INTRODUCTION:

Asthma is a very common chronic condition affecting all age classes especially children. Approximately, 235 million people are affected with asthma worldwide, with an annual death figure of 250000, most of them in the developing countries[1]. The reasons beyond this increase may mainly related urbanization [2]. Different measure in terms of therapies whether chemicals [3-5] or herbal [6-7] or enhancing life style [8-9] have been shown to alleviate the symptoms. However, prevention of asthma obligates finding the precise causes of the disease.

So far, the exact causes of asthma are beyond the current knowledge. Genetic and environmental factors such as air pollution and life style have been well documented [10-11] and undoubtedly associate with much variation in the prevalence of the disease. Usually, the environmental factors require genetic background to exert their effects, and this explains the variation in asthma incidence among population exposed to the same environmental circumstances [12]. Thus, determining the genetic factors of asthma has the priority in order to recognize the most prone subjects. Single nucleotide polymorphism has emerged as one the most widespread genetic factors affecting the incidence or different diseases [13-14].

Interleukin-10 (IL-10) is one of the most key regulator cytokines for immune response. This cytokine inhibits the synthesis of a range of cytokine produced by Th1 (interferon-γ (IFN-γ), IL-2, tumor necrosis factor-β (TNF-β) and eventual shifting of immune response towards Th2 – profile [15-16]. A number of studies reported low serum levels of IL-10 in asthmatic patients. So, it is reasonable to assume that genetic factors influencing these cytokines have direct effect on asthma susceptibility. The promoter region of IL-10 gene involves several SNPs, among which the SNP C-597A which was found to affect IL-10 expression, and hence it is a perfect candidate for investigation for its role in asthma [17]. This study aimed to investigate the relationship IL-10 C-597A gene polymorphism and the occurrence of asthma in Iraqi patients.

SUBJECTS AND METHODS:

Study Population:

A total of 45 asthma out-patients were included in the study. Age range was from 16 to 48 years, 18 (40%) males and 27 (60%) females. These patients were attending Al Zahra'a Consultative Center for Allergy and Asthma, and Consultative Clinic for Chest and Respiratory Diseases/Baghdad during the period from January to April, 2015. The diagnosis of asthma was performed by a specialist physician. Inclusion criteria involve having current symptoms of asthma, suffered from four or more wheezing episodes during the last year, and being on inhalation steroid therapy. In addition, age- and sex-matched 40 apparently healthy individuals were recruited to represent the control group. The exclusion criteria for both patients and control were any history of respiratory illness like chronic obstructive pulmonary disease (COPD), tuberculosis, pneumonia, or bronchitis, and any other comorbid illness such as diabetes mellitus, hypertension, or pregnant females. Data including age, gender, smoking status, first relative family history for asthma and dwelling were obtained from each participant by direct interview. Informed consent was obtained from all participants and the study was approved by ethical committee of Colleges of Medicine-Al-Nahrain University.

Blood Samples, DNA Extraction and Gene Amplification:

From each subject, 5ml of blood was drawn from vein puncture in an EDTA tube. DNA was extracted from these samples using ready kit (ZymoBead™ Genomic DNA Kit, USA). For PCR amplification of IL-10 gene, the primer set was forward primer: 5’- CCTAGGTCACAGTGACGTGG-3’ and reverse primer: 5’- GGTGAGCACTACCTGACTAGC-3’, with 412bp fragment length amplicon [18]. The following PCR protocol was adapted: an initial denaturation at 95 ̊C for 5 min followed by 35 cycles of 95 ̊C for 50 sec, 53 ̊C for 50 sec and 72 ̊C for 1 min. The final extension was achieved at 72 ̊C for 7 min.

Polymerase chain reaction products from patients and controls were directly sequenced using Big Dye Terminator method/ Sandor Life Sciences Pvt. Ltd /India. The obtained sequences were aligned with normal sequence from GenBank and examined for the presence of SNPs.

Statistical Analysis:

The Statistical Package for the Social sciences (SPSS, version 14) was used for statistical analysis. Continuous variables were expressed as mean± standard deviation (SD). Risk association between the genotypes and asthma susceptibility was estimated by the calculation the adjusted odds ratio (OR) and 95% confidence intervals (CI) using binary logistic regression. Chi- square was used for testing the deviation from Hardy- Weinberg equilibrium as well as for comparing between categorical variables. A p-value < 0.05 was considered statistically significant.

RESULTS:

Demographic Data:

Table 1 shows the demographic data of the study population. The only demographic factor which had significant association with asthma is family history. Among asthma patients there was 48.89% who had one or more first relative with asthma compared to only 5% among controls who had such relative (P<0.001).

Table 1: Demographic data of asthma patients and controls

Risk Factors

Cases N=45

Control N=40

P-value

Mean age in years (SD)

34.61 (4.11)

36.68 (4.08)

0.229

Family history

Yes

23 (51.11 %)

22 (48.89%)

38 (95%)

2(5%)

< 0.001

Sex

Male

Female

18(40%)

27(60%)

18(45%)

22(55%)

0.403

Smoking

Never

Smoker (ex/current)

37 (82.22%)

8 (17.78%)

38 (95%)

2(5%)

0.090

Dwelling

Urban

Rural

29 (64.44%)

16(35.56%)

27 (67.5%)

13 (32.5%)

0.082

Genotyping:

Gel electrophoresis of PCR product for IL-4 and IL-10 genes are shown in figures (1)

Figure (1): Gel electrophoresis for IL-10 PCR products visualized under U. V light after staining with ethidium bromide. M: 100-1000bp ladder; lane 1-6: positive amplification of the gene from DNA extracted from blood samples of asthma patients and controls. The size of product is 412bp.

This polymorphism had only two genotypes in patients and controls (CC and CA, figure 2). The frequency of these genotypes among asthma patients were 60% and 40% respectively compared to 95% and 5% respectively among controls (table 2) with highly significant difference (OR= 12.667, 95%=2.71-59.198, P=0.001).

Homozygous wild genotype (CC)

Heterozygous genotype (CA)

Figure 2: Different pattern of IL-10 C-597A polymorphism

Allele analysis confirmed the genotype results in that the mutant allele (A) was more frequent among asthma patients than controls (20% vs 7.5%) with highly significant difference (OR=9.750, 95%CI=2.185-43.504)

Table (2): Genotypes and allele frequencies of the SNP IL-10-C-597A

Variables

Cases

N=45

Control

N=40

P-value

OR(95%CI)

C-597A

Genotypes

HWE

27 (60%)

18(40%)

0 (0%)

0.093

38 (95%)

2 (5%)

0 (0%)

0.871

0.001

1.0

12.667

(2.71-59.198)

Alleles

72 (80%)

18 (20%)

78 (92.5%)

2 (7.5%)

0.001

1.0

9.750

(2.185-43.504)

HWE: Hardy Weinberg Equilibrium, OR: odds ratio, CI: confidence interval

DISCUSSION:

The present study revealed highly significant association between the heterozygote CA genotype and the susceptibility to asthma (OR =12.667, 95% CL=2.17-59.198, P=0.001). That means individuals carrying CA genotype have 12.667-fold risk to develop asthma compared to those carrying CC genotype. Allelic analysis boosted this result, as the mutant allele (A) was more frequent among asthmatic patients than controls with significant difference (OR=9.75, 95% CL=2.185 -43.504). Of note, none among asthmatic patients or in controls had AA genotype.

These results agree with that of Gaddam et al. [19] and Chatterjee et al. [20] in Indian population and Guglielmi et al. [21] in French women. Furthermore, a meta-analysis involving 4716 asthmatic patients and 5093 controls, Zheng et al. [22] showed that -597AA genotype was significantly associated with increased asthma susceptibility. However, another meta-analysis including 1372 asthmatic patients and 1266 has failed to show any association [23].

IL -10 plays an important role in asthma pathogenesis [24]. This cytokine inhibits the synthesis of broad range of other cytokines such as interferon-γ (IFN- γ), IL-2, tumor necrosis factor-β (TNF-β), IL-1 and TNF-α [25-26]. It is because this anti-inflammatory trait, IL-10 is considered to have anti-asthmatic feature, and decreased serum levels of IL-10 is always associated with asthma. The C-597A polymorphism is located within a putative negative regulatory region on the promoter region of IL-10 gene. This region represents a binding site for the transcription factor signal transducer and activator of transcription 3(STAT 3). Similar to T allele in IL-4C-590T, A allele of IL-10 C-597A increases the accessibility of STAT 3 to its site in the promoter region but in contrast to IL-4-T590, IL-10 - A 597 has negative regulation activity. Thus, allele A is associated with reduced production of IL-10 [23].

In a recent study Wang et al. [27] demonstrated that administration of IL-10 to mice with allergic rhinitis alleviate allergic inflammatory response. This effect was evidenced by the decreased number of infiltrated eosinophils and mast cells and a reduction in Th2 and Th17 response^]. This, at least partially explains the highly significant association between allele A of the SNP IL-10 –C-597A and the prevalence of asthma in the current study.

These data highly suggest the role of the SNP IL-10 C-597A as a risk factor for asthma. However, more studies are required to draw solid conclusion.

ACKNOWLEDGEMENT:

The authors highly appreciate the effort of all staff in Al Zahra'a Consultative Center for Allergy and Asthma, and Consultative Clinic for Chest and Respiratory Diseases/Baghdad during samples collection.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 04.07.2017 Modified on 21.08.2017

Research J. Pharm. and Tech. 2017; 10(9): 2867-2870.

DOI: 10.5958/0974-360X.2017.00505.4