Cytokine 62 (2013) 389–394

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Cytokine

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High risk association of IL-1 receptor antagonist (IL-1RN) VNTR polymorphismwith asthma in a North Indian population: A pilot study

Niti Birbian a, Jagtar Singh a,⇑, Surinder Kumar Jindal b

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

a r t i c l e i n f o a b s t r a c t

Article history:Received 26 September 2012Received in revised form 13 March 2013Accepted 18 March 2013Available online 16 April 2013

Keywords:IL-1 receptor antagonist (IL-1RN)IL-1RN VNTR polymorphismAsthmaRiskNorth Indian population

1043-4666/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.cyto.2013.03.019

⇑ Corresponding author. Tel.: +91 172 2534065, +92541407.

E-mail addresses: nitibirbian@yahoo.com (N. B(J. Singh), skjindal@indiachest.org (S.K. Jindal).

Background: A pilot case-control study was conducted to evaluate the role of IL-1 receptor antagonist (IL-1RN) VNTR penta-allelic polymorphism in asthma that has been associated with various inflammatorydiseases worldwide. This is the first case-control study conducted in India, investigating the role of IL-1RN VNTR polymorphism in asthma pathogenesis.Methods: A case-control study was performed with a total of 824 adult subjects, inducting 410 asthmapatients and 414 healthy controls from North India. The genotypes were identified by polymerase chainreaction.Results: Statistical analysis for the IL-1RN VNTR polymorphism revealed that the IL-1RN�2 allele was sig-nificantly associated with asthma with OR = 1.45, 95% CI (1.15–1.85) and p = 0.001. The IL-1RN�2/2 geno-type posed a risk towards asthma with OR = 1.66, 95% CI (0.97–2.86) and p = 0.048. Most of thephenotypic traits were significantly associated with the disease.Conclusions: IL-1RN�2 allele is a high risk factor for asthma in the studied North Indian population.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Over the last decade, the prevalence of atopic diseases such asasthma, dermatitis and allergic rhinitis has been on a rise globally,and understanding the mechanisms of onset and severity of al-lergy, has offered a great challenge to researchers and scientistsworldwide, due to the complex interplay of genetic as well as envi-ronmental factors [1]. According to the second National FamilyHealth Survey (NFHS 2), conducted in the year 1998–99, asthmais the leading disease in India as compared to other diseases [2].

Either a single-bp substitution of one nucleotide for another or avariable number of tandem repeat (VNTR), which is a short, repet-itive DNA sequence, may result in genetic variations in an individ-ual [3], which may further alter the rate of gene transcription,stability of mRNA or the quantity and activity of the encoded pro-tein [4]. Individuals with different copy numbers of these tandemrepeats differ in the potential protein binding sites.

Interleukin-1 (IL-1), an important pro-inflammatory cytokine,enhances the expression of a wide variety of cytokine genes andalso down regulates the expression of various housekeeping aswell as receptor genes. A variety of cell types such as macrophages,monocytes and keratinocytes produce IL-1. Chromosome 2

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1 9876775160; fax: +91 172

irbian), jagtar72@gmail.com

(q12–q14) contains the IL-1 gene complex which encodes for thepro-inflammatory cytokines IL1-a and IL-1b as well as the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1RN) whichcounteracts and inhibits them. The IL-1RN protein encoded bythe IL-1RN gene has anti-inflammatory activity and hence reducesbronchial hyper responsiveness (BHR) [5,6]. Thus, a balance be-tween IL-1 and IL-1RN is an important parameter for normal anddisease states which determines the severity of an inflammatoryresponse to an environmental stimulus that is disturbed as a resultof the IL-1RN VNTR polymorphism [7].

The IL-1RN gene has a penta-allelic polymorphic site in intron 2region due to the presence of an 86-bp variable number of tandemrepeat (VNTR) sequence [8,9]. The IL-1RN gene polymorphismshave been associated with severity of or susceptibility to variousinflammatory disorders [10].

The present study aimed at investigating the role of IL-1RNVNTR polymorphism in asthma.

2. Material and methods

2.1. Ethical clearance

Ethical clearance for conducting the study on humanblood samples was granted by the ‘‘Ethics Committee, PGIMER,Chandigarh’’. The study was conducted strictly in accordance withthe ethical guidelines for bio-medical research on human subjects

390 N. Birbian et al. / Cytokine 62 (2013) 389–394

proposed by the ‘‘Central Ethics Committee on Human Research(CECHR) ICMR-2000’’ and of those contained in the ‘‘Declarationof Helsinki’’. The selection of asthma patients was based on physi-cian’s diagnosis. However, only the patients fulfilling the criteria ofGINA (Global Initiative for Asthma) guidelines for diagnosis ofbronchial asthma were recruited in the study [11].

2.2. Inclusion criteria

This is the first case-control study conducted in India to evalu-ate the role of IL-1RN VNTR polymorphism in asthma pathogenesisby recruiting a total of 824 adult subjects. The patients were re-cruited from different states of North India such as Punjab, Harya-na, Chandigarh, Uttar Pradesh, Himachal Pradesh, Uttaranchal,Jammu & Kashmir, Rajasthan and New Delhi. A total of 410 asthmapatients visiting the Out Patient Department, Pulmonary Medicine,Post Graduate Institute of Medical Education and Research (PGI-MER), Chandigarh, were enrolled in the study, out of which, 323subjects were asthma patients with allergic rhinitis. Informed con-sent was duly obtained from the asthma patients participating inthe study, and a detailed proforma of the asthma patients with acomplete questionnaire regarding the clinical symptoms of the dis-ease, i.e. wheeze/whistling, cough, shortness of breath (SOB), al-lergy, early morning or night symptoms, along with spirometrytests was assessed. Complete information of the patient regardingname, age, sex, history of the disease, occupation was taken intoaccount (Table 1).

Table 1Characteristics of the study population.

Asthma patients410 (%)

Controls414 (%)

SexMales 183 (44.6) 351 (60.3)Females 227 (55.4) 231 (39.7)

Age 38.1 ± 16.2 41.9 ± 16.6

Allergic rhinitis 323 (78.8) 0No rhinitis 87 (21.2) 414

Allergic to at least 2 provoking factors 366 (89.3) 0Non-allergic 44 (10.7) 414

Ever-smoker 65 (15.9) 0Non-smoker 345 (84.1) 414

Spirometry data (n = 190) n.d.FVC observed 2.56 ± 0.96FVC predicted 3.19 ± 0.73FEV1 observed 1.94 ± 0.82FEV1 predicted 2.68 ± 0.77FEV1/FVC observed % 75.00 ± 13.71FEV1/FVC predicted % 83.12 ± 5.84

Weight (kg) 57.6 n.d.

Height (cm) 158.7 n.d.

BSA (m2) 1.58 n.d.

BMI (kg/m2) 22.7 n.d.Underweight (<18.5) 17.1Normal weight (18.5–24.9) 22.2Over weight (25.0–29.9) 26.8Obesity (30.0 or >30.0) 32.7

IgE (IU/ml) (n = 219) (n = 150)4066.55 2354.40

FVC, Forced Vital Capacity; FEV1, Forced Expiratory Volume in 1 s.Spirometry test, weight, height, BSA and BMI have been recorded for 190 asthmapatients and mean values in each category have been calculated.IgE levels were confirmed for 219 asthma patients and 150 controls and given asaverage in IU/ml.n.d. – not determined.

A total of 414 age-matched, normal and completely healthycontrols were inducted in the study. Some of the healthy volun-teers were blood donors at various blood donation camps, educa-tional institutes, employee groups. Completely healthy controlsubjects with no history of asthma, rhinitis, eczema, allergic skindiseases or any other co-morbid illness were recruited in the study.Care was taken that both the asthma patients as well as the controlsubjects were free from any other systemic immune or inflamma-tory conditions.

2.3. Exclusion criteria

Asthma patients with history of any other pulmonary ailmentsuch as tuberculosis, Chronic Obstructive Pulmonary Disease(COPD), bronchitis and emphysema were excluded from the study.No ABPA (allergic bronchopulmonary aspergillosis) patients weretaken in the study. Any subject having a first degree relative withasthma or allergy has not been recruited as a control in the presentstudy. Not only the respiratory or allergic skin disorders, any sub-ject with other diseases such as diabetes, high blood pressure orwith drinking and smoking habits have also not been included ascontrols in the study. Each control was first enquired for all ofthe above conditions at the time of taking their written informedconsent and 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 inaccordance with the British Thoracic Society/Association of Respi-ratory Technicians and Physiologists (BTSs/ARTP) guidelines [12].The subjects were asked to relax, avoid any kind of exercise at least30 min prior to the test and avoid smoking or using bronchodilatorat least 4 h prior to the test. After placing the mouth-piece in thesubject’s mouth, the procedure involved a maximal forced expira-tory and then a forced inspiratory manoeuvre. Three acceptablemanoeuvres, all within 5% of each other were recorded as flow-vol-ume curve.

2.5. Immunological investigation

Total serum IgE concentration (IU/ml) was assessed for 219asthma patients and 150 control subjects with an ELISA reader (Ta-ble 1). The BAL (bronchoalveolar lavage) fluid of subjects wasexamined for acid fast bacillus, aspergillosis and malignancy soas to distinguish the asthma patients from patients suffering fromtuberculosis (TB), allergic bronchopulmonary aspergillosis(ABPA) and lung cancer, respectively. However, the Th1/Th2 cyto-kine profiling of BALF was not performed. No significant differencefor IgE level among cases and controls was observed for thepolymorphism.

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 wascalculated using the measures set by the US National Heart Lungand Blood Institute (NHLBI) [13]. Apart from BMI (kg/m2), height(cm), weight (kg) and Body Surface Area (BSA) (m2) have also beenrecorded for the patients and have been given as mean values inTable 1.

2.7. Sample collection

Blood samples were collected in EDTA coated vials, and storedat �80 �C until genomic DNA extraction was done. Genomic DNA

N. Birbian et al. / Cytokine 62 (2013) 389–394 391

was isolated from the thawed blood samples by the Sodium SalineCitrate Buffer Method [14] and checked for DNA on 0.8% agarosegel by electrophoresis.

2.8. Genotyping of IL-RN VNTR polymorphism

The amplification of the IL-1RN VNTR polymorphism was donewith F 50-TCCTGGTCTGCAGGTAA-30 and R 50-CTCAGCAACACTCC-TAT-30 primers [15]. PCR was carried out in a thermal cycler, in atotal 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 eachdNTP, 0.125 U Taq polymerase and 2 ll genomic DNA. The PCRconditions were: initial denaturation at 94 �C for 5 min, followedby 35 cycles at 94 �C for 1 min, 60 �C for 1 min, 72 �C for 1 min,and final extension step at 72 �C for 10 min. The PCR products ofIL-1RN�1 (410 bp = 4 repeats), IL-1RN�2 (240 bp = 2 repeats), IL-1RN�3 (500 bp = 5 repeats), IL-1RN�4 (325 bp = 3 repeats) and IL-1RN�5 (595 bp = 6 repeats) were observed by electrophoresis on2% agarose gels stained with ethidium bromide and visualized byUV transillumination (Fig. 1).

The European Molecular Genetics Quality Network (EMQN)good practice guidelines have been followed. A few PCR vials withall the PCR contents except the DNA, were also included per PCRbatch as ‘‘negative controls’’. No contamination was observed andthere were no ‘‘false positives’’. To minimize the risk of contamina-tion, sterilized and autoclaved solutions and equipment were usedduring DNA isolation. The ingredients for PCR were well stored at�20 �C and were thawed just before use [16]. Retyping of sampleswas done at random to check for the homology of results.

2.9. Statistical analysis

The allelic distribution of the IL-1RN VNTR polymorphism be-tween the asthma patients and healthy control subjects were ana-lyzed statistically using Chi2 test. The data was analyzed using SPSS17.0 software and Epi Info version 3.4.3. Fisher’s exact test wasused wherever applicable. Statistical significance was assumedfor p < 0.05.

3. Results

In the present study, a total of 824 subjects, including 410 adultasthma patients and 414 adult healthy controls were genotyped forthe IL-1RN VNTR polymorphism.

Statistical analysis of the results indicated that the allelic distri-bution of the more common IL-1RN�1 allele was higher among thecontrol subjects (78.9%) as compared to the asthma patients(72.3%) while the IL-1RN�2 allele was more prevalent among theasthma patients (26.1%) than in the controls (19.6%), conferring asignificant risk towards asthma with OR = 1.45, 95% CI (1.15–1.85) and p = 0.001. The IL-1RN�3 allele was slightly less prevalentin asthma patients (1.0%) than in controls (1.2%). The IL-1RN�4 al-lele was also marginally more present among the asthma patients(0.6%) in contrast to the healthy controls (0.4%). However, the IL-1RN�5 allele was completely absent from the studied population(Table 2).

The genotypic frequencies revealed that the IL-1RN�1/1 genotypewas more prevalent among the control subjects (65.2%) than in asth-matics (54.6%). The IL-1RN�1/2 genotype was significantly presentmore among the asthmatics (32.7%) than the control subjects(24.9%) with OR = 1.57, 95% CI (1.13–2.17) and p = 0.004. The IL-1RN�2/2 genotype was also significantly more prevalent amongthe asthmatics (9.8%) than the controls (7.0%) with OR = 1.66, 95%CI (0.97–2.86) and p = 0.048. It was also observed that asthma indi-viduals with at least one copy of the IL-1RN�2 allele (�1/2+�2/2) were

at significant increased risk than the controls (31.9%) with OR = 1.59,95% CI (1.18–2.14), p = 0.005, (�1/2+�2/3) with OR = 1.55, 95% CI(1.12–2.15), p = 0.005, (�1/2+�1/3) with OR = 1.53, 95% CI (1.11–2.10), p = 0.006, (�1/2+�1/4) with OR = 1.59, 95% CI (1.15–2.19),p = 0.003, (�1/2+�4/4) with OR = 1.56, 95% CI (1.13–2.16), p = 0.004and (�2/2+�1/4) with OR = 1.73, 95% CI (1.02–2.93), p = 0.030. It isconcluded that �2 allele in a genotypic fashion such as �1/2 or �2/2confers significant risk towards asthma.

The other genotypes with the rarer IL-1RN alleles such as IL-1RN�2/3, IL-1RN�1/3, IL-1RN�1/4 and IL-1RN�4/4 were present invery less frequencies, neither pre-disposing any significant risknor protection to the studied population. The IL-RN�3/3 genotypewas completely absent from the population (Table 2).

Further categorizing the asthma patients on the basis of pheno-typic characteristics of the disease (Table 3), such as sex (male/fe-male), occurrence (seasonal/throughout), severity (wheeze onexertion/wheeze at rest), family history (positive/nil), rhinitis (po-sitive/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 dailysymptoms (morning SOB/nocturnal SOB/anytime during daySOB), significant high risk association was observed between theIL-1RN�2 allele and almost all the phenotypes of asthma (all p-val-ues <0.05), except for the nil traits of cough, sputum production,rhinitis, allergy and anytime pattern of SOB, where no associationwas observed.

BMI of 190 asthma patients has been recorded in Table 1. Themean values have been calculated for 4 different categories: under-weight (<18.5 kg/m2), normal weight (18.5–24.9 kg/m2), over-weight (25.0–29.9 kg/m2) and obesity (30.0 or >30.0 kg/m2). Itwas observed that the wild IL-1RN�1 allele was most prevalentamong the asthma patients with normal BMI while the mutantIL-1RN�2 allele was most prevalent among the asthma patientswho were either underweight or overweight (data not shown).

4. Discussion

Ever since the completion of the human genome project, a lot ofresearch is being carried out worldwide to investigate the role ofvarious gene polymorphisms implicated in asthma pathogenesisso as to streamline the genes involved in the complex interplaywith the environmental factors and stimuli.

The present research is the first study to evaluate the role of IL-1RN VNTR polymorphism in a North Indian population with asth-ma, which has revealed that this gene polymorphism poses ahighly significant risk towards the disease. The results obtainedfrom the current study supported the above hypothesis with theobservations that both the allelic as well as the genotypic frequen-cies of the IL-1RN VNTR polymorphism confer significant risk to-wards asthma and hence in turn reveal a major protective role ofthe functional IL-1RN gene.

In the overall scenario, our study supported the findings byother researchers to the fact that IL-1RN�1 (4 repeats) and the IL-1RN�2 (2 repeats) are the most common alleles in the human pop-ulation [15,17] wherein the IL-1RN �2 allelic as well as the IL-1RN�2/2 genotypic frequencies were found to be significantly associ-ated with asthma (p = 0.05). However, no significant difference inthe distributions of the IL-1RN�3,�4 and �5 was observed betweenthe cases and the controls.

IL-1RN�2 allele (2 repeats) is critical in the molecular pathways ofseveral diseases and it has been associated with autoimmune dis-eases such as multiple sclerosis, ulcerative colitis, rheumatoidarthritis, vestibulitis, alopecia areata, psoriasis, diabetic nephropa-thy, idiopathic recurrent miscarriages, Grave’s disease as well as sys-temic lupus erythematosus [15,17–26]. A study has also reported

Fig. 1. PCR products of IL-1RN VNTR polymorphism on 2% agarose gel. Lanes 1, 2, 3, 4, 8, 9, 15: IL-1RN�1/1 genotype, lanes: 6, 13, 14, 16: IL-1RN�1/2 genotype, lane 12: IL-1RN�2/2 genotype, lane 5: IL-RN�2/3 genotype, lane 7: IL-1RN�1/4 genotype, lane 10: IL-1RN�4/4 genotype, lane 11: IL-1RN�1/3 genotype, lane 17: 100 bp ladder (Fermentas).

Table 2Distribution of IL-1RN VNTR genotypic and allelic frequencies among asthma patients and controls.

Frequencies Asthma patients 410 (%) Controls 414 (%) OR 95% CI p-Value

Genotype frequenciesIL-1RN�1/1 224 (54.6) 270 (65.2) Ref (1.0)�1/2 134 (32.7) 103 (24.9) 1.57 (1.13–2.17) 0.004�2/2 40 (9.8) 29 (7.0) 1.66 (0.97–2.86) 0.048�1/2+�2/2 174 (42.5) 132 (31.9) 1.59 (1.18–2.14) 0.005�2/3 0 (0.0) 1 (0.2) 0.0 (0.0–47.18) 1.000F

�1/2+�2/3 134 (32.7) 104 (25.1) 1.55 (1.12–2.15) 0.005�2/2+�2/3 40 (9.8) 30 (7.2) 1.61 (0.94–2.75) 0.064

�1/3 8 (2.0) 9 (2.2) 1.07 (0.37–3.08) 0.888�1/2+�1/3 142 (34.7) 112 (27.1) 1.53 (1.11–2.10) 0.006�2/2+�1/3 48 (11.8) 38 (9.2) 1.52 (0.94–2.48) 0.072�2/3+�1/3 8 (2.0) 10 (2.4) 0.96 (0.34–2.70) 0.939�1/4 3 (0.7) 1 (0.2) 3.62 (0.29–190.52) 0.335F

�1/2+�1/4 137 (33.4) 104 (25.1) 1.59 (1.15–2.19) 0.003�2/2+�1/4 43 (10.5) 30 (7.2) 1.73 (1.02–2.93) 0.030�2/3+�1/4 3 (0.7) 2 (0.4) 1.81 (0.20–21.8) 0.663F

�1/3+�1/4 11 (2.7) 10 (2.4) 1.33 (0.51–3.44) 0.526�4/4 1 (0.2) 1 (0.2) 1.21 (0.02–94.96) 1.000F

�1/2+�4/4 135 (32.9) 104 (25.1) 1.56 (1.13–2.16) 0.004�2/2+�4/4 41 (10.0) 30 (7.2) 1.65 (0.97–2.81) 0.050�2/3+�4/4 1 (0.2) 2 (0.4) 0.60 (0.01–11.66) 1.000F

�1/3+�4/4 9 (2.2) 10 (2.4) 1.08 (0.40–2.95) 0.861�1/4+�4/4 4 (0.9) 2 (0.4) 2.41 (0.34–26.83) 0.418F

Allele frequenciesIL-1RN�1 593 (72.3) 653 (78.9) Ref (1.0)IL-1RN�2 214 (26.1) 162 (19.6) 1.45 (1.15–1.85) 0.001IL-1RN�3 8 (1.0) 10 (1.2) 0.88 (0.32–2.43) 0.791IL-1RN�4 5 (0.6) 3 (0.4) 1.84 (0.35–11.86) 0.490F

F: Fisher’s exact test applied.

392 N. Birbian et al. / Cytokine 62 (2013) 389–394

that stimulation of granulocyte–macrophage colony-stimulatingfactor (GM-CSF), resulted in enhanced �2 allele associated IL-1RNproduction [27].

While a recent data has suggested a major role of the IL-1RNVNTR polymorphism in various types of cancer [28,29], a Swedishstudy on patients with myasthenia gravis reports no associationbetween IL-1RN VNTR polymorphism and the disease [30].

In a Finnish atopic population study, IL-1RN�2/2 homozygosityhas been associated with an increased risk of skin prick test(SPT) positivity with OR = 2.69, 95% CI (1.32–5.47) [31] and evenin our study the IL-1RN�2/2 genotype is significantly more preva-

lent among the asthmatics (9.8%) than the controls (7.0%) withOR = 1.66, 95% CI (0.97–2.86) and p = 0.048. Another investigationinvolving random German and Swedish asthma patients has con-cluded that IL-1RN gene polymorphisms are associated with asth-ma (p < 0.05) [32]. A Turkish pediatric study has reported anenhanced serum spIgE level in asthmatic children with the IL-1RN�2/2 genotype. However, unlike the present study, the �1/1genotype posed a greater risk towards asthma with OR = 1.54,95% CI (1.07–2.23) and p = 0.02. While in the Turkish study, the�1/2 genotype conferred protection with OR = 0.49, 95% CI (0.29–0.83) and p = 0.004, in our study it predisposed towards risk of

Table 3Phenotypic characteristics of the study population and IL-1RN VNTR polymorphism.

Phenotypic traits n �1/1 �1/2 �2/2 IL-1RN�1 IL-1RN�2 OR 95% CI p-Value

Controls 402 270 103 29 643 161Males 262 175 67 20 417 107 Ref (1.0)Females 140 95 36 9 226 54

Asthma patientsSex

Males 178 97 62 19 256 100 1.52 (1.10–2.11) 0.008Females 220 127 72 21 326 114 1.46 (1.00–2.15) 0.040

OccurrenceThroughout 227 160 86 31 406 148 1.46 (1.12–1.89) 0.003Seasonal 121 64 48 9 176 66 1.50 (1.06–2.11) 0.016

SeverityWheeze on exertion 208 111 78 19 300 116 1.54 (1.16–2.05) 0.001Wheeze at rest 190 113 56 21 282 98 1.39 (1.03–1.87) 0.025

Family historyFamily history (Nil) 279 157 93 29 407 151 1.48 (1.14–1.93) 0.002Family History (+ve) 119 67 41 11 175 63 1.44 (1.01–2.04) 0.033

RhinitisRhinitis (Nil) 86 52 27 7 131 41 1.25 (0.83–1.88) 0.262Rhinitis (+ve) 312 172 107 33 451 173 1.53 (1.19–1.97) 0.001

AllergyAllergy (Nil) 43 22 18 3 62 24 1.55 (0.91–2.62) 0.086Allergy (+ve) 355 202 116 37 520 190 1.46 (1.14–1.87) 0.001

Smoking statusNon-smoker 335 188 115 32 491 179 1.46 (1.13–1.87) 0.002Ever-smoker 63 36 19 8 91 35 1.54 (0.98–2.40) 0.047

CoughCough (Nil) 72 50 17 5 117 27 0.92 (0.57–1.48) 0.723Cough (+ve) 326 174 117 35 465 187 1.61 (1.25–2.06) 0.001

Sputum productionSputum (Nil) 94 63 27 4 153 35 0.91 (0.60–1.40) 0.662Sputum (+ve) 304 161 107 36 429 179 1.67 (1.29–2.15) 0.001

Pattern of daily symptomsMorning/night SOB 303 166 103 34 435 171 1.57 (1.22–2.03) 0.001Anytime SOB 95 58 31 6 147 43 1.17 (0.78–1.74) 0.423

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asthma with OR = 1.57, 95% CI (1.13–2.17) and p = 0.004. The �5allele was the only significant allele in the Turkish study (p = 0.05)and �5/5 genotype was significantly more protective in nature withOR = 0.42, 95% CI (0.39–0.48) and p = 0.03 while it is completely ab-sent from our studied North Indian population [33]. Another Egyp-tian paediatric study has revealed that the �1 allele played as a riskfactor for asthma with the overall more prevalence in the asthmacases with OR = 7.5, 95% CI (3.4–14.0) and p = 0.05. Moreover, the�1/1 genotype predisposed towards risk of asthma with a higher fre-quency in asthmatic children with OR = 1.5, 95% CI (1.3–1.8) andp < 0.0001 whereas unlike the present study the �1/2 genotype con-fers protection with OR = 0.2, 95% CI (0.09–0.46) and p < 0.0001 [34].While another Finnish [35] study conducted on patients with aller-gic rhinitis, the homozygous �2/2 genotype predisposed towardsgreater risk of allergic rhinitis with OR = 2.8, 95% CI (1.3–6.2) andp < 0.05 while in our study the �2/2 genotype also predisposed to-wards greater risk of rhinitis with OR = 1.53, 95% (1.19–1.97) andp = 0.001. A Japanese [6] study has also reported the role of �2 allelein bronchial asthma, predisposing towards risk with OR = 5.71, 95%CI (1.63–19.80) and p = 0.007 while another Japanese study finds noassociation of the IL-1RN VNTR polymorphism with the disease alto-gether [36].

In contrast to the risk posed by IL-1RN polymorphism towardsasthma in most of the research conducted worldwide, an Americanstudy has reported no association of the IL-1RN genotypes with therate of lung function decline in smokers [37]. Furthermore, aJapanese study also reports no association of this polymorphismwith COPD [38].

A research conducted on South African White and Blackasthmatic populations also reports no significant association ofIL-1RN VNTR polymorphism and asthma [39]. Similar results wereobserved for a study conducted on a Taiwanese asthmatic popula-tion as well, thereby negating any association with asthma [40].

A large number of case-control studies have been conducted inthe recent past and the results have been found to vary profoundlywith differences in the asthmatic populations studied across theworld, mostly not revealing similar significances, but leading to adefinite conclusion that asthma is a complex polygenic disease,which certainly does not follow classical Mendelian pattern ofinheritance [41]. As a result, it makes it all the more crucial to iden-tify the genetic makeup associated with the complexity of asthma.

The role of IL-1RN VNTR polymorphism in asthma is a much de-bated scenario. However, the reason for the contrasting results ofthe studies on this gene polymorphism’s association with asthma,as obtained in different populations globally, can be attributed tothe differences in the ethnicities as well as to the complex inter-play of the multiple genes and environmental factors involved inthe mechanism of asthma pathogenesis.

However, the present study concludes that the IL-1RN VNTRpolymorphism is a high risk factor for asthma and is useful markerfor asthma in the studied North Indian population.

Acknowledgements

J. Singh is highly grateful to the Department of Science & Tech-nology, New Delhi under the Ministry of Science & Technology,

394 N. Birbian et al. / Cytokine 62 (2013) 389–394

India, for providing research Grant (SR/FT/LS-018/2008) for thestudy.

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