High risk association of IL-4 VNTR polymorphism with asthma in a North Indian population

Cytokine 66 (2014) 87–94

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High risk association of IL-4 VNTR polymorphism with asthma in a North Indian population Niti Birbian a, Jagtar Singh a,⇑, Surinder Kumar Jindal b, Ranbir Chander Sobti a a b

Department of Biotechnology, Panjab University, Chandigarh, India Department of Pulmonary Medicine, PGIMER, Chandigarh, India

a r t i c l e

i n f o

Article history: Received 15 June 2013 Received in revised form 19 December 2013 Accepted 7 January 2014 Available online 1 February 2014 Keywords: IL-4 VNTR polymorphism Asthma Risk North Indian population

a b s t r a c t Background: A case-control study was conducted to evaluate the role of IL-4 VNTR polymorphism in asthma that has been associated with various inflammatory diseases worldwide. This is the first case-control study conducted in India, investigating the role of IL-4 VNTR polymorphism in asthma pathogenesis. Methods: A case-control study was performed with a total of 824 adult subjects, inducting 410 asthma patients and 414 healthy controls from North India. The genotypes were identified by polymerase chain reaction. Results: Statistical analysis for the IL-4 VNTR polymorphism revealed that the Rp1 allele was significantly associated with asthma with OR = 1.47, 95% CI (1.11–1.94) and p = 0.005. The Rp1/Rp1 homozygous mutant genotype posed a high risk towards asthma with OR = 2.39, 95% CI (0.96–6.14) and p = 0.040. The Rp2/Rp1 heterozygous genotype also posed a risk towards asthma with OR = 1.39, 95% CI (1.00– 1.94) and p = 0.040. Most of the phenotypic traits were significantly associated with the disease. Conclusions: IL-4 VNTR polymorphism is a high risk factor for asthma in the studied North Indian population. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Increasing urbanization has increased the prevalence of atopic diseases such as asthma, dermatitis and allergic rhinitis globally, and understanding the mechanisms of onset and severity of allergy has offered a great challenge to researchers and scientists worldwide, due to the complex interplay of genetic as well as environmental factors [1,2]. According to the second National Family Health Survey (NFHS 2), conducted in the year 1998–99, asthma is the most common disease in India as compared to tuberculosis, diabetes, malaria, thyroid and other diseases [3]. In the presence of an allergen, naive CD4+ helper T (TH) cells get differentiated and are classified into two types: TH1 and TH2, on the basis of their cytokine secretions. TH1 cells promote cell-mediated immunity by producing Interferon-c (IFN-c) and Interleukin-2 (IL-2) while TH2 cells promote humoral immunity by inducing antibody production and secrete IL-4, IL-10 and IL-13 [4]. A TH1/ TH2 imbalance may trigger autoimmune or inflammatory responses [5–8]. IL-4 plays a critical role in the regulation of naive TH0 cell differentiation during an immune response, as during ⇑ Corresponding author. Tel.: +91 172 2534065, +91 9876775160; fax: +91 172 2541407. E-mail addresses: [email protected] (N. Birbian), [email protected] (J. Singh), [email protected] (S.K. Jindal), [email protected] (R.C. Sobti). 1043-4666/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cyto.2014.01.002

antigen challenge, T cells differentiate either as TH1 or TH2 cells, the mechanisms of which are very much dependent on cytokines. IL-4 promotes TH2 differentiation by inhibition of TH1 cell differentiation [9,10]. TH2 cells in turn further express IL-4 and IL-13 as the main cytokines, which are implicated in the pathogenesis of asthma and atopy [11,12], and promotion of acute inflammatory processes and airway remodelling [13]. IL-4 is an anti-inflammatory cytokine that reduces the effect of pro-inflammatory cytokines and is involved in the isotype switching from IgM/IgG to IgE by the B lymphocytes [14,15]. IgE mediated immune responses are further enhanced by IL-4 as it upregulates FCeRII (CD23) on B lymphocytes and phagocytes as well as FCeRI on mast cells and basophils [16]. The IgE dependent mast cell activation triggers immediate allergic reactions. Moreover, IL-4 also induces mucin gene expression that results in hypersecretion of mucus [17]. It also upregulates eotaxin expression from fibroblasts that leads to inflammation and asthma [18]. IL-4 further promotes cellular inflammation by recruiting vascular cell adhesion molecule 1 (VCAM-1) on vascular endothelium through which it directs the migration of T lymphocytes, monocytes, basophils and eosinophils to the inflammatory loci [19]. In murine model studies conducted on IL-4 transgenic mice, expression of IL-4 in the lungs has shown to cause epithelial cell hypertrophy and macrophage, lymphocyte, eosinophil and neutrophil accumulation in the lung airways [20]. In another study, it has

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been observed that IL-4 induces AHR and goblet-cell hyperplasia, independent of IL-13 [21]. In asthma patients, enhanced IL-4 levels have been detected in airway smooth muscle cells [13,22]. IL-4 is also involved in stimulating mucus-producing cells and fibroblasts, proving its role in airway remodelling [17,23,24]. Moreover, inhalation of human recombinant IL-4 has shown to induce airway eosinophilia and BHR in atopic asthma patients [25]. Furthermore, bronchial biopsies of asthma patients show elevated levels of IL-4 expression at mRNA and protein level as compared to the healthy control subjects [26–28]. IL-4 gene is located on chromosome 5q31–q33 and three polymorphisms are known to exist in it, two in the promoter region 590C/T and +33C/T and the third is a variable number of tandem repeat (VNTR) of 70 bp located within the third intron [29]. The IL-4 VNTR polymorphism is characterised by a rare Rp1 allele (2 repeats = 183 bp), a frequent Rp2 allele (3 repeats = 253 bp) and Rp3 (4 repeats), which is a rarer allele. It has been suggested that specific number of VNTR copies affect the transcriptional activity of the gene, thereby modifying the resulting immune response. The rarer Rp1 allele (2 repeats) is more responsive to transcription, leading to IL-4 over-expression, thereby altering the TH1/TH2 balance by upregulation of the TH2 immune response and downregulation of the TH1 immune response [30]. A variable number of tandem repeat (VNTR) is a short, repetitive DNA sequence, which may result in genetic variations in an individual [31] and may further alter the rate of gene transcription, stability of mRNA or the quantity and activity of the encoded protein. Individuals with different copy numbers of the repeat sequences differ in the number of potential protein binding sites, thereby altering the amount of cytokine production [32]. Numerous studies have been conducted across the globe to evaluate the role of IL-4 VNTR gene polymorphism in various auto-immune and non-infectious diseases. IL-4 VNTR polymorphism has been significantly associated with cerebral malaria in a Ghanian study [33]. In a Malaysian as well as an Indian study, the IL-4 VNTR polymorphism has also been significantly associated with end stage renal disease [34,35]. A study conducted on rheumatoid arthritis (RA) shows that the IL-4 VNTR gene polymorphism plays a protective role in the disease [36] while another Swiss study reports that the rarer Rp2 allele is associated with it [37], no association between the polymorphism and type 2 diabetes mellitus has been observed in an Indian study [38]. Results of a study conducted on patients with multiple sclerosis (MS), show that the IL-4 Rp1 allele is associated with late onset of MS and therefore might represent a modifier of age of onset rather than a susceptibility factor for patients with MS [30]. In a Chinese study conducted on patients with systemic lupus erythematosus (SLE), the role of IL-4 VNTR polymorphism remained unclear and further studies on a larger sample size is required [39]. In the present study, the hypothesis that IL-4 VNTR polymorphism is associated with asthma was examined. This is the first study that examined the role of IL-4 VNTR polymorphism in the North Indian population.

2. Materials and methods 2.1. Ethical clearance Ethical clearance for conducting the study on human blood samples was granted by the ‘‘Ethics Committee, PGIMER, Chandigarh’’. The study was conducted strictly in accordance with the ethical guidelines for bio-medical research on human subjects proposed by the ‘‘Central Ethics Committee on Human Research

(CECHR) ICMR-2000’’ and of those contained in the ‘‘Declaration of Helsinki’’. The selection of asthma patients was based on physician’s diagnosis. However, only the patients fulfilling the criteria of GINA (Global Initiative for Asthma) guidelines for diagnosis of bronchial asthma were recruited in the study [40].

2.2. Inclusion criteria This is the first case-control study conducted in India to evaluate the role of IL-4 VNTR polymorphism in asthma pathogenesis by recruiting a total of 824 adult subjects. The patients were recruited from different states of North India such as Punjab, Haryana, Chandigarh, Uttar Pradesh, Himachal Pradesh, Uttaranchal, Jammu & Kashmir, Rajasthan and New Delhi. A total of 410 asthma patients visiting the Out Patient Department, Pulmonary Medicine, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, were enrolled in the study, out of which, 323 subjects were asthma patients with allergic rhinitis. Informed Consent was duly obtained from the asthma patients participating in the study, and a detailed proforma of the asthma patients with a complete questionnaire regarding the clinical symptoms of the disease, i.e. wheeze/whistling, cough, shortness of breath (SOB), allergy, early morning or night symptoms, along with spirometry tests was assessed. Complete information of the patient regarding name, age, sex, history of the disease, occupation was taken into account (Table 1). A total of 414 age-matched, normal and completely healthy controls were inducted in the study. Some of the healthy volunteers were blood donors at various blood donation camps, educational institutes, employee groups. Completely healthy control subjects with no history of asthma, rhinitis, eczema, allergic skin diseases or any other co-morbid illness were recruited in the study. Care was taken that both the asthma patients as well as the control subjects were free from any other systemic immune or inflammatory conditions.

2.3. Exclusion criteria Asthma patients with history of any other pulmonary ailment such as tuberculosis, Chronic Obstructive Pulmonary Disease (COPD), bronchitis and emphysema were excluded from the study. No ABPA (Allergic Bronchopulmonary Aspergillosis) patients were taken in the study. Any subject having a first degree relative with asthma or allergy has not been recruited as a control in the present study. Not only the respiratory or allergic skin disorders, any subject with other diseases such as diabetes, high blood pressure or with drinking and smoking habits have also not been included as controls in the study. Each control was first enquired for all of the above conditions before the collection of blood samples.

2.4. Lung function test Spirometer device Spiro 232 (PK Morgan, Rainham, Kent, UK) was used for plethysmography which was performed strictly in accordance with the British Thoracic Society/Association of Respiratory Technicians and Physiologists (BTS/ARTP) guidelines [41]. The subjects were asked to relax, avoid any kind of exercise at least 30 min prior to the test and avoid smoking or using bronchodilator at least 4 h prior to the test. After placing the mouth-piece in the subject’s mouth, the procedure involved a maximal forced expiratory and then a forced inspiratory manoeuvre. Three acceptable manoeuvres, all within 5% of each other were recorded as flow-volume curve.

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N. Birbian et al. / Cytokine 66 (2014) 87–94 Table 1 Characteristics of the study population. Asthma patients 410 (%)

Controls 414 (%)

p

183 (44.6) 227 (55.4)

271 (65.5) 143 (34.5)

0.031* 0.141

41.9 ± 16.6 0 414 0 414 0 414

n.d.¥ 0.006* 0.144 0.006* 0.180 0.010* 0.018*

Childhood pneumonia No pneumonia

38.1 ± 16.2 323 (78.8) 87 (21.2) 366 (89.3) 44 (10.7) 65 (15.9) 345 (84.1) (n = 208) 42 166

0 414

0.126 0.020*

Spirometry data  FVC Observed FVC Predicted FEV1 Observed FEV1 Predicted FEV1/FVC Observed % FEV1/FVC Predicted %

(n = 190) 2.56 ± 0.96 3.19 ± 0.73 1.94 ± 0.82 2.68 ± 0.77 75.00 ± 13.71 83.12 ± 5.84

n.d.¥

n.d.¥

Weight (Kg)  Height (cm)  BSA (m2) 

57.6 158.7 1.58

n.d.¥ n.d.¥ n.d.¥

n.d.¥ n.d.¥ n.d.¥

BMI (kg/m2)  Underweight (<18.5) Normal weight (18.5–24.9) Over weight (25.0–29.9) Obesity (P30.0)

22.7 17.1 22.2 26.8 32.7

n.d.¥

n.d.¥

IgE (IU/ml)§

(n = 219) 4066.55 ± 5742.24

(n = 150) 2354.40 ± 1486.61

0.283

Sex Males Females Age Allergic rhinitis No rhinitis Allergic to at least 2 provoking factors Non-allergic Ever-smoker Non-smoker

*   § ¥

FVC, Forced vital capacity; FEV1, Forced expiratory volume in 1 s. Spirometry test, weight, height, BSA and BMI have been recorded for 190 asthma patients and mean values in each category have been calculated. IgE levels were confirmed for 219 asthma patients and 150 controls and given as average in IU/ml. n.d. –not determined.

2.5. Immunological investigation Total serum IgE concentration (IU/ml) was assessed for 219 asthma patients and 150 control subjects with an ELISA reader (Table 1). The BAL (bronchoalveolar lavage) fluid of subjects was examined for acid fast bacillus, aspergillosis and malignancy so as to distinguish the asthma patients from patients suffering from tuberculosis (TB), allergic bronchopulmonary aspergillosis (ABPA) and lung cancer, respectively. However, the TH1/TH2 cytokine profiling of BALF was not performed. No significant difference for IgE level among cases and controls was observed for the polymorphism. 2.6. Body mass index (BMI) Body mass index (BMI), a measure of body fat based on height (cm) and weight (kg) that applies to adult men and women was calculated using the measures set by the U.S. National Heart Lung and Blood Institute (NHLBI) [42]. The mean values have been calculated for asthmatics for 4 different categories: underweight (<18.5 kg/m2), normal weight (18.5–24.9 kg/m2), overweight (25.0–29.9 kg/m2) and obesity (P30.0 kg/m2). Apart from BMI (kg/m2), Height (cm), Weight (kg) and Body Surface Area (BSA) (m2) have also been recorded for the patients and have been given as mean values in Table 1. 2.7. Sample collection Blood samples were collected in EDTA coated vials, and stored at 80 °C until genomic DNA extraction was done. Genomic DNA

was isolated from the thawed blood samples by the Sodium Saline Citrate Buffer Method [43] and checked for DNA on 0.8% agarose gel by electrophoresis.

2.8. Genotyping of IL-4 VNTR polymorphism The amplification of the IL-4 VNTR polymorphism was done with F 50 – TAGGCTGAAAGGGGGAAAGC-30 and R 50 -CTGTTCACCTCAACTGCTCC-30 primers [29]. PCR was carried out in a thermal cycler, in a total volume of 25 ll containing: 10X PCR Buffer, 3 mM MgCl2, 1 mg/ml nuclease free BSA, 50 pmol of each primer, 10 mM of each dNTP, 0.125 U Taq polymerase and 2 ll genomic DNA. The PCR conditions were: initial denaturation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30s, 56 °C for 30 s, 72 °C for 1 min, and final extension step at 72 °C for 10 min. The PCR products were analyzed directly on 2% agarose gel stained with ethidium bromide and visualized by UV transillumination. A 253 bp product (three repeats) indicated the presence of homozygous wild Rp2/Rp2 genotype while a 183 bp product (two repeats) marked the presence of the homozygous mutant Rp1/Rp1 genotype. The presence of 183 bp and 253 bp products in a lane indicated the heterozygous Rp2/Rp1 genotype (Fig. 1). The European Molecular Genetics Quality Network (EMQN) good practice guidelines have been followed. A few PCR vials with all the PCR contents except the DNA, were also included per PCR batch as ‘‘negative controls’’. No contamination was observed and there were no ‘‘false positives’’. To minimize the risk of contamination, sterilized and autoclaved solutions and equipment were used during DNA isolation. The ingredients for PCR were well stored at

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Fig. 1. PCR products of IL-4 VNTR gene polymorphism on 2% agarose gel. Lanes 2, 4, 5, 6, 7, 8, 9, 12, 13, 14, 16: homozygous wild Rp2/Rp2 (253 bp), lanes 1, 3, 11: heterozygous Rp2/Rp1 (253 bp and 183 bp), lanes 10, 15: homozygous mutant Rp1/ Rp1 (183 bp), lane 17: 100 bp ladder.

20 °C and were thawed just before use [44]. Retyping of samples was done at random to check for the homology of results. 2.9. Statistical analysis The allelic distribution of the IL-4 VNTR polymorphism between the asthma patients and healthy control subjects were analyzed statistically using Chi2 test. The data was analyzed using SPSS 17.0 software and Epi Info version 3.4.3. Fisher’s exact test was used wherever applicable. Statistical significance was assumed for p < 0.05.

3. Results In the present study, a total of 824 subjects, including 410 adult asthma patients and 414 adult healthy controls were genotyped for the IL-4 VNTR polymorphism. Statistical analysis of the allelic distribution of the wild Rp2 allele was higher among the control subjects (87.0%) as compared to the asthma patients (82.0%) while the mutant Rp1 allele was more prevalent among the asthma patients (18.0%) than in the controls (13.0%), conferring a significant risk towards asthma with OR = 1.47, 95% CI (1.11–1.94) and p = 0.005 (Table 2). The genotypic frequencies revealed that the homozygous wild Rp2/Rp2 genotype was more prevalent among the control subjects (75.8%) than in asthmatics (68.1%). The heterozygous Rp2/Rp1 genotype was significantly present more among the asthmatics (27.8%) than the control subjects (22.2%), posing a risk towards asthma with OR = 1.39, 95% CI (1.00–1.94) and p = 0.040. However, the homozygous mutant Rp1/Rp1 genotype was more prevalent among the asthmatics (4.1%) than the controls (1.9%) with OR = 2.39, 95% CI (0.96–6.14) and p = 0.040. It was also observed that Rp2/Rp1 + Rp1/Rp1 in combination resulted in a significant

increased risk with OR = 1.47, 95% CI (1.07–2.03) and p = 0.012 (Table 2). Further categorizing the asthma patients on the basis of phenotypic characteristics of the disease (Table 3), such as sex (male/female), occurrence (seasonal/throughout), severity (wheeze on exertion/wheeze at rest), family history (positive/nil), rhinitis (positive/nil), allergy to at least 2 provoking factors (positive/nil), smoking status (non-smoker/ever-smoker), longstanding cough (positive/nil), sputum production (positive/nil), pattern of daily symptoms (morning SOB/nocturnal SOB/anytime during day SOB), significant high risk association was observed between the Rp1 allele and male asthmatics, throughout occurrence of asthma, wheeze at rest, nil family history and positive traits of rhinitis, allergy, cough, sputum, night SOB, in non-smokers as well as eversmokers (p < 0.05). Significant high risk association was observed between the Rp1 allele and male asthmatics with OR = 1.50, 95% CI (1.02– 2.22) and p = 0.031, patients with throughout occurrence of asthma with OR = 1.54, 95% CI (1.14–2.09) and p = 0.003, wheeze at rest with OR = 1.68, 95% CI (1.20–2.34) and p = 0.001, nil family history with OR = 1.63, 95% CI (1.21–2.20) and p = 0.001 and positive rhinitis with OR = 1.49, 95% CI (1.11–2.00) and p = 0.006, allergy with OR = 1.47, 95% CI (1.10–1.95) and p = 0.006, cough with OR = 1.48, 95% CI (1.10–1.98) and p = 0.006, sputum production with OR = 1.52, 95% CI (1.13–2.04) and p = 0.004, night SOB with OR = 1.50, 95% CI (1.11–2.02) and p = 0.007, non-smokers with OR = 1.40, 95% CI (1.05–1.88) and p = 0.018 and ever smokers with OR = 1.83, 95% CI (1.12–2.98) and p = 0.010. However, no association was observed for female asthmatics, seasonal occurrence, wheeze on exertion, positive family history, nil rhinitis, nil allergy, nil cough, nil sputum production, morning SOB as well as anytime SOB (all p > 0.05). In 208 asthma patients, who had ever suffered from childhood pneumonia, it was observed that IL-4 VNTR polymorphism conferred significant risk towards asthma in patients with no history of childhood pneumonia with OR = 1.50, 95% CI (1.05–2.15) and p = 0.020. However, no association of asthma was observed for positive childhood pneumonia history with OR = 1.57, 95% CI (0.84–2.89) and p = 0.126 (Table 4). 4. Discussion Candidate gene association studies are the most widely employed approaches to investigate the susceptibility towards asthma. A typical case-control gene polymorphism study investigates the relationship between the genetic markers and the disease by comparison of the distribution of the genotype and allele frequencies of the studied genes among the patient and the control groups. The present research is the first study to evaluate the role of IL-4 VNTR gene polymorphism in a North Indian population with asthma, which has revealed that the polymorphism poses a significant

Table 2 Distribution of IL-4 VNTR genotype and allele frequencies in asthma patients and controls.

*

Asthma patients

Controls

410 (%)

414 (%)

v2

OR

(95% CI)

p

Genotype frequencies Rp2/Rp2 Rp2/Rp1 Rp1/Rp1 Rp2/Rp1 + Rp1/Rp1

279 (68.1) 114 (27.8) 17 (4.1) 131 (31.9)

314 (75.8) 92 (22.2) 8 (1.9) 100 (24.1)

4.20 4.22 6.21

1.39 2.39 1.47

Ref. (1.0) (1.00–1.94) (0.96–6.14) (1.07–2.03)

0.040* 0.040* 0.013*

Allele frequencies Rp2 Rp1

672 (82.0) 148 (18.0)

720 (87.0) 108 (13.0)

7.87

1.47

Ref. (1.0) (1.11–1.94)

0.005*

p < 0.05.

91

N. Birbian et al. / Cytokine 66 (2014) 87–94 Table 3 Phenotypic characteristics and IL-4 VNTR polymorphism.

*

v2

Phenotypic traits

n

Rp2/Rp2

Rp2/Rp1

Rp1/Rp1

Rp2

Rp1

Controls Males Females

414 271 (65.5) 143 (34.5)

314 (75.8) 209 (77.1) 105 (73.4)

92 (22.2) 57 (21.0) 35 (24.5)

8 (2.0) 5 (1.9) 3 (2.1)

720 (87.0) 475 (87.6) 245 (85.7)

108 (13.0) 67 (12.4) 41 (14.3)

IgE 65,000 IU/ml 5,000–10,000 IU/ml P10,000 IU/ml

150 145 (96.7) 5 (3.3) 0 (0)

109 (72.7) 104 (71.7) 5 (100) 0 (0)

35 (23.3) 35 (24.1) 0 (0) 0 (0)

6 6 0 0

(4.0) (4.1) (0) (0)

253 (84.3) 243 (83.8) 10 (100) 0 (0)

47 (15.7) 47 (16.2) 0 (0) 0 (0)

Asthmatics Sex Males Females

183 (44.6) 227 (55.4)

125 (68.3) 154 (67.8)

52 (28.4) 62 (27.3)

6 (3.3) 11 (4.9)

302 (82.5) 370 (81.5)

64 (17.5) 84 (18.5)

4.65 2.17

1.50 1.36

(1.02–2.22) (0.89–2.08)

0.031* 0.141

Occurrence Seasonal Throughout

128 (31.2) 282 (68.8)

88 (68.8) 191 (67.7)

38 (29.7) 76 (27.0)

2 (1.6) 15(5.3)

214 (83.6) 458 (81.2)

42 (16.4) 106 (18.8)

1.85 8.53

1.31 1.54

(0.87–1.96) (1.14–2.09)

0.173 0.003*

Severity Wheeze on exertion Wheeze at rest

216 (52.7) 194 (47.3)

149 (69.0) 130 (67.0)

64 (29.6) 50 (25.8)

3 (1.4) 14 (7.2)

362 (83.8) 310 (79.9)

70 (16.2) 78 (20.1)

2.34 10.16

1.29 1.68

(0.92–1.81) (1.20–2.34)

0.126 0.001*

Family history Nil +ve

285 (69.5) 125 (30.5)

187 (65.6) 92 (73.6)

84 (29.5) 30 (24.0)

14(4.9) 3 (2.4)

458 (80.4) 214 (85.6)

112 (19.6) 36 (14.4)

11.11 0.31

1.63 1.12

(1.21–2.20) (0.73–1.72)

0.001* 0.581

Rhinitis Nil +ve

87 (21.2) 323 (78.8)

58 (66.7) 221 (68.4)

28 (32.2) 86 (26.6)

1 (1.1) 16 (5.0)

144 (82.8) 528 (81.7)

30 (17.2) 118 (18.3)

2.13 7.63

1.39 1.49

(0.87–2.21) (1.11–2.00)

0.144 0.006*

Allergy Nil +ve

44 (10.7) 366 (89.3)

29 (65.9) 250 (68.3)

14 (31.8) 100(27.3)

1 (2.3) 16 (4.4)

72 (81.8) 600 (82.0)

16 (18.2) 132 (18.0)

1.79 7.43

1.48 1.47

(0.80–2.73) (1.10–1.95)

0.180 0.006*

Smoking status Non-smoker Ever smoker

345 (84.1) 65 (15.9)

238 (69.0) 41 (63.1)

94 (27.2) 20 (30.8)

13 (3.8) 4 (6.1)

570 (82.6) 102 (78.5)

120 (17.4) 28 (21.5)

5.57 6.66

1.40 1.83

(1.05–1.88) (1.12–2.98)

0.018* 0.010*

Cough Nil Longstanding cough

74 (18.0) 336 (82.0)

50 (67.6) 229 (68.1)

22 (29.7) 92 (27.4)

2 (2.7) 15 (4.5)

122 (82.4) 550 (81.8)

26 (17.6) 122 (18.2)

2.17 7.46

1.42 1.48

(0.86–2.32) (1.10–1.98)

0.141 0.006*

Sputum production Nil +ve

95 (23.2) 315 (76.8)

66 (69.5) 213 (67.6)

27 (28.4) 87 (27.6)

2 (2.1) 15(4.8)

159 (83.7) 513 (81.4)

31 (16.3) 117 (18.6)

1.40 8.38

1.30 1.52

(0.82–2.05) (1.13–2.04)

0.236 0.004*

Pattern of daily symptoms Morning SOB 20 (4.9) Night SOB 292 (71.2) Anytime SOB 98 (23.9)

15 (75.0) 202 (69.2) 62 (63.3)

5 (25.0) 73 (25.0) 36 (36.7)

0 (0) 17 (5.8) 0 (0)

35 (87.5) 477 (81.7) 160 (81.6)

5 (12.5) 107 (18.3) 36 (18.4)

– 7.39 3.72

0.95 1.50 1.50

(0.28–2.52) (1.11–2.02) (0.97–2.31)

0.921 0.007* 0.054

IgE 65,000 IU/ml 5,000–10,000 IU/ml P10,000 IU/ml

144 (65.8) 102 (65.8) 37 (64.9) 5 (71.4)

68 (31.0) 48 (31.0) 18 (31.6) 2 (28.6)

7 5 2 0

356 (81.3) 252 (81.3) 92 (80.7) 12 (85.7)

82 (18.7) 58 (18.7) 22 (19.3) 2 (14.3)

1.15 0.65 – –

1.24 1.19 – –

(0.82–1.87) (0.76–1.86) – –

0.283 0.420 – –

(95% CI)

p

219 155 (70.8) 57 (26.0) 7 (3.2)

(3.2) (3.2) (3.5) (0)

OR

(95% CI)

p

Ref. (1.00)

p < 0.05.

Table 4 Occurrence of childhood pneumonia in asthmatics and IL-4 VNTR polymorphism.

Controls asthmatics Childhood pneumonia (+) Nil *

n

Rp2/Rp2

Rp2/Rp1

Rp1/Rp1

Rp2

Rp1

414

314 (75.8)

92 (22.2)

8 (2.0)

720 (87.0)

108 (13.0)

42 166

29 109

10 53

3 4

68 271

16 61

v2

OR

Ref. (1.00) 2.34 5.41

1.57 1.50

(0.84–2.89) (1.05–2.15)

0.126 0.020*

p < 0.05.

risk towards asthma. This case-control study evaluated the role of gene polymorphisms in asthma pathogenesis by inducting a total of 824 adult subjects with 410 asthma patients and 414 healthy controls. The subjects were recruited from different states of North India such as Punjab, Haryana, Chandigarh, Uttar Pradesh, Himachal Pradesh, Uttaranchal, Jammu & Kashmir, Rajasthan and New Delhi. Strict exclusion criteria were followed while selecting both the categories of subjects to prevent bias and confounding due to population stratification.

The mean age (±SD) of the asthma patients was 38.1 ± 16.2 and of controls was 41.9 ± 16.6 years. The frequency of male asthma patients was 44.6% and of females was 55.4% while frequency of male control subjects was 65.5% and of females was 34.5%. Out of the total asthmatics, 78.8% suffered from allergic rhinitis while 21.2% had no rhinitis. 89.3% asthmatics were allergic to at least 2 provoking factors while the remaining 10.7% were not allergic. Taking into account the smoking history of asthmatics, 15.9% patients were ever-smokers with a smoking history while 84.1%

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asthmatics were non-smokers with no smoking history. However, control subjects with no smoking history were enrolled. Spirometry for 190 asthma patients was conducted with mean values for FEV1/FVC observed% was 75.00 ± 13.71 and FEV1/FVC predicted % 83.12 ± 5.84. A limitation of the study is that since the spirometry data for controls was not available, statistics could not be applied. For the 190 asthma patients undergoing spirometry, mean values for weight- 57.6 kg, height- 158.7 cm and BSA1.58 m2 were observed. The mean BMI was 22.7 kg/m2 with 26.3% underweight, 45.3% normal weight, 23.2% overweight and 5.3% obese patients. Total serum IgE levels were evaluated for 219 asthmatics with a mean value of 4066.55 IU/ml and 150 controls with 2354.40 IU/ml. IL-4 VNTR polymorphism genotyping was done by direct PCR. The % age of Rp2 repeat in the total sample size was 84.5%, while the % age of Rp1 repeat was 15.5%. Both genotypic as well as the allelic frequencies were significantly associated with asthma, indicating that the IL-4 VNTR polymorphism poses as a risk towards the disease in the studied North Indian population. IL-4 plays a pivotal role in asthma and atopy, leading to the differentiation of naive TH0 cells to B- cell activating TH2 cells and surface expression of CD23 and MHC class II. It also leads to IgM switching to IgE [45,46]. Examination of bronchoalveolar lavage (BAL) of asthmatic patients has shown enhanced IL-4 levels [47]. A major role of IL-4 has also been suggested in asthma and atopy in humans [48–50]. Murine studies on mice deprived of IL-4 gene [51] and on transgenic mice with over-expression of IL-4 [52], have demonstrated a key role of IL-4 in allergy and autoimmunity. A more studied IL-4 590C/T polymorphism has shown a higher luciferase activity and increased total IgE levels [53]. A study conducted on patients with atopic allergy in Philippines reported that the IL-4 590C/T allele predisposed towards risk of allergy with OR = 2.06, 95% CI (1.285–3.301) and p = 0.002. Moreover, the IL-4 590 TT genotype was associated with increased IgE levels (P1000 IU/ml) with OR = 3.968, 95% CI (1.499–10.5) and p = 0.016 [54]. A Taiwanese study has shown that the 589T allele is higher in asthmatics (p < 0.0001) and in asthmatics with severe AHR (p < 0.05) as compared to the controls [55]. An Iranian study has shown a strong association of IL-4 590C/ T, 33C/T and IL-4 1092G/T polymorphism with bronchial asthma. While the allelic frequencies for the IL-4 +33C/T alone were associated with protective effect towards asthma (p < 0.000), IL-4 1092G/T was associated with risk towards asthma (p = 0.0005), the genotypic frequencies of IL-4 590C/T (p = 0.03) and IL-4 +33C/T (p = 0.0001) conferred protection from asthma and IL-4 1092 G/T polymorphism predisposed towards risk of asthma (p = 0.0001). Moreover, the mean total serum IgE level (258.8 IU/ ml) in asthmatics with TTT/GCC haplotypes was significantly higher than in other haplotypes (95.4 IU/ml) with p = 0.004 [56]. Another Iranian study has shown association of IL-4 590C/T polymorphism and asthma susceptibility (p = 0.02). Furthermore, the polymorphism was also associated with asthma severity (p = 0.07) [57]. While an Iranian study suggests an influence of IL-4 590C/T polymorphism and asthma [58], another study shows no association of IL-4 590C/T polymorphism and asthma susceptibility [59]. In a study conducted on Australian and British populations, the 590C/T polymorphism was weakly associated with wheeze (p = 0.029) and specific IgE to house mites (p = 0.013), however the British population did not show any association with the polymorphism [60]. Data from a Japanese paediatric study has suggested that IL-4 590C/T polymorphism may be associated with asthma (p = 0.023) but not with total serum IgE [61]. A Canadian paediatric study has shown the association of IL-4 589T allele (RR = 4.1) and

that the TT genotype predisposed to increased risk of rhinitis development (RR = 2.4) [62]. A Chinese study on mite-sensitized allergic persistent rhinitis showed that the CT/CC genotypes had decreased risk of allergic rhinitis with OR = 0.64, 95% CI (0.45–0.92) and lower serum total IgE (p = 0.0001) [63] while another Chinese study has reported no significant association between IL-4 589C/T polymorphism and asthma (p > 0.05) and IgE levels (p > 0.05) among asthmatics and controls [64]. A South Indian study has suggested a role of IL-4 590C/T polymorphism in atopic asthma (p = 0.044) [65]. A Chinese study has shown that the IL-4 +33 CT and TT genotypes led to higher asthma risk and total serum IgE levels as compared to the wild CC genotype (p < 0.01) [66]. A Japanese study has shown association of IL-4 +33C/T polymorphism with total serum IgE levels (p < 0.05) [67]. A Spanish study has reported no association of the allelic or genotypic frequencies distribution of IL-4 33C/T polymorphism with asthma [68]. A study conducted on COPD patients in two diverse, Japanese and Egyptians populations, also revealed no significant association with IL-4 VNTR polymorphism [69]. An Indian study conducted on COPD patients also revealed no significant IL-4 VNTR allelic or genotypic association with the disease [70]. A very few studies have investigated the role of IL-4 VNTR polymorphism in asthma. A study conducted in Venezuela has shown significant difference in the genotypic distribution of IL-4 VNTR Rp2/Rp2 homozygotes among asthmatics and controls (p = 0.0137) and has been associated with susceptibility to asthma [71]. Despite the numerous observational, cohort and case-control candidate gene association studies conducted worldwide to investigate the susceptibility towards asthma, the complete and exact causative agents have not yet been characterised. Only one-third of the genetic predisposition underlying asthma has been unveiled. Several studies have concluded that asthma is a complex disease due to interplay of multiple genetic and environmental factors. Identifying the genes underlying the mechanism of asthma may clarify the reason for the rise of asthma with progressive urbanization. Candidate gene studies like the present one, by targeting the new genes may lead to a better classification of asthma. The gene polymorphism studies may also help to identify the protective environmental factors and those that confer risk. To play a relevant role in clinical asthma, the susceptibility genes need to be investigated in case-control studies with different levels of disease severity, populations and risk factors, so that genotype can be used as a disease predictor just like other epidemiological risk factors. All the potential gene polymorphisms implicated in asthma should be examined in various representative populations from different regions of the world, so that different genes interacting with different environmental conditions may help in understanding the different therapeutic implications. Novel therapies can be initiated on the basis of the newer genetic findings for more effect strategies for disease prevention and cure, just like the anti-IL4R therapy that has been developed against asthma. TH2 cytokines, such as IL-4, IL-13 and signal transducer and activator of transcription factor-6 (STAT-6) play a key role in the airway hyperresponsiveness (AHR), airway inflammation and mucus production. It has been suggested that targeting molecules against these cytokines may prove to be beneficial in asthma therapy. Since IL-4 and IL-13 share IL-4Ra as a common receptor, approaches of employing anti-IL-4Ra therapy might be promising in the treatment of moderate to severe asthma [72]. IL-4 is the key cytokine that regulates the inflammatory cascade in asthma, right from the eosinophilic chemotaxis to the differentiation of naive TH0 cells to TH2 cells [73,74], which leads to secretion of TH2 cytokines such as IL-3, IL-4, IL-5, IL-13, GMCSF, further

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aggravating airway inflammation, a key trait of asthma [75–77]. Hence, keeping in view the major regulatory role of IL-4 in asthma, human ‘anti-IL-4 mAb’ has been developed by Amgen (CA, USA; AMG-317) to block the action of IL-4 and IL-13 cytokines and is currently being administered to patients with moderate to severe asthma [78]. Another experimental treatment involves subcutaneous injection or nebulization of cynomolgus monkeys with the IL-4 variant termed as ‘pitrakinra’, which has functional mutations preventing the binding of IL-4 and IL-13 to IL-4Ra complex, preventing allergen induced airway eosinophilia and AHR [79]. 5. Conclusions The findings of this study aim to shed some light on the role of the IL-4 VNTR polymorphism in asthma. Moreover, this is the first North Indian study conducted on IL-4 VNTR polymorphism which reveals it as a risk towards asthma. Most of the phenotypic traits of the disease were also associated with the polymorphism. Acknowledgements J. Singh is thankful to the University Grants Commission, New Delhi, India, for providing grant support for the study [UGC Grant F. No. 40-161/2011 (SR)].

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