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- EM, expectation maximisation
- IL, interleukin
- LD, linkage disequilibrium
- SNP, single nucleotide polymorphism
- SPT, skin prick test
The interleukin (IL)-1 gene cluster within chromosome 2 contains genes coding for both anti- and proinflammatory cytokines, including IL-1alpha, IL-1beta, IL-1Ra, and several novel cytokines designated IL-1F5 - IL-1F10.1 Proinflammatory cytokines IL-1alpha and beta are involved in the enhancement of inflammation and host defence. Anti-inflammatory IL-1Ra counteracts the function of IL-1alpha and IL-1beta. These cytokines are produced by a variety of cell types, for example, monocytes, magrophages, and keratinocytes. All IL-1 family genes are polymorphic and several of these polymorphisms have been shown to be associated with either susceptibility to or severity of inflammatory conditions and diseases.2
Atopy is a familial syndrome underlying atopic eczema, allergic rhinoconjuctivitis, and bronchial asthma. The skin prick test (SPT) is used to examine specific IgE mediated allergic responses and the results are generally in line with anamnestic data on atopy.3 Both IL-1alpha and beta have been shown to play a role in the regulation of allergen induced early and late reactions.4,5 IL-1alpha has also been found to accelerate cutaneous inflammation,6 whereas IL-1beta is involved in the regulation of contact sensitisation.7 In asthmatic bronchial epithelium, the expression of both IL-1beta and IL-1Ra is increased.8
Cytokines function as a network and thus the net effect on allergic inflammation may be dependent on allele combinations of individual cytokine genes. Recently in our population based case control study, we showed that IL1A (SNP +4845) genotype influences the SPT results.9 The carriers of the rarer allele 2 had a significantly lower SPT positive rate than that of non-carriers. Now we have expanded the study by analysing two additional SNPs (IL1B SNP +3954 C>T and IL1RN VNTR in intron 2) in the IL-1 gene cluster from the same cohort.
MATERIALS AND METHODS
Altogether 254 females and 151 males (mean age 60 years, age range 31–89 years) participated in the study. The subjects studied were the controls of a Finnish population based case-control study conducted to investigate the risk factors of adult asthma. More detailed information on the subjects is described elsewhere.9 All subjects gave informed consent for participation. Approval for this study was obtained from the ethical committee of Tampere University Hospital.
Atopy was determined by skin prick test performed by specially trained nurses with a panel of 22 common allergen extracts, including dog, cat, birch, cow dander, horse, mugwort, alder, meadow foxtail, timothy, barley, oats, wheat, rye, Alternaria alternata, Acarus siro, Aspergillus fumigatus, Cladosporium herbarum, Der farinae, Der pteronyssinus, Lepidoglyphus destructor, Tyrophagus putrescentiae, and with both a negative control (saline) and a positive control (histamine) (ALK A/S, Copenhagen, Denmark). The patient was considered prick test positive if at least one allergen elicited a weal with a diameter at least 3 mm larger than that of the negative control. Allergy testing by the skin prick method was carried out on 99.3% of subjects (252 females and 150 males).
We have recently shown that a single nucleotide polymorphism (SNP) at position +4845 (G>T) of the interleukin 1A (IL1A) gene is associated with reactivity in the skin prick test (SPT); the carriers of the rarer allele (allele 2) had a significantly lower SPT positive rate than that of non-carriers.
To examine whether the allele combinations or haplotypes of this complex would mediate functionally different responses, we now genotyped two additional polymorphisms of the IL-1 gene complex.
It was shown that in addition to IL1A*1 homozygosity, IL1B*1 homozygosity, and IL1RN*2 homozygosity were associated with a higher risk of SPT positivity (OR 1.62 (95% confidence intervals 1.070 to 2.460) and 2.69 (1.320 to 5.470) respectively). The genotype combination containing homozygotes of IL1A*1, IL1B*1, and IL1RN*2 was associated with the highest risk of SPT+ (OR 4.07 (1.720 to 9.630)). Moreover, haplotype analysis with expectation maximisation algorithm showed that IL1A*1, IL1B*1, and IL1RN*2 belonged to the same haplotype and its presence increased the risk of SPT positivity (OR 1.80 (1.290 to 2.530)).
We conclude that the IL1A*1/IL1B*1/IL1RN*2 haplotype contains the decisive allele or allele combinations which increase the risk of atopy.
Genomic DNA was isolated from EDTA whole blood samples using standard methods. IL1A SNP+4845 (G/T polymorphism in exon 5; G is marked as allele 1 and T is marked as allele 2), IL1B SNP +3954 (C/T polymorphism; C is marked as allele 1 and T is marked as allele 2), and IL1RN VNTR (86 bp repeat sequence in intron 2; dup 2–5) were analysed as described previously.9–11
To detect a possible deviation of the genotype frequencies from the Hardy-Weinberg equation, the exact test using the Markov chain algorithm was used (Arlequin program, version 2.0. A software for population genetics data analysis. Schneider S, Roessli D, Excoffier L, Genetics and Biometry Laboratory, Geneva, Switzerland). The haplotypes and their frequencies were calculated according to genotyped data using the expectation maximisation (EM) algorithm (Arlequin program). This approach has been shown to have high predictive value and it is in good concordance (90–98%) with pedigree data or simulated sample set analysis.12,13 The genotype distributions did not deviate from expected under the Hardy-Weinberg equation (HWE), which is a prerequisite for EM algorithm based haplotype analysis. Odds ratios and their 95% confidence intervals (CI) were calculated using CIA software (version 1.1, copyrighted by M J Gardner and British Medical Journal, 1989). Linkage disequilibrium (LD) between each locus was calculated as described previously.14 The theoretical maximum D’ values between loci with alleles A/a and B/b (allele frequencies CA, Ca, CB, and Cb and haplotype frequencies CAB, CAb, CaB, and Cab) were obtained by dividing CAB-CACB by min (CACb, CaCB), if D>0, and min (CACB, CaCb), if D <0. The likelihood ratio test was used to assess the significance of the linkage between loci.
The percentage of skin test positive subjects was 38.0. Participants were divided into SPT+ subjects (that is, ⩾1 positive reaction) and SPT− subjects. The genotypic data of these three SNPs is shown in table 1. IL1A genotype 1.1, IL1B 1.1, and IL1RN 2.2 were associated with an increased risk of SPT positivity, whereas IL1A genotype 1.2 and IL1B 1.2 were associated with diminished risk. After combining IL1A, IL1B, and IL1RN genotypes, it appeared that the distribution of genotype combinations differed significantly between SPT− and SPT+ groups (p<0.001, chi-square test, df=18). The genotype combination 1.1, 1.1, 2.2 markedly increased the risk of skin test positivity (OR 4.07 (95 % CI 1.720 to 9.630)). The opposite effect was observed with the genotype combination 1.2, 1.2, 1.1 (table 2).
It has been shown that IL-1 complex loci are in strong linkage disequilibrium.15 As expected, this was also true in our material. All the linkages between the pairs of loci (IL1A-IL1B, IL1A-IL1RN, IL1B-IL1RN) were significant in both groups. When LDs were compared between SPT+ and SPT− groups, a significant difference in IL1A-IL1RN linkage was observed (p<0.001, chi-square test, df=3). The linkage between IL1A and IL1RN was significantly stronger in SPT+ (D’= 0.87) than in SPT− (D’=0.40) groups, indicating that these three loci form haplotypes. When the haplotype frequency analyses were carried out with the EM algorithm, the haplotype distribution differed significantly between SPT− and SPT+ groups (p<0.001, chi-square test, df= 10). SPT positivity was associated with a common haplotype, IL1A*1/IL1B*1/IL1RN*2 (table 2). A rare (2.9%) haplotype IL1A*2/IL1B*1/IL1RN*2 markedly decreased the risk of SPT positivity.
In this study, we showed that the IL-1 gene complex on the whole has an effect on the risk of atopy. This report extends our previous finding9 concerning the association of IL1A with the skin test responses for common allergens.
In the present study the alleles associated with an increased susceptibility for atopy, that is, IL1A*1, IL1B*1, and IL1RN*2 were shown to belong to the same IL-1 gene complex haplotype. With respect to IL-1 family gene analysis, the analysis of haplotypes is essential. Owing to the unique structure of the IL-1 gene complex, each single haplotype will contain a distinct combination of alleles from genes with proinflammatory (IL1A, IL1B) and anti-inflammatory (IL1RN) significance. It is thus probable that each distinct haplotype will mediate inflammatory responses with individual strength and nature, and in the presence of co-dominance the net response of a subject will be dependent on the inherited combination of maternal and paternal haplotypes. However, at present the possibility that the observed effect is the result of an allele of an unknown gene locus located in this haplotype cannot be excluded.
The biological function of the individual IL-1 gene complex alleles studied here has been analysed extensively. In vitro findings have shown that allele 2 in IL1A (SNP −889 which is >99% disequilibrium with SNP +4845) increases the production of IL-1beta in healthy subjects,16 whereas IL1RN*2 has been associated with both high17 and low18 IL-1beta release depending on the study. In addition, IL1RN*2 is associated with high levels of IL1Ra in plasma17 but low levels produced by epithelial cells.19 In these studies, however, the genetic linkage of IL-1 gene family members has not been taken into account. The biological significance of IL1A (SNP +4845), IL1B (SNP +3954), and IL1RN VNTR haplotype/genotype combination cannot be deduced from the published data.
It should be noted that although the odds ratio associated with the presence of the IL1A*1, IL1B*1, and IL1RN*2 haplotype is low (1.80), it may be a substantial risk factor because of the high prevalence of this haplotype (22.9%). The risk of atopy in genotype combination was also shown to be greater than that of haplotypes. This is probably because of the dose effect. A protective haplotype was found in a small subgroup of subjects (2.9%). The importance of this rare haplotype, however, is doubtful owing to its low frequency in the general population.
According to current dogma, atopy is caused by an excessive Th2 response and its increase in developed countries has been attributed to the lack of Th1 inducing microbial contacts (the hygiene hypothesis). Yazdanbakhsh et al20 have recently presented a model where the pro- versus anti-inflammatory axis is decisive in the formation of regulatory T cells which keep the inflammatory T cells (both Th1 and Th2) under control. This model explains some of the inconsistencies of the simple Th1/Th2 balance model. IL-1 is known to be one of the key inflammatory mediators in primary response to microbes. The genetic variability within the IL-1 gene complex could therefore affect this agonist/antagonist balance directly or via stimulation of other inflammatory cytokines.21
This work was supported by the Medical Research Fund of Tampere University Hospital, Rehabilitation Funds of the Finnish Social Insurance Institution, and the Tampere Tuberculosis Foundation The authors would like to thank Dr Arpo Aromaa, National Public Health Institute, Helsinki, Finland and Dr Timo Klaukka, Social Insurance Institution, Helsinki, Finland for taking part in designing and assembling the study.