Background

Asthma is a disease of the lower airways that is remarkably heterogeneous between affected individuals [1]. Asthma symptoms are caused by inflammation, which results in narrowing of the airways, mucous secretion, and are characterized by recurrent attacks of paroxysmal dyspnea, with wheezing due to spasmodic contraction of the bronchi [2, 3]. Asthma is very common, with approximately 10% of people in the western world diagnosed with asthma at some stage in their life. The causes of asthma are not fully understood. Both genetic and environmental factors are involved, but how these factors interact to confer risk is still largely unknown.

Many biological pathways, and genes in those pathways, have been implicated in asthma pathogenesis. Variants in over 100 genes have been associated with asthma, but all with small individual effect sizes. It is likely that many genes act in concert to determine individual-specific risks for asthma [4]. Genes involved in immunological pathways are important in asthma pathogenesis. Therefore, based on results of previous candidate gene and genome-wide studies, we selected 15 single nucleotide polymorphisms (SNPs) in 10 cytokine and chemokine genes for genotyping in Pakistani asthma cases and controls. The selection of genes is purely rely on the immunological pathways.

Methods

Patient population and study design

Ethical statement

The ethical review committee of the parent organization approved this project (ERC-08-01).

Informed consent

Written informed consent was obtained from all participants.

Asthmatic subjects were recruited from cities of Islamabad and Lahore of Pakistan. These all subjects belong to ethnic groups from Pakistan. Cases and controls all are from same ethnicity. There are total 533 samples included in the present study in which 333 Pakistani adult subjects were with an asthma diagnosis provided by a pulmonologist. Cases of asthma were selected for sample collection from the outpatient clinics of Rawalpindi, Islamabad and Lahore. Chest specialists based on clinical examination diagnosed the patients. Both patients and controls were from a similar ethnic background, and belonged to various castes and tribes from northern Punjab and the Northwest Frontier Province of Pakistan. Normal subjects, as control, were selected from general healthy population. Two hundred non-asthmatic healthy controls were recruited from the general population to be similar to the cases with respect to ethnicity and proportions of males and females.

Blood sample collection, DNA extraction, and genotyping

A venous blood sample was obtained from each study participant, and genomic DNA was extracted from whole blood using a standard phenol chloroform extraction protocol [5]. We selected SNPs in genes that are involved in the immune system and implicated in asthma risk, as reported in previous studies. Ten SNPs were genotyped using a Sequenom iPLEX assay and five SNPs were genotyped using TaqMan assays and analyzed on an ABI 7900 HT Fast Real Time PCR (Applied Biosystems, USA). All genotyping was performed at the University of Chicago USA.

Quality checks and statistical analyses

Hardy-Weinberg equilibrium (HWE) was determined in the entire sample and separately in the cases and controls. All SNPs were in HWE. Multiple logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for each SNP.

Results

Demographic and genotyping characteristics

The asthma cases included 148 (44.5%) males and 185 (55.5%) females; the mean age was 40 ± SE = 0.93 years. The controls included 88 males (44.0%) males and 112 (56.0%)) females with mean age of 30 ± SE = 0.97 years.

Of the 15 SNPs included in our study, all were in Hardy-Weinberg equilibrium. The genotyping methods, call rates, minor allele frequencies, and Hardy-Weinberg P-value calculated as a group are shown for all SNPs in Table 1.

Table 1 Single Nucleotide Polymorphisms (SNPs) Genotyped in Asthma cases and controls
Full size table

Allelic and genotypic associations

Four SNPs showed evidence for an association with asthma at a P < 0.05. The allele and genotype frequencies in cases and controls for these four SNPs and results of all analyses are shown in Table 2.

Table 2 Association of Genotype and Allele Frequencies with Asthma Among Cases and Controls
Full size table

Homozygosity for the minor alleles at SNPs in two other genes, IL10 and IL13, was associated with asthma risk. There were more GG homozygotes at rs1800896 in IL10 in cases compared to controls (recessive risk model p = 0.04) and more TT homozygotes at rs1800925 in IL13in cases compared to controls (recessive risk model, P = 0.009).

Discussion

This candidate gene study was conducted in Pakistani asthma patients and healthy controls. This study was carried out to validate in our population various cytokines and chemokines associations with asthma. This is the first study to report associations between these SNPs and asthma in Pakistani population to see the effect of these variations in pathogenesis if we look at the immunological pathways.

In the present study, we investigated the association between 15SNPs in 10 candidate genes (Table 1) for asthma in the Pakistani population. All of the genes included in the present study were either directly or indirectly involved in pathways affecting the immunological process. The lack of very strong associations in our data could be due to the relatively small size of the study sample and the fact that the subjects in our study were adults whereas most of the previous genetic studies were performed largely in children with asthma [3, 6,7,8]. The present data is a helpful in future genetic studies of adult asthmatics.

Asthma is caused by interaction of multiple genes, some of which have a protective effect and others contribute to the pathogenesis of the disease, with each gene having its own tendency to be influenced by the environment. In asthmatic individuals, antigen presentation is thought to result in the polarization of T-cell differentiation towards a Th2 pattern, whereas T-cells from non-atopic, non-asthmatic individuals show the opposing Th1 (interferon-γ and IL-2) pattern of cytokine secretion [9]. Activated Th2cells secrete cytokines such as IL-4, IL-13 and IL-5. IL-13 has a pivotal role in asthma pathogenesis: it activates a receptor complex that is composed of the IL-4Rα and IL-13Rα1 on many cell types in the airway wall and is thought to mediate many processes that are relevant to asthma pathology as a result of activation of this receptor complex [10]. Th17 lymphocytes also play a role in the pathogenesis of several autoimmune and inflammatory diseases [11]. Studies have also demonstrated that the proportion of Th17/Th2 cells is extremely low in healthy subjects, whereas their numbers appeared to be significantly higher in the circulation of patients with chronic severe asthma [12].

Homozygosity for the minor alleles at IL13 was associated with asthma risk. There were more TT homozygotes at rs1800925 in IL13in cases compared to controls (recessive risk model, P = 0.009). Interleukin (IL)-13 is a critical mediator in the pathogenesis of allergic inflammation [13]. This cytokine upregulates major histocompatibility complex class II expression and promotes IgEisotype switching. This cytokine is found to be critical to the pathogenesis of allergen-induced asthma but operates through mechanisms independent of IgE [14]. Multiple genetic variants in the promoter (C-1112 T: rs1800925; A-1521C: rs1881457) and coding regions (G2044A: rs20541) have been associated with atopic asthma and non-atopic asthma, increased risk of sensitization to food and outdoor allergens, and bronchial hypersensitiveness in multiple studies [15, 16]. It has also been reported that the rs1800925 (−1112C/T) polymorphism resulted in enhanced promoter activity [17]. In spite of the importance of IL-13 in asthma [18,19,20], some studies failed to show an association between IL13 polymorphisms and asthma phenotypes [21, 22], possibly because of different exposures to environmental risk factors such as tobacco smoke exposure. Beghe [23] reported associations of rs1800925, rs1295685 and rs20541 in IL13 with both atopy and asthma. These are different SNPs which are located in IL13, some or which are not in LD. All three SNPs were included in our study of Pakistani cases and controls, but only rs1800925 showed evidence of association with asthma. In all studies, including ours, the T allele was associated with susceptibility. Previous functional study on SNP rs1800925 showed that -1112 T allele enhanced IL13 promoter activity in CD4+ Th2 lymphocytes. Increased expression of IL13 -1112 T in Th2 cells was associated with attenuated STAT6-mediated transcription repression [24]. This offers a possible reason for the significant P-value for the T allele of rs18000925 in our Pakistani subject population.

Homozygosity for the minor alleles at SNPs IL10 was associated with asthma risk. There were more GG homozygotes at rs1800896 in IL10 in cases compared to controls (recessive risk model P = 0.04). IL-10 is important in immuno-regulation and considered to be an immunosuppressive factor. Low levels of IL-10 expression have been reported to play a role inthe pathogenesis of asthma [25, 26]. In contrast, high levels of IL-10 from regulatory T-cells have a protective effect against airway hyperreactivity and inflammation [27]. In our study, we replicated the association between asthma and a SNP (rs1800896) in the promoter region of IL10. This polymorphism conferred susceptibility to asthma in East Asians and adult asthmatics [28]. It lies within a putative ETS-like transcription factor binding site, and it has been suggested that a G allele at this position results in higher expression levels of IL-10 transcript [29, 30].The association with the G allele in the Pakistani population is consistent with results of studies in Indian [31], Egyptian [6] and other populations [32,33,34], although the opposite allele was reported in a Korean population [35].

Conclusion

The GG genotype at rs1800896 (IL10) and the TT genotype at rs1800925 (IL13) are susceptibility genotypes for asthma in the Pakistani population. These results are consistent with previous results on Caucasians and other related population. This adds up new information of these cytokines in Pakistani population as this is not reported in our population previously and this data can be helpful infuture prospects of genetic studies in other world. Further functional genomicsstudies on large number of samples will be needed to replicate these associations and determine the influence of these genes on asthma pathogenesis in Pakistani population, and their roles in gene-gene and gene-environment interactions.

Summary at glance

Asthma is a chronic disease of the airways. Its causes are not understood. Both genetic and environmental factors are involved. In the present study15 SNPs in 10cytokine and chemokine genes were genotyped in Pakistani asthmatic cases and controls. For genotyping the Sequenom Mass ARRAY iPLEX platform and TaqMan assay were used. Polymorphism in IL10 and IL 13 are associated with asthma susceptibility in Pakistani population.