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ASSOCIATION OF INTERLEUKIN-13 (IL-13) GENE POLYMORPHISMS WITH IL-13 PRODUCTION IN MALAYSIAN ASTHMATICS: A PILOT STUDY Tan, L.K.#, Raj, V.L.*, Liam, C.K.*, Naidu, R.+, Radhakrishnan, A.K.# # International Medical University, Kuala Lumpur, Malaysia *University Malaya, Kuala Lumpur, Malaysia; +Monash University Malaysia, Selangor, Malaysia Table 2: Results of the Hardy-Weinberg equilibrium (HWE) test Total subjects INTRODUCTION IL-13 SNPs Asthma is one of the most common respiratory disorders encountered in both children and adults. Clinically, it is characterised by intermittent and reversible airway obstruction, bronchohyperresponsiveness (BHR) and airway inflammation1. Asthma is currently a worldwide problem, with over 300 million people worldwide suffer from asthma2. The development of asthma appears to be determined by the interaction between host susceptibility and a variety of environmental exposures3. High serum immunoglobulin E (IgE) levels have been reported to be correlated with the clinical expression of allergy and asthma and a strong genetic component has been shown to contribute to this association4. Genetic studies in several populations have identified a region on chromosome 5q31-q33 that contains the asthma susceptibility gene in several populations5,6,7,8. This region contains a cluster of pro-inflammatory cytokines genes that play an important role in immune regulation. Two members of this gene cluster, interleukin (IL)-4 and IL-13 have been shown to have a role in the pathogenesis of asthma8,9. These cytokines, produced by T-helper-2 (TH2) cells induce class-switching of B-lymphocytes to produce immunoglobulin E (IgE) class of antibodies. Several single nucleotide polymorphisms (SNPs) of the human IL-13 gene have been reported [7,8]. Two of these SNPs (-1111, 4257), are reported to have the strongest relationship with the incidence of asthma in a Dutch population study8. This is a pilot study conducted on the Malaysian population to investigate the allelic frequency of two SNPs (-1111, 4257) in the human IL-13 gene and its impact on IL-13 production. This study can provide valuable insight into the overall mechanisms that cause susceptibility to asthma. The outcome of this study may provide a basis for future studies and contribute to early detection and diagnosis of asthma, as well as development of more effective therapeutic approaches. MATERIALS AND METHODS χ2 p-value -1111C>T 1.132 0.287 4257G>A 0.955 0.328 Table 2: Genotype data obtained from RFLP and sequencing was first tested with HWE test before further genetic analysis to ensure that the genotype distributions have no statistically significant departures from the HWE. P-values for both SNPs were >0.05. Genotype distributions of both SNPs in all subjects were in HWE. Table 3: The IL-13 SNP allele frequencies for asthmatic patients and control subjects. Allele frequency Frequencies Asthmatic patients (n=87) Control subjects (n=94) C 91 (52.3%) 125 (66.5%) -1111 4257 T 83 (47.7%) 63 (33.5%) G 95 (54.6%) 105 (55.9%) A 79 (45.4%) 7.561 0.006 1 0.057 0.811 1 IL-13 Production (pg/mL) IL-13 Production Lymphocyte Culture (72 hours) Mann-Whitney test Asthmatic patients (mean ± standard deviation) Control subjects (mean ± standard deviation) p-value CC 345.41 ± 117.71 222.63 ± 93.30 <0.001 CT 252.96 ± 119.40 208.31 ± 107.64 0.089 TT 295.92 ± 95.70 158.05 ± 65.40 0.001 GG 264.16 ± 93.50 209.44 ± 91.54 0.068 GA 285.62 ± 93.50 215.25 ± 103.46 0.011 AA 300.41 ± 116.75 182.26 ± 92.72 0.008 DNA Extraction -1111C>T Quantification of IL-13 production by ELISA Restriction Fragment Length Polymorphism (RFLP) DF* Table 4: The relationship between the SNPs in the human IL-13 gene and IL-13 production Genotype Polymerase Chain Reaction (PCR) p-value Table 3: A significant association between allele frequencies in the promoter region (-1111) were found with asthmatic patients and control subjects (p=0.006). The mutant T-allele may have an effect on the development of asthma in Malaysian asthmatics. These results matched previous studies done in the Dutch7,8 and African American populations9. Recruitment of asthmatics and control subjects (5 ml blood) Allele Frequencies 83 (44.1%) Chi-square test 4257G>A DNA sequencing DISCUSSION RESULTS AND DISCUSSION Allele frequencies of the Malaysian population for the -1111C wild-type allele and the 4257G wild-type allele of the IL-13 gene were 0.665 and 0.558 respectively in control subjects and 0.523 and 0.546 respectively in asthmatics. Table 1: Racial distribution of asthmatic patients and control subjects recruited Malay Chinese Indian Total Asthmatic patients 29 31 27 87 Control subjects 30 31 33 94 1 2 3 PCR Product 4 5 6 bp 1 2 3 PCR Product CC TT CT GG 4 AA 5 It was hypothesised that a higher frequency of mutant alleles of SNPs in IL-13 gene would be found in asthmatic patients compared to control subjects. The findings for SNP at promoter region -1111 supported this hypothesis with statistically significant (p=0.006) results. Although the results for SNP at position 4257 also supported this hypothesis but the association observed was not statistically significant (p=0.811). 6 bp SNP at site -1111 resulted in patients carrying homozygous wild-type CC and homozygous mutant AA alleles had higher IL-13 production. The results also showed that the 4257A mutant allele is associated with significantly higher IL-13 production in asthmatics compared with control subjects. GA REFERENCES 372 343 (a) Hpy99I (-1111) 236 210 178 (b) NlaIV (4257) FIGURE-1: The restriction enzyme Hpy99I recognises the SNP at site -1111C>T; NlaIV recognises the SNP at site 4257G>A. In both gels, Lanes 1 and 6 represent 100 bp DNA ladder as molecular marker. Lane 2 represent undigested PCR products. Lane 3 represents RFLP patterns seen when only the respective wild-type alleles are present. Lane 4 shows RFLP patterns when only the respective homozygous mutant alleles are present. Lane 5 shows the restriction patterns observed when respective heterozygous alleles are present. [1] J-C Renauld. New insights into the role of cytokines in asthma. J Clin Pathol 2001; 54: 577-589. [2] World Health Organisation: Asthma Fact Sheet. [Online] 2007 June 10 [cited 2007 April 4]. Available from: URL: http://www.who.int/mediacentre/factsheets/fs307/en/ [3] Cookson B. The alliance of genes and environment in asthma and allergy. Nature 1999; 402: B5-11. [4] Burrows B, Martinez FD, Halonen M, Barbee RA, Cline MG. Association of asthma with serum IgE levels and skin-test reactivity to allergens. N Engl J Med 1989; 320: 271-277. [5] Van der Pouw Kraan TCTM, van Veen A, Boeije LCM, van Tuyl SAP, de Groot ER, Stapel SO, Bakker A, Verweij CL, Aarden LA, and van der Zee JS. An IL-13 promoter polymorphism associated with increased risk of allergic asthma. Genes Immun 1999; 1: 61-65. [6] Graves PE, Kabesch M, Halonen M, Holbergc CJ, Baldini M, Fritzsch C, Weiland SK, Erickson RP, von Mutius E, and Martinez FD. A cluster of seven tightly linked polymorphisms in the IL-13 gene is associated with total serum IgE levels in three populations of white children. J Allergy Clin. Immunol 2000; 105: 506-513. [7] Wills-Karp, Luyimbazi MJ, Xu X, Schofield B, Neben TY, Karp CL, and Donaldson DD. Interleukin-13: central mediator of allergic asthma. Science 1999; 282: 2258-2261. [8] Howard TD, Whittaker PA, Zaiman AL, Koppelman GH, Xu J, Hanley MT, Meyers DA, Postma DS, Bleecker ER. Identification and association of polymorphisms in the interleukin-13 gene with asthma and atopy in a Dutch population. Am J Respir Cell Mol Biol 2001; 25(3): 377-84 [9] Moissidis, Chinoy B, Yanamandra K, Napper D, Thurman T, Bocchini J, Bahna SL. Association of IL-13, RANTES, and leukotriene C4 syntase gene promoter polymorphisms with asthma and/or atopy in African Americans. Journal of Allergy and Clinical Immunology 2005; 113(2): S195.