populations and a review of low-affinity Fcgamma receptor polymorphisms in control and disease populations. Blood 1999;94:4220–32. [9] Sanders LAM, Van de Winkel JGJ, Rijkers GT, et al. FcÁ receptor IIa (CD32) heterogeneity in patients with recurrent bacterial respiratory tract infections. J Infect Dis 1994;170:854–61.
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[10] Carlsson LE, Santoso S, Baurichter G, et al. Heparin induced thrombocytopenia: new insights into the impact of the FcÁRIIa R-H131 polymorphism. Blood 1998;92:1526–31. [11] Burgess JK, Lindeman R, Chesterman CN, Chong BH. Single amino acid mutation of FcÁ receptor is associated with the development of heparininduced thrombocytopenia. Br J Haematol 1995;91:761–6. [12] Salmon JE, Millard S, Schachter LA, et al. FcÁRIIA alleles are heritable risk factors for lupus nephritis in African Americans. J Clin Invest 1996; 97:1348–54. [13] Ando A, Martin TR, Galli SJ. Effect of chronic treatment with the c-kit ligand, stem cell factor, on immunoglobulin E-dependent anaphylaxis in mice. J Clin Invest 1993;92:1639–49. [14] Martin TR, Galli SJ, Katona IM, Drazen JM. Role of mast cells in anaphylaxis. Evidence for the importance of mast cells in the cardiopulmonary alterations and death induced by anti-IgE in mice. J Clin Invest 1989;83:1375–83. [15] Miyajima I, Dombrowicz D, Martin TR. Systemic anaphylaxis in the mouse can be mediated largely through IgG and FcgRIII. Assessment of the cardiopulmonary changes, mast cell degranulation, and death associated with active IgE- or IgG1-dependent passive anaphylaxis. J Clin Invest 1997;99:915–25. [16] Dombrowicz D, Flamand V, Miyajima I, et al. Absence of Fcepsilon RI alpha chain results in upregulation of FcgRIII-dependent mast cell degranulation, and anaphylaxis. Evidence of competition between Fc epsilon RI and FcgRIII for limiting amounts of FcR B and γ chain. J Clin Invest 1997;99:901–14. [17] Global initiative for asthma (GINA). Global strategy for asthma management and prevention. NHLBI/WHO workshop report. National Institute of Health. National Heart, Lung and Blood Institute. Publication No: 023659, Revised 2002. [18] Wilson A, Dempsey OJ, Sims EJ, et al. Evaluation of treatment response in patients with seasonal allergic rhinitis using domiciliary nasal peak unspiratory flow. Clin Exp Allergy 2000;30:833–8.
[19] Meltzer EO, Malmstrom K, Lu S, et al. Concomitant montelukast and loratadine as treatment for seasonal allergic rhinitis: clinical trial. J Allergy Clin Immunol 2000;105:917–22. [20] Dreborg S, Frew A. Allergen standardization and skin tests. EAACI Position Paper. Allergy 1993;48(Suppl 14):49–82. [21] Koene HR, Kleijer M, Swaak AJ, et al. The FcgammaRIIIa: 158F allele is a risk factor for systemic lupus erythematosus. Arthritis Rheum 1998;41: 1813–8. [22] Rascu A, Repp R, Westerdaal NA, Kalden JR, van de Winkel JG. Clinical relevance of Fc gamma receptor polymorphisms. Ann N Y Acad Sci 1997;815:282–95. [23] Ozturk C, Aksu G, Berdeli A, Kutukculer N. Fc gamma RIIa, IIIa and IIIb polymorphisms in Turkish children susceptible to recurrent infectious diseases. Clin Exp Med 2006;6:27–32. [24] Karassa FB, Trikalinos TA, Ioannidis JP. Role of the Fcgamma receptor IIa polymorphism in susceptibility to systemic lupus erythematosus and lupus nephritis: a meta-analysis. Arthritis Rheum 2002;46: 1563–72. [25] Bredius RG, Derkx BH, Fijen CA, et al. Fc gamma receptor IIa (CD32) polymorphism in fulminant meningococcal septic shock in children. J Infect Dis 1994;170:848–53. [26] Platonov AE, Shipulin GA, Vershinina IV, et al. Association of human Fc gamma RIIa (CD32) polymorphism with susceptibility to and severity of meningococcal disease. Clin Infect Dis 1998;27:746–50. [27] Berdeli A, Celik H, Ozyurek R, Aydin H. Involvement of immunoglobulin FcγRIIA and FcγRIIIB gene polymorphisms in susceptibility to rheumatic fever. Clin Biochem 2004;37:925–9. [28] Takizawa F, Adamczewski M, Kinet JP. Identification of the low affinity receptor for immunoglobulin E on mouse mast cells and macrophages as FcγRII and FcγRII. J Exp Med 1992;176:469–76. [29] Watanabe T, Okano M, Hattori H, et al. Roles of FcgammaRIIB in nasal eosinophilia and IgE production in murine allergic rhinitis. Am J Respir Crit Care Med 2004;169:105–12. [30] Pawlik A, Carlsson L, Meisel P, et al. The FcRγIIa polymorphism in children with atopic diseases. Int Arch Allergy Immunol 2004;133: 233–8.
The FcγRIIa polymorphism in Turkish children with asthma bronchial and allergic rhinitis Figen Gulen a,⁎, Remziye Tanac a , Serdar Altinoz a , Afig Berdeli b , Dost Zeyrek a , Huseyin Koksoy a , Esen Demir a a
Aegean University School of Medicine, Department of Paediatrics, Division of Allergy and Pulmonology, TR-35100, Bornova/azmir, Turkey b Aegean University School of Medicine, Molecular Research Laboratory, Turkey Received 14 September 2006; received in revised form 27 October 2006; accepted 8 November 2006 Available online 5 January 2007
Abstract Objective: The aim of the present study was to evaluate the FcγRIIa polymorphism in Turkish children with atopic asthma and allergic rhinitis. Design and methods: In this study, 372 atopic children (192 asthma bronchial, 180 allergic rhinitis) between ages of 5 and 16 years old (11.3 ± 2.9) who were followed at Aegean University Paediatric Allergy and Pulmonology Outpatient Clinics and 234 healthy subjects as the control group were included. The evaluation of subjects included routine biochemical blood analysis and allergic workup based on the following laboratory determinants. The FcγRIIa polymorphism was determined using the polymerase chain reaction method. Results: Distribution of R131R genotype was significantly different among patient groups compared to controls (for asthmatic children OR: 2.64 95%CI: 1.22–5.79, p = 0.006; for allergic rhinitis OR: 2.58 95%CI: 1.18–5.71, p = 0.009). Frequency of 131R allele was significantly different among patient groups compared to controls (for asthmatic children OR: 1.66 95%CI: 1.22–2.26, p = 0.0007; for allergic rhinitis OR: 1.93 95%CI: 1.42–2.63, p = 0.00001). Conclusion: This study shows that FcγRIIa gene 131R allele represents an important genetic risk factor for bronchial asthma and allergic rhinitis susceptibility. © 2007 Published by The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: FcγRIIa gene; Polymorphism; Allergic rhinitis; Asthma bronchial; Children
Introduction Allergic diseases are considered to be multifactorial inflammatory diseases involving complex interactions between genetic and environmental influences [1]. The impact of genetic factors on the pathogenesis of allergy is currently an area of intense investigation. Epidemiological studies find more and more genes that are likely to contribute to allergic inflammation [2]. Fc receptors (FcR) are glycoproteins found on the surface of hamatopoietic cells that bind the Fc portion of immunoglobulin and provide a link between the humoral and cellular immune systems. Interaction of FcR with immunoglobulin provides the host with protection against infection by means of phagocytosis, antibody-dependent cellular cytotoxicity, release of inflammatory ⁎ Corresponding author. Fax: +90 232 342 6990. E-mail address: [email protected] (F. Gulen).
mediators, and enhancement of antigen presentation [3]. The efficacy of IgG-induced FcγR function displays inter-individual heterogeneity due to genetic polymorphisms of three FcγR subclasses: FcγRIIa, FcγRIIIa, and FcγRIIIb. Three FcγR subclasses display functionally relevant genetically determined polymorphisms [4]. FcγRII is found on the surface of monocytes, macrophages, neutrophils, platelets, basophils, eosinophils, and other cells [5]. FcγRIIa receptors exhibit a genetically determined polymorphism (FcγRIIa-R131 and FcγRIIa-H131), resulting in differential ability to recognize and be activated by human and murine IgG isotypes. This polymorphism results from a single base substitution from guanidine to adenine at nucleotide 494 of the coding region in exon 4, which leads to an amino acid change from arginine to histidine at position 131 of the second extracellular domain [6]. The H131 allele (494A) codes for histidine at position 131 and has a high affinity for human IgG2 and a low affinity for
0009-9120/$ - see front matter © 2007 Published by The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2006.11.014
F. Gulen et al. / Clinical Biochemistry 40 (2007) 392–396 Table 1 Characteristics of the study and control groups
Diagnosis Asthma (%) Allergic rhinitis (%) Age, years ± SD (range) Gender Girls Boys IgE(kU/L), mean ± SD Eosinophils (per mm3)
393
Patients and methods
Study group, n: 372
Control group, n: 234
p
192 (51.6) 180 (48.4) 11.5 ± 3.1 (5–16)
11.7 ± 2.6 (6–16)
=0.11 =0.53
140 232 401 ± 119 362 ± 211.4
100 134 33.6 ± 17.4 138 ± 102.8
<0.001 =0.001
murine IgG1, while the R131 allele (494G) has arginine at position 131 and has little or no affinity for human IgG2 but has a high affinity for murine IgG1. To a lesser extent, the two genotypes also differ in their ability to ligate human IgG3, with the homozygous FcγRIIa-H131 genotype having the higher affinity for IgG3. Thus, this polymorphism may influence susceptibility to diseases, especially in situations where IgG2, and to a lesser extent IgG3, are the predominant antibody subclasses produced. The phagocytic capacity of leukocytes with R131R for IgG2 and IgG3 opsonized bacteria is lower than the ones with H131H [5,7,8]. The FcγRIIa genotype might influence the ability to clear encapsulated organisms [9]. The FcγRIIa genotype is also related to a risk of other immune-mediated diseases, including heparin-associated thrombocytopenia [10,11] and immune complex clearance in systemic lupus erythematosus [12]. Fcγ receptor II (FcγRIIa) polymorphism may be relevant to FcγRIIa function. This might be linked to variability in immune response and therefore related to the pathogenesis of atopic diseases. The important role of FcγR in anaphylaxis has been shown in mice, unfortunately there are no available data showing the relevance of FcγR receptors in humans anaphylaxis. The anaphylaxis reaction in mice has been considered to be a typical immediate hypersensitivity response determined by the activation of mast cells via antigen-induced aggregation of an IgE-sensitized high affinity receptor for IgE (FcεRI) causing the release of systemic mediators [13,14]. In addition to modulating IgG-triggered hypersensitivity responses, FcγRII and III on mast cells are potent regulators of IgE-mediated responses and reveal the existence of a regulatory pathway for IgE triggering of effector cells through IgG FcR that could contribute to the etiology of the atopic response [15,16]. The aim of the present study was to evaluate the FcγRIIa polymorphism in Turkish children with atopic asthma and allergic rhinitis.
The study consisted of 372 atopic children (192 asthma bronchial, 180 allergic rhinitis; 140 girls, 232 boys; age range 5– 16 years). The children were followed in the Aegean University Paediatric Allergy and Pulmonology Outpatient Clinics. The control group comprised 234 ethnically matched unrelated volunteers (100 girls, 134 boys; age range 6–16 years). All subjects were Caucasians. The inclusion criteria were as follows: –patients with allergic rhinitis or bronchial asthma –a positive spIgE against respiratory allergens –a positive SPT against respiratory allergens. Diagnosis of allergy Evaluation of clinical status The asthmatic patients were evaluated according to GINA criteria [17] and allergic rhinitis patients were evaluated according to symptom scoring system [18,19]. Evaluation of subjects included routine serum biochemical analysis and allergic workup based on the following laboratory determinants: eosinophilic granulocyte count in the peripheral blood, serum total IgE level, specific IgE levels against some inhaled allergens, and skin prick test. The eosinophilic granulocyte count was considered to be elevated when it exceeded 4% of total white blood cell count. Total IgE and specific IgE levels were estimated with enzyme immune assay method by using a 3M diagnostic System Kit. Total IgE levels were related to the age-specific limits provided by the manufacturer. Values that exceeded 100 kU/L were considered to be high. The presence of allergenspecific IgE antibodies was estimated with selected aeroallergens, i.e. house dust, ascarids (Dermatophagoides pteronyssinus and Dermatophagoides farinae), feathers, cat and dog hair, and pollens of Graminaceae, weeds, and trees. Pharmacia CAP system was used; 0.35 kU/L was regarded as positive. Skin prick test All patient's skin prick test was performed by a pediatric allergy specialist. Standard respiratory allergen panel included tree pollen, grass pollen, dust mite, fungus, animal dander, grains pollen, wild grass, flower pollen, and latex (Stallergenes S.A., France). Allergens were first dropped on the anterior side
Table 2 Genotype distribution of FcγRIIa-131 in asthmatic children and controls FcγRIIa genotypes HH HR RR a
Asthma group observed n
%
80 88 24
41.7 45.8 12.5
Hardy–Weinberg expectancy.
Asthma group expected, n a 78.6 88.4 24.8
Control group observed n
%
130 92 12
55.6 39.3 5.1
Control group expected, na
OR
95%CI
p
131.6 87.7 14.6
1 1.31 2.64
– 0.87–1.96 1.22–5.79
1 0.175 0.006
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Table 3 Genotype distribution of FcγRIIa-131 in children with allergic rhinitis and controls FcγRIIa genotypes HH HR RR a
Allergic rhinitis group observed n
%
62 96 22
34.4 53.3 12.2
Allergic rhinitis group expected, n a 66.9 85.6 27.3
Control group observed n
%
130 92 12
55.5 39.3 5.1
Control group expected, na
OR
95%CI
p
131.6 87.7 14.6
1 1.76 2.5
– 1.17–2.67 81.18–5.71
1 0.004 0.009
Hardy–Weinberg expectancy.
of the forearm, and then pierced by stallerpoint. After 20 min, the test was evaluated. Histamine solution of 1 mg/mL was used as positive control and serum physiologic was used as negative control. Results were evaluated according to EAACI criteria [20]. The result of the control test was considered to be positive if the diameter of the blister was 3 mm larger than that of a negative control or if some reaction occurred that was at least half the size of that with histamine. Results were evaluated according to EAACI criteria [20]. The children were classified into three groups as follows: children with atopic bronchial asthma (192 subjects); children with allergic rhinitis (180 subjects); healthy children as control group (234 subjects). Exclusion criteria Children with recurrent respiratory tract infections, bronchitis obliterans, and chronic rhinopharyngitis were excluded from the study. Patients with clinical and laboratory findings of other chronic systemic diseases or immune deficiency were also excluded. DNA purification Genomic DNA from patients and healthy controls was extracted from peripheral blood leukocytes with QIAamp DNA Blood Mini Kits (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. FcγRIIa genotyping FcγRIIa genotyping was performed by using the method defined by Flesch BK et al. [21]. We used a 25-μL PCR mixture containing 2.5 μI of genomic DNA, 2.5 μI of 10× PCR buffer (Applied Biosystems, Foster City, CA, USA), 2 mM MgCl2, 200 μmol/L of each dNTP (Promega, Madison, USA), and 0.5 U AmpliTag DNA Polymerase (Applied Biosystems). In addition, 0.5 μmol/L of H131-specific sense primer (5′ATCC-CAGAAATTCTCCCA-3′) from the second extracellular domain or 0.5 μmol/L R 131-specific sense primer (5′-ATCCCAGAAATTCTCCCG-3′) was used. All primer oligonucleotides were synthesized by Qiagen Operon Co. and 0.5 μmol/I common anti-sense primer from an area downstream of the intron (5′-CAATTTTGCTGCTATGGGC-3′). The resulting fragment was 253 bp in length. As internal PCR control, we used 0.125 μmol/I human growth hormone (HGH)-1 forward primer (5′-CAGTGCCTTCCCAACCATTCCCTTA-3′) and
0.125 μmol/I HGH-II reverse primer (5′-ATCCACTCACGGATTTCTGTTGTGTTTC-3′), which resulted in a 439-bp fragment. A thermal cycler (GeneAmp 9700, Applied Biosystems) was used to perform a hot-start PCR as follows; 5 min at 95 °C, 10 cycles of 1 min at 95 °C, 2 min at 57 °C, and 1 min at 72 °C; thereafter, to enhance the sensitivity, we used 22 cycles of 1 min at 95 °C, 2 min at 54 °C, and 1 min at 72 °C and a final extension step for 5 min at 72 °C. The PCR amplification products were separated on a 2% agarose gel and visualized by ethidium bromide staining. To validate accuracy and reproducibility of the results obtained using the above described techniques, we randomly ran 20% of samples from both groups (study and control groups) for FcγRIIa genotypes by using directly DNA sequencing technique with ABI PRISM 310 Genetic Analyzer System (Applied Biosystems). The study was approved by the local ethics committee and informed consent was obtained from all parents of the children. Statistical analysis Statistical analysis of the data was performed with SPSS 11.0 computer program. Differences in demographic factors were tested with the chi-square test. Genotypic distribution and allelic frequencies were statistically compared by Fisher's exact test calculated on contingency tables using the GraphPad PRISM program (version 4.0 for Windows, GraphPad Software, San Diego, CA, USA). Odd ratios with 95% confidence intervals (95%CI) were calculated using the same software. Deviation from the Hardy–Weinberg expectancy was examined with the chi-square test. P < 0.05 is accepted as statistically significant. Results The characteristics of the study population are given in Table 1. Genotypic distributions of this case-control study did not Table 4 The frequency of FcγRIIa alleles in asthmatic children and controls FcγRIIa alleles
H R
Asthma
Control group
n
(%)
n
(%)
248 136
64.6 35.4
352 116
75.2 24.8
OR
95%CI
p
1.66
1.22–2.26
0.0007
F. Gulen et al. / Clinical Biochemistry 40 (2007) 392–396 Table 5 The frequency of FcγRIIa alleles in children with allergic rhinitis and controls FcγRIIa alleles
H R
Allergic rhinitis
Control group
n
(%)
n
(%)
220 140
61.1 38.9
352 116
75.2 4.8
OR
95%CI
p
1.93
1.42–2.63
0.00001
deviate significantly from the Hardy-Weinberg equilibrium expectations in each group (p > 0.05). Genotype distribution of FcγRIIa in the asthmatic children and control group is presented in Table 2. R131R genotype (12.5%) was significantly more frequent in asthmatic children compared to control group (5.1%) (OR: 2.64 95%CI: 1.22–5.79, p = 0.006). Genotype distribution of FcγRIIa in the allergic rhinitis group and the control group is presented in Table 3. R131R genotype was significantly more frequent in the allergic rhinitis group (12.2%) compared to the control group (5.1%) (OR: 2.58 95%CI: 1.18–5.71, p = 0.009). The frequencies of alleles were as follows: 131R allele 136 (35.4%), 131H allele 248 (64.6%) in asthmatic children, and 131R allele 140 (38.9%), 131H allele 220 (61.1%) in allergic rhinitis. 131R allele was more frequent among asthmatic children and children with allergic rhinitis, compared to the control group (OR: 1.66 95%CI: 1.22–2.26, p = 0.0007 and OR: 1.93 95%CI: 1.42–2.63, p = 0.00001, respectively) (Tables 4 and 5). Discussion Fcγ receptor (FcγR) induced leukocyte functions including antibody-dependent cellular cytotoxicity, phagocytosis, superoxide generation, degranulation, cytokine production, and regulation of antibody production are all essential for host defense. Three FcγR subclasses display functionally relevant genetically determined polymorphisms [4]. FcγR polymorphisms are now considered to be heritable risk factors for autoimmune and infectious diseases and support for a relevant role of these polymorphisms has been obtained in previous studies [22,23]. There is convincing evidence that FcγRIII and FcγRII alleles are risk factors for systemic lupus erythematosus [21,24]. In some other reports, it has been shown that patients carrying the FcγRIIa-R131R polymorphism suffer from different severe infections and infection complications [25,26,27]. Allergic diseases are considered to be multifactorial inflammatory diseases involving complex interactions between genetic and environmental influences [1]. The FcγR may also be involved in anaphylactic response. The ability of the immune system to respond appropriately to foreign antigen depends on a balance of activating and inhibitory signals. Recently, the role of cell surface inhibitory receptors in attenuating immune responses, thereby preventing pathologic conditions including autoimmunity and atopy, has been recognized [28]. The important role of FcγR in anaphylaxis has been shown in mice, unfortunately there are no available data showing the relevance of FcγR receptors in humans anaphylaxis [13,14].
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In addition to modulating IgG-triggered hypersensitivity responses, FcγRII and III on mast cells are potent regulators of IgE-mediated responses and reveal the existence of a regulatory pathway for IgE triggering of effector cells through IgG FcR that could contribute to the etiology of the atopic response [15,16]. In small number of studies examining the relation between atopic diseases and FcγR, FcγRIIb−/− mice were found to be more sensitive to allergic rhinitis [29], but the study of Pawlik et al. found no association between the FcγRIIa genotypes and atopic diseases. The study included 140 atopic children, 77 with food allergy and 126 healthy subjects as the control group. The distribution of FcγRIIa genotypes in atopic children did not differ from that of healthy controls [30]. This study examined the hypothesis that FcγRIIa polymorphism may be a heritable factor influencing susceptibility to atopic asthma and allergic rhinitis in Turkish children. The study consisted of 372 atopic children (192 asthma bronchial, 180 allergic rhinitis) and 234 healthy subjects. Distribution of R131R genotype was significantly different among patient groups compared to controls (for asthmatic children OR: 2.64 95%CI: 1.22–5.79, p = 0.006; for allergic rhinitis OR: 2.58 95%CI: 1.18–5.71, p = 0.009). Frequency of 131R allele was significantly different among patient groups compared to controls (for asthmatic children OR: 1.66 95%CI: 1.22–2.26, p = 0.0007; for allergic rhinitis OR: 1.93 95%CI: 1.42–2.63, p = 0.00001). As a conclusion, this study shows that FcγRIIa gene 131R allele represents an important genetic risk factor for bronchial asthma and allergic rhinitis susceptibility. References [1] Holgate St. Genetic and environmental interaction in allergy and asthma. J Allergy Clin Immunol 1999;104:1139–46. [2] Vercelli D. Genetic polymorphism in allergy and asthma. Curr Opin Immunol 2003;15:609–13. [3] Van de Winkel JGJ, Capel PJA. Human IgG Fc receptor heterogenity: molecular aspects and clinical implications. Immunol Today 1993;14: 215–20. [4] van Sorge NM, van der Pol WL, van de Winkel JG. Fcgamma R polymorphisms: implications for function, disease susceptibility and immunotherapy. Tissue Antigens 2003;61:189–202. [5] Parren PWHI, Warmerdam PAM, Boeije LCM, et al. On the interaction of IgG subclasses with the low affinity FcγRIIa (CD32) on human monocytes, neutrophils, and platelets: analysis of a functional polymorphism to human IgG2. J Clin Invest 1992;90:1537–46. [6] Warmerdam PAM, Van de Winkel JGJ, Gosselin EJ, Capel PJA. Molecular basis for a polymorphism of human Fcγ receptor II (CD32). J Exp Med 1990;172:19–25. [7] Sanders LA, Feldman RG, Voorhorst-Ogink MM, et al. Human immunoglobulin G (IgG) Fc receptor IIa (CD32) polymorphism and IgG2-mediated bacterial phagocytosis by neutrophils. Infect Immun 1995;63:73–81. [8] Lehrnbecher T, Foster CB, Zhu S, et al. Variant genotypes of the lowaffinity Fcgamma receptors in two control populations and a review of low-affinity Fcgamma receptor polymorphisms in control and disease populations. Blood 1999;94:4220–32. [9] Sanders LAM, Van de Winkel JGJ, Rijkers GT, et al. FcÁ receptor IIa (CD32) heterogeneity in patients with recurrent bacterial respiratory tract infections. J Infect Dis 1994;170:854–61.
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