TF Leung, IHS Chan, GWK Wong, NLS Tang, SL Ma, CWK Lam, TF Fok, PC Ng

Abstract

Interleukin (IL)-1 gene family encodes for pleiotropic pro-inflammatory and anti-inflammatory cytokines. Genome screens mapped asthma phenotypes to chromosome 2q12-21 where IL-1 gene cluster is located. This study investigated the relation between asthma traits and polymorphisms at positions -31 and -511 of IL-1β gene (IL1B) in Chinese children. Plasma total IgE and allergen-specific IgE concentrations were measured by immunoassays. IL1B promoter genotypes were characterised by restriction fragment length polymorphism. One hundred and fifty-eight patients and 56 controls were recruited. Significant inter-ethnic variations in allele frequencies of IL1B were found between Chinese and other populations. Neither IL1B polymorphisms was associated with asthma. However, patients homozygous for IL1B -31C had lower FEV₁ (p=0.03) and FVC (p=0.008). More subjects with IL1B -31C/-511T haplotype had increased plasma total IgE (OR 1.61, 95%CI 1.02-2.54; p=0.04) and decreased FEV₁ (OR 1.78, 95%CI 1.06-3.02; p=0.03) and FVC (OR 1.87, 95%CI 1.09-3.22; p=0.02). In conclusion, IL1B promoter polymorphisms are associated with poorer lung function and increased plasma total IgE concentration in Chinese children.

Introduction

Asthma is characterised by chronic airway inflammation caused by a complex interaction between genetic and environmental factors. A key element in the inflammatory response of the asthmatic airways is the production of pro-inflammatory cytokines, notably interleukin (IL)-1β, and to a lesser extent, tumour necrosis factor-α.¹ The human airway smooth muscle releases IL-1β, which, together with IL-5 from type 2 helper T lymphocytes, modulates bronchial hyperresponsiveness (BHR).² On the other hand, IL-1Ra possesses anti-inflammatory properties, and inhibits in vivo BHR to histamine and airway inflammation in sensitised animals.² The imbalance between pro- and anti-inflammatory cytokines, especially IL-1β and IL-1Ra, might thus be an important determinant of asthma.^3,4 Genome-wide searches mapped asthma and atopy to a number of chromosomal regions,^5-10 including chromosome 2q12-21 where the genes encoding IL-1α (IL1A), IL-1β (IL1B) and IL-1 receptor antagonist (IL-1Ra, IL1RN) were located.¹¹ These genetic and functional characteristics of IL-1 support the candidacy of IL-1 cluster as an asthma susceptibility locus. Using candidate gene approach, IL1RN polymorphisms were associated with various asthma and atopy phenotypes.^3,12,13 Interestingly, a gender-specific effect was seen between IL1B -511 polymorphism and asthma susceptibility in Finnish adults.¹³ On the other hand, the effects of different polymorphic sites in IL1B on asthma severity and atopy have not been studied. The aim of this study is to investigate the correlation between IL1B polymorphisms at positions -31 and -511 and physician-diagnosed asthma, atopy and spirometric parameters of asthma severity in Chinese children.

Patients and Methods

Study Population

This case-control study recruited children aged 5 years or above, with asthma as diagnosed according to the criteria proposed by the American Thoracic Society¹⁴ and onset of disease before 15 years of age, who were followed in the general paediatric clinic of a university teaching hospital in Hong Kong. Both parents were ethnic Chinese by self-reporting. In brief, these patients had typical asthma symptoms that were relieved by bronchodilators and the presence of reversibility and/or hyperreactivity on spirometry. Controls were selected among children attending the hospital for non-allergic and non-immunologic diseases. Subjects or their parents gave informed written consent, and the Clinical Research Ethics Committee of our university approved this study.

Plasma IgE Measurements

Plasma total IgE concentration was measured by micro-particle immunoassay (IMx analyser, Abbott Laboratories, Abbott Park, IL), and results were expressed following logarithmic transformation (IgE_log). Specific IgE antibodies to locally prevalent allergens^15,16 D. pteronyssinus, cat, dog, mixed cockroaches and mixed molds were measured by fluorescent enzyme immunoassay (Pharmacia Diagnostics AB, Uppsala, Sweden), with concentration >=0.35 kIU/l being positive. Atopy was defined as the presence of at least one type of allergen-specific IgE.

Spirometry

All asthmatic patients underwent spirometry (COMPACT II, Vitalograph, Buckingham, England) to measure their lung functions.

PCR Assays for IL1B Polymorphisms

Genomic DNA was extracted from peripheral venous blood using High Pure Viral Nucleic Acid Kit (Boehringer Mannheim, Indianapolis, IN). IL1B polymorphisms at -31 and -511 were determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism.^3,17,18 Table 1 describes the PCR primers and assay conditions used in this study. In particular, we created a Hae III restriction site at position -31 (1903 bp of the complete DNA sequence) in IL1B promoter with the use of a pair of novel mismatched primers: 5'-CTC CTA CTT CTG CTT TTG AAG GC-3' and 5'-GAG CAA TGA AGA TTG GCT GA-3' (GenBank accession number X04500).^19,20 The final PCR products were digested using appropriate restriction enzymes (New England Biolabs, Hitchin, Herts, UK) and visualised on agarose gel containing ethidium bromide.

Restriction enzyme digestions were performed each time in a 96-well plate containing 88 samples and 8 positive controls with PCR products of known genotypes. Ten random samples from each IL1B genotype were also sequenced using BigDye Terminator Cycle sequencing kits and ABI-310 autosequencer (Applied Biosystems, Foster City, CA). All genotypes from restriction enzyme digestion were confirmed.

Statistical Analysis

The clinical phenotypes of asthma and atopy between patients and control subjects were compared using χ² or Fisher Exact test. The distributions of different genotypes in the two polymorphic markers between asthmatics and controls were compared using χ². The clinical, laboratory and spirometric variables between various subject or genotype groups were compared by Student t test or ANOVA as appropriate. Linkage disequilibrium between the two polymorphic markers was assessed by Haploview (Daly Lab, Cambridge, MA). Two-locus haplotype frequencies for IL1B -31 and -511 were maximum likelihood estimates (EH program, Laboratory of Statistical Genetics at Rockefeller University, New York, NY). All comparisons were made two-sided. A p-value <0.05 was considered to be significant.

Results

Clinical Characteristics

One hundred fifty-eight asthmatic children and 56 non-allergic control subjects were recruited. Table 2 summarises the clinical characteristics of our asthmatic patients. Their mean±SD ages at evaluation were 10.2±3.6 years and 11.1±4.0 years respectively (p=0.121), with 61% of patients and 59% of controls being males (p=0.746).

Plasma Total and Allergen-specific IgE Concentrations

The mean±SD of IgE_log in kIU/l in asthmatic patients was significantly higher than in non-allergic controls (2.62±0.60 vs 2.12±0.74, p<0.0001). Table 3 summarises the pattern of allergic sensitisation. Atopy was found in 141 (90%) of patients and 33 (59%) of control children.

Sensitisation to D. pteronyssinus and cat were significant risk factors for asthma diagnosis in our subjects.

IL1B Polymorphisms and Asthma or Atopy Phenotypes

The allele frequencies of IL1B polymorphic markers are listed in Table 1, and the allele-genotype distributions of polymorphic markers -31 and -511 followed Hardy-Weinberg equilibrium (p=0.395 and 0.409, respectively). IL1B -31 and -511 were in strong linkage disequilibrium (D'=0.971; r²=0.891), with -31T being linked to -511C and vice versa. There was no significant association between the diagnosis of asthma, whether classified as atopic or nonatopic, and polymorphisms in IL1B (results not shown). Neither were these polymorphisms associated with plasma total IgE concentration or the presence of any allergen-specific IgE in this study (results not shown).

IL1B Polymorphisms and Spirometric Variables

Table 4 summarises the relationship between spirometric variables in our patients and IL1B polymorphisms. There was significant correlation between IL1B -31 polymorphism and the measured FEV₁ (104% vs 99% vs 93% for wild-type, heterozygous and mutant respectively, p=0.03) and FVC (127% vs 118% vs 113%, p=0.008).

Association between Clinical Phenotypes and IL1B Haplotypes

Table 5 summarises the results of haplotype analysis for IL1B -31 and -511 polymorphisms. Significantly more subjects with mutant alleles at both loci (-31C/-511T) had increased plasma total IgE concentrations (OR 1.61, 95%CI 1.02-2.54; p=0.04) and reduced FEV₁ (OR 1.78, 95%CI 1.06-3.02; p=0.03) and FVC (OR 1.87, 95%CI 1.09-3.22; p=0.02) as compared to those with wild-type alleles (-31T/-511C). However, IL1B promoter haplotype was not associated with a diagnosis of asthma or atopy.

Discussion

The present study shows that asthmatic patients homozygous for -31C in IL1B promoter have poorer lung function (FEV₁ and FVC) as compared to other genotypes. On the other hand, IL1B -511 is not associated with asthma phenotypes, plasma IgE or lung function in our Chinese children. Haplotype analysis confirms the association between IL1B -31/-511 and spirometric variables. This IL1B promoter haplotype is also associated with plasma total IgE concentration, with subjects having -31C/-511T have increased total IgE.

The pathogenesis of asthma is mediated by CD4⁺ T lymphocytes that produce a type 2 cytokine profile.^1,21 The surge in these interleukins, in the presence of pro-inflammatory cytokines such as IL-1β, IL-6 and tumour necrosis factor-α, results in airway inflammation and BHR.^1,2 The imbalance in pro-inflammatory cytokine IL-1β and anti-inflammatory IL-1Ra could be seen in patients with severe asthma.⁴ Genome-wide screens have also mapped asthma to chromosome 2q12-21 containing the IL-1 gene cluster.^5,7,9 Polymorphisms in this gene cluster were associated with asthma susceptibility in the Caucasian population.^12,13 One of the objectives of this study is thus to investigate the usefulness of IL1B polymorphisms in predicting asthma severity in Chinese children. To the best of our knowledge, this study shows for the first time that IL1B polymorphism is linked to FEV₁ and FVC (i.e. asthma severity). Haplotype analysis of IL1B T-31C and C-511T confirmed this association. Our results also reveal that the haplotype with mutant alleles at both -31 and -511 is associated with increased plasma total IgE concentrations whereas genotype analyses for each of these polymorphic sites were negative. Haplotype analysis is thus a more powerful method than single nucleotide polymorphism in identifying susceptibility loci for asthma phenotypes.

Our asthmatic patients carrying the C allele at IL1B -31 had significantly lower FEV₁ compared to those with T allele. Interestingly, this restriction site at IL1B -31 is located at the TATA box that is essential for normal gene transcription.¹⁹ Thus, this single-base substitution probably results in diminished IL1B transcription. Because IL-1β is pro-inflammatory,^1,2 this mutation would theoretically decrease airway inflammation and lead to better performance on spirometric measures that contradicts results obtained in this study. One possible explanation is that IL1B T-31C and C-511T, or other polymorphisms in close linkage, may alter the production of both IL-1β and IL-1Ra. As reported also by Tillie-Leblond et al., these changes can upset the balance between IL-1β and IL-1Ra that in turn serves as an important mechanism in the pathogenesis of asthma.⁴ The ability of leukocytes to produce IL-1b and IL-1Ra from subjects having different IL1B -31 (and -511) polymorphisms is not tested in the present study. Further studies should try to characterise any functional consequence associated with T→C substitution at position -31 of IL1B, and to delineate the relationship between IL1B haplotypes and plasma concentrations of IL-1β and IL-1Ra.

DPP10, encoding a homolog of dipeptidyl peptidases that cleave terminal dipeptides from cytokines and chemokines, has recently been identified as a novel gene influencing asthma.²² In this study, Allen and colleagues initially investigated the association between asthma and polymorphisms in the IL-1 gene cluster. Although such association could not be detected, they observed that asthma susceptibility in these subjects was highly significantly associated with the microsatellite D2S308 in the neighbouring region. Using positional cloning on a comprehensive, high-density, single-nucleotide polymorphism linkage disequilibrium map, the authors identified DPP10 as a novel asthma candidate gene. Interestingly, this gene is also located on chromosome 2q14, about 800 kb distal to the IL-1 cluster, which harbors various promising candidate genes that mediate inflammatory responses.^11,23-25 Thus, it is possible that our observed associations between asthma-related traits and IL1B polymorphisms may be due to the linkage with DPP10 or other candidate genes in this region. Future studies should extend our findings by including the genotyping of other polymorphic markers in the adjacent region of IL1B and its gene cluster.

Our research group as well as others reported significant inter-ethnic variations in the allele frequencies of a number of asthma candidate genes in Chinese as compared with Caucasians or even Japanese.^26-32 With regard to IL1B polymorphisms (Table 1), the frequency of -511C was detected in 48% of our subjects that is similar to those in Japanese and Caucasian^3,18,20 whereas -31T was present in 49% of our Chinese children and 38% of Caucasians.²⁰ Thus, results of Caucasian genetic studies may not be generalisable to the oriental population. Population-based studies are needed to investigate the allele frequencies and clinical importance of our IL1B markers in Chinese subjects.

Acknowledgements

This project was supported by a Direct Grant for Research of the Chinese University of Hong Kong, and a donation from Zindart (De Zhen) Foundation Ltd, Hong Kong.

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