Abstract

Introduction

Asthma, one of the most common chronic respiratory diseases of childhood, is characterized by reversible airflow obstruction due to chronic inflammation of the airways (¹1. Accordini S, Corsico A, Cerveri I, Gislason D, Gulsvik A, Janson C, et al. The socio-economic burden of asthma is substantial in Europe. Allergy 2008; 63: 116-124, doi: 10.1111/j.1398-9995.2007.01523.x.
https://doi.org/10.1111/j.1398-9995.2007... ). It is thought to be caused by a combination of genetic and environmental factors (²2. Sengler C, Lau S, Wahn U, Nickel R. Interactions between genes and environmental factors in asthma and atopy: new developments. Respir Res 2002; 3: 7, doi: 10.1186/rr179.
https://doi.org/10.1186/rr179... ). In the last decade, analysis of single nucleotide polymorphisms (SNPs) has become the newest approach for detection and localization of the genetic determinants of asthma (³3. Denham S, Koppelman GH, Blakey J, Wjst M, Ferreira MA, Hall IP, et al. Meta-analysis of genome-wide linkage studies of asthma and related traits. Respir Res 2008; 9: 38, doi: 10.1186/1465-9921-9-38.
https://doi.org/10.1186/1465-9921-9-38... ,⁴4. Zhang Y, Zhang J, Huang J, Li X, He C, Tian C, et al. Polymorphisms in the transforming growth factor-beta1 gene and the risk of asthma: A meta-analysis. Respirology 2010; 15: 643-650, doi: 10.1111/j.1440-1843.2010.01748.x.
https://doi.org/10.1111/j.1440-1843.2010... ).

Elevated levels of total immunoglobulin (Ig)E and allergy-specific IgE are hallmarks of allergic inflammation (⁵5. Oettgen HC, Geha RS. IgE regulation and roles in asthma pathogenesis. J Allergy Clin Immunol 2001; 107: 429-440, doi: 10.1067/mai.2001.113759.
https://doi.org/10.1067/mai.2001.113759... ). Many genetic studies have shown that the C-109T polymorphism ofMS4A2 encoding the β chain of the high-affinity IgE receptor is associated with increased plasma IgE levels (⁶6. Kim SH, Bae JS, Holloway JW, Lee JT, Suh CH, Nahm DH, et al. A polymorphism of MS4A2 (-109T>C) encoding the beta-chain of the high-affinity immunoglobulin E receptor (FcepsilonR1beta) is associated with a susceptibility to aspirin-intolerant asthma. Clin Exp Allergy 2006; 36: 877-883, doi: 10.1111/j.1365-2222.2006.02443.x.
https://doi.org/10.1111/j.1365-2222.2006... ) and the release of proinflammatory factors in asthmatic airways (⁷7. Kim YK, Park HW, Yang JS, Oh SY, Chang YS, Shin ES, et al. Association and functional relevance of E237G, a polymorphism of the high-affinity immunoglobulin E-receptor beta chain gene, to airway hyper-responsiveness. Clin Exp Allergy 2007; 37: 592-598, doi: 10.1111/j.1365-2222.2007.02680.x.
https://doi.org/10.1111/j.1365-2222.2007... ,⁸8. Cui T, Wang L, Wu J, Xie J. The association analysis of FcepsilonRIbeta with allergic asthma in a Chinese population. Chin Med J 2003; 116: 1875-1878.). Interleukin (IL)-13 may promote the differentiation and survival of eosinophils and mast cells and induce the isotype switching of IgE. The T allele of the IL13 C1923T locus is significantly associated with increased risk of asthma (⁹9. Lachheb J, Chelbi H, Ammar J, Hamzaoui K, Hamzaoui A. Promoter polymorphism of the IL-18 gene is associated with atopic asthma in Tunisian children. Int J Immunogenet 2008; 35: 63-68, doi: 10.1111/j.1744-313X.2007.00738.x.
https://doi.org/10.1111/j.1744-313X.2007... ), andrs1295686 is associated with the dysregulation of total IgE (¹⁰10. Granada M, Wilk JB, Tuzova M, Strachan DP, Weidinger S, Albrecht E, et al. A genome-wide association study of plasma total IgE concentrations in the Framingham Heart Study. J Allergy Clin Immunol 2012; 129: 840-845, doi: 10.1016/j.jaci.2011.09.029.
https://doi.org/10.1016/j.jaci.2011.09.0... ). ADRB2 R16G, a polymorphism of the β2-adrenergic receptor gene, may be strongly associated with airway hyperresponsiveness following activation by β2-adrenoceptor agonists (¹¹11. Tiotiu A, Todea D, Tiotiu D, Sandor V. [The distribution and polymorphism of beta-2-adrenoreceptors in the bronchi]. Pneumologia 2005; 54: 177-180.). Association of the IL-4 and IL-4 receptor alpha chain (IL4RA) gene with asthma has also been reported in some studies (¹²12. Li Y, Guo B, Zhang L, Han J, Wu B, Xiong H. Association between C-589T polymorphisms of interleukin-4 gene promoter and asthma: a meta-analysis. Respir Med 2008; 102: 984-992, doi: 10.1016/j.rmed.2008.02.008.
https://doi.org/10.1016/j.rmed.2008.02.0... ,¹³13. Risma KA, Wang N, Andrews RP, Cunningham CM, Ericksen MB, Bernstein JA, et al. V75R576 IL-4 receptor alpha is associated with allergic asthma and enhanced IL-4 receptor function. J Immunol 2002; 169: 1604-1610.).

In our research, we genotyped six SNP loci from the five candidate genes (IL13, IL4, IL4RA,MS4A2, ADRB2) in Mauritian Indian and Chinese Han populations in order to determine their association with asthma.

Material and Methods

Subjects

This case-control association study included 382 unrelated Mauritian Indian children, 193 with asthma and 189 healthy controls, and 384 unrelated Chinese Han children, 192 with asthma and 192 healthy controls. The children with asthma were recruited at the Sir Seewoosagur Ramgoolam National Hospital of Mauritius and the Asthma Clinic of Shanghai Xin Hua Hospital in China. They were 3 to 12 years of age, evenly divided between males and females and fulfilled the guidelines of the American Thoracic Society for the diagnosis of asthma (¹⁴14. American Thoracic Society. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. This official statement of the American Thoracic Society was adopted by the ATS Board of Directors, November 1986. Am Rev Respir Dis 1987; 136: 225-244, doi: 10.1164/ajrccm/136.1.225.
https://doi.org/10.1164/ajrccm/136.1.225... ). The control group consisted of unrelated healthy volunteers, 18 to 22 years of age, evenly divided between males and females, had no symptoms or history of allergy or other pulmonary diseases, and had no first-degree relatives with a history of asthma or atopy. All subjects provided written informed consent.

The study design was approved by the Ethics Committees of the Ministry of Life and Quality of Life of the Republic of Mauritius and Shanghai Xin Hua Hospital in China, and was conducted according to the Declaration of Helsinki and all subsequent revisions.

Genotyping

Genomic DNA was collected and isolated from oral mucosa swabs using a DNA extraction kit (Tiangen, China). The six SNP loci from the Chinese Han population were detected using polymerase chain reaction (PCR)-restriction fragment length polymorphism (¹⁵15. Kumar R, Barbacid M. Oncogene detection at the single cell level. Oncogene 1988; 3: 647-651.,¹⁶16. Aron Y, Swierczewski E, Lockhart A. A simple and rapid micromethod for genomic DNA extraction from jugal epithelial cells. Application to human lymphocyte antigen typing in one large family of atopic/asthmatic probands. Allergy 1994; 49: 788-790, doi: 10.1111/j.1398-9995.1994.tb02105.x.
https://doi.org/10.1111/j.1398-9995.1994... ). PCR amplification of the corresponding genomic region surrounding each SNP locus was performed in a TaKaRa PCR thermal cycler (TaKaRa TP600, China). The reaction was performed in a final volume of 10 μL including 2.05 μL commercial PCR master mix (TaKaRa Ex Taq), 5 pmol of each primer, and 10 ng genomic DNA. Cycling conditions included 1 cycle at 95°C for 5 min, 40 cycles at 95°C for 30 s, melting temperature for 45 s, 72°C for 1 min, and a final extension at 72°C for 10 min. To avoid contamination, negative controls were included in each PCR. PCR-amplified products were detected by 2% agarose gel electrophoresis. Six different restriction enzymes were used to digest the PCR products of the six loci. A 10-μL reaction mixture containing 0.5 U restriction enzyme (NEB, China) and 5 μL PCR products was incubated for 16 h. Digested products were analyzed by 4% agarose gel electrophoresis and photographed using the Automatic Digital Gel Imaging system (Tanon-3500, China).

The six SNP loci from the Mauritian Indian population were genotyped by TaqMan real-time quantitative PCR (ABI 9700, Applied Biosystems,USA). The reaction was performed on a 384-well plate. Each well contained 6-FAM (6-carboxyfluorescein)- and VIC (4,7,2′-trichloro-7′-phenyl-6-carboxyfluorescein)-labeled probes. The total volume of 10 μL included 10 ng genomic DNA, 5 μL TaqMan Universal Master Mix (Applied Biosystems), 0.2 μL TaqMan SNP Genotyping Assay Mix, and 2.5 μL RNase. Cycling conditions included 1 cycle of 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 60 s. The Assay IDs of rs569108, rs1441586, rs1042713, rs1805010, rs2243250, rs1295686 were C___900116_10, C___1842226_10, C___2084764_20, C___2769554_10, C___16176216_10, and C___8932053_10, respectively. Negative controls were included in each PCR to avoid contamination. After PCR, each sample was automatically analyzed by measuring allele-specific final fluorescence in the ABI Prism 9700HT detection system, using the SDS 2.2 software for allele discrimination (Applied Biosystems).

Statistical analysis

Hardy-Weinberg equilibrium and genotype distributions between asthma children and healthy controls were analyzed by the chi-square test. All statistical analyses were done with the SPSS 18.0 software (IBM Corporation, USA). Two-tailed P values of 0.05 or less were considered to be statistically significant.

Results

Genotype distributions with asthma in Mauritian Indian population

As shown in Table 1, there was a significant difference in the distribution of IL13 C1923T in asthma and control groups (P=0.033), and the frequency of IL13 C1923T T/T in the asthma group was significantly higher than in the control group [odds ratio (OR)=2.119, 95% confidence interval (95%CI)=1.048-4.285]. No statistically significant differences were found in genotype distributions of the other five loci (MS4A2 E237G,MS4A2 C-109T, ADRB2 R16G, IL4RA I75V, and IL4 C-590T) between the two groups (P>0.05).

Genotype distributions with asthma in Chinese Han population

Significant differences were found in the distribution of MS4A2 C-109T (P=0.001) and ADRB2 R16G (P=0.000) between the asthma and control groups. The asthma group had significantly higher frequencies of MS4A2 C-109T T/T (OR=1.961, 95%CI=1.31-2.94), and ADRB2 R16G A/A (OR=2.575, 95%CI=1.66-3.99) than the control group. There were no statistically significant differences in genotype distributions of the other four loci (MS4A2 E237G,IL4RA I75V, IL4 C-590T, and IL13 C1923T) between the 2 groups (P>0.05; Table 2).

Discussion

In the first part of this study, we genotyped six SNP loci from five candidate genes in Mauritian Indian children suffering from asthma. A significant difference in the distribution of IL13 C1923T was found between the asthma and control groups. The frequency of IL13 C1923T T/T homozygote in the asthma group was significantly higher than in the control group. The results indicate that IL13 C1923T may be associated with asthma in Mauritian Indian children and the homozygous IL13 C1923T T/Talleles may be responsible for the development of asthma.

We also genotyped these six SNPs in the Chinese Han population and found significant differences in the distribution of MS4A2 C-109T and ADRB2 R16G between the asthma and control groups, suggesting that these two SNPs may be related to asthma in Chinese Han children. Compared with the control group, the asthma group had significantly higher frequencies of MS4A2 C-109T T/T and ADRB2 R16G A/A, indicating that C-109T T/T and R16G A/A homozygotes might be responsible for the development of asthma.

In our study, no significant association of MS4A2 E237G,IL4RA I75V and IL4 C-590T with asthma was shown in the two ethnic populations, which contrasts with findings in other ethnic groups. One study performed in central China reported that the T allele of theIL13 C1923T locus was significantly associated with increased risk of pediatric asthma (¹⁷17. Wu X, Li Y, Chen Q, Chen F, Cai P, Wang L, et al. Association and gene-gene interactions of eight common single-nucleotide polymorphisms with pediatric asthma in middle China. J Asthma 2010; 47: 238-244, doi: 10.3109/02770900903509099.
https://doi.org/10.3109/0277090090350909... ). However, in our study the IL13 C1923T polymorphism was associated with asthma only in the Mauritian Indian and not in the Chinese Han population. We did not find a common locus predisposing to asthma in the two ethnically different populations.

In conclusion, our study indicates IL13 C1923T as a predisposing locus to asthma in Mauritian Indian children and MS4A2 C-109T T/Tand ADRB2 R16G A/A as associated with asthma in Chinese Han children.

Research supported in part by grants from the National Natural Science Foundation of China (#30972750 and #30872805), the Key Technology R&D Program from the Science and Technology Commission of Shanghai (#10DZ1951000) and a grant from Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine in 2012 (#12QYJ03).

References

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