A hexanucleotide repeat upstream of eotaxin gene promoter is associated with asthma, serum total IgE and plasma eotaxin levels

Background

Eotaxin (CCL11) is a small protein produced in the lungs of patients with asthma, and is a potent chemoattractant for eosinophils.

Aim

To elucidate the role of eotaxin in asthma by an association study of functional and novel eotaxin polymorphisms in case–control and family‐based study designs.

Methods

Eotaxin +67G/A, –384A/G and –426C/T single‐nucleotide polymorphisms and a hexanucleotide (GAAGGA)_n repeat 10.9 kb upstream of the gene were genotyped in a cohort of age, sex and ethnically matched patients with asthma (n = 235) and healthy controls (n = 239), and also in a study population of 230 families with asthma recruited from north/northwest India. Total serum IgE (TsIgE) and plasma eotaxin levels were measured using ELISA.

Results

+67G/A polymorphism was found to be significantly associated with asthma in case–control (p = 0.009) and family‐based studies (p = 0.006). Its functional role, as it was correlated with plasma eotaxin levels (p = 0.006), was also demonstrated. Further, –384C/T single‐nucleotide polymorphism was found to be significantly associated with log₁₀ TsIgE (p = 0.016 in case–control and p = 0.018 in families) and eotaxin levels (p = 0.007). Most interestingly, for the first time, a highly significant association of the newly studied (GAAGGA)_n hexanucleotide repeat with asthma (p = 3×10⁻⁶), log₁₀TsIgE (p = 0.006) and eotaxin levels (p = 0.004) was observed. G_A_C_8 was also identified as an important risk haplotype associated with high TsIgE and plasma eotaxin levels.

Conclusions

This study provides further evidence that eotaxin polymorphisms are associated with the development of asthma by regulating eotaxin levels and reinforces towards the scanning of other chemokine genes present at 17q21 locus for their association with asthma and related phenotypes.Eosinophils play a major role in the pathogenesis of allergic diseases including asthma by releasing various granular proteins, reactive NO, cytokines and chemokines at the site of inflammation, thus causing tissue damage.¹ Interplay between chemokines and their receptors are considered to be crucial for the trafficking of eosinophil and other lymphocytes from the circulation to the bronchoalveolar spaces of the patients with asthma.² Eotaxin (chemokine, CC motif, ligand; CCL11) is a predominant eosinophil chemoattractant, which binds to chemokine receptor 3.^3,4 Chemokine receptor 3 is also present on the Th2 CD4⁺ lymphocytes, basophils, dendritic cells and mast cells.^5,6 Thus, the role of eotaxin in asthma is not confined to eosinophil migration and activation only, but extended to many other effector cells involved in disease pathogenesis. It has also been observed that along with interleukin 5, eotaxin prolongs the viability of eosinophils.⁷ Eotaxin mRNA and protein are found to be elevated in the induced sputum, bronchial epithelium and airways of the patients with moderate to severe asthma.^8,9,10 Bronchoalveolar lavage fluid of the patients with asthma also showed increased levels of eotaxin after allergen inhalation.¹¹ In addition, higher plasma eotaxin levels have been observed in subjects with symptoms of acute asthma and airflow obstruction than subjects with stable asthma. Plasma eotaxin levels have been correlated with severity of asthma even in the presence of steroid treatment.^12,13Eotaxin gene is present on chromosome 17q21.1, where linkage with asthma and related phenotypes has been previously reported in various ethnic populations.^14,15 Previous studies on eotaxin gene polymorphisms and asthma remain inconclusive as most of them failed to establish a strong association.^{16,17,18,19,20} However, in few other studies, eotaxin single‐nucleotide polymorphisms (SNPs) have been correlated significantly with asthma‐associated phenotypes including lung function, serum IgE, circulating blood eosinophils, eosinophil migration and activation, and plasma eotaxin levels.^{16,17,18,21,22} Studies have also been undertaken to demonstrate the functional implication of various promoter and exonic variants of the eotaxin.^20,22 Notably, the variant and the direction of association are inconsistent across different ethnic populations owing to some inherent reasons. However, no such studies have yet been undertaken in an ethnically divergent Indian population.Importantly, the 17q11–17q21 chromosomal region harbours many other important chemokines including RANTES, MCP1, MCP3 and so on, and also the gene for inducible nitric oxide synthase. Recently, we have reported the association of the gene for inducible nitric oxide synthase microsatellite repeats with asthma and related phenotypes in an Indian population, demonstrating the importance of 17q region in asthma predisposition.²³ The results of our previous study and the well‐documented role of eotaxin in asthma prompted us to undertake an association study of eotaxin gene with asthma and associated phenotypes in case–control and family‐based study designs. We have also measured the plasma eotaxin level and attempted to correlate it with eotaxin gene variants.