Introduction

Atopic asthma, allergic rhinitis and atopic eczema/dermatitis syndrome (AEDS) are typical symptoms of atopy, which could be defined as a tendency to produce IgE antibodies in response to low doses of allergens (Johansson et al. 2001). Pathogenesis of these allergic diseases is considered to be multifactorial, involving complex interactions between genetic and environmental influences (Holgate at al. 1999). Using two main approaches, genome-wide screens and candidate genes studies, many authors have attempted to elucidate genetic factors involved in the pathogenesis of bronchial asthma and other atopic diseases. Among a variety of other loci, the gene for IKAP (IKK complex-associated protein) on chromosome 9q31 could be considered as a candidate gene.

The first reason is the possible participation of IKAP in the NF-κB activating cascade. NF-κB comprises a family of inducible transcription factors that play an important role in the regulation of inflammatory and immune responses, cellular proliferation and apoptosis. IKAP was suggested to be a scaffold protein and regulator for three different kinases (IKK-α, IKK-β and NIK) (Cohen et al. 1998). Activation of these kinases leads to the phosphorylation of inhibitory proteins known as IκB, resulting in their degradation. Thus, disinhibited NF-κB proteins can translocate from cytoplasm to nucleus, where they activate transcription of specific cellular genes. This way, in response to over 150 different stimuli, active NF-κB factors can control expression of inflammatory cytokines, chemokines, immunoreceptors, cell adhesion molecules, and enzymes whose products contribute to the pathogenesis of the inflammatory process (Pahl 1999).

Secondly, the IKAP gene lies on chromosome 9q, where, using a genome-wide linkage study approach, Wjst et al. (1999) identified one of four regions that could be linked to bronchial asthma and total and specific immunoglobulin E serum levels (P<1×10-2 for all three cases) in German and Swedish population.

Takeoka et al. (2001) identified six SNPs within the coding region of the IKAP gene in the Japanese population: T819C, G2295A, A2446C, A2490G, T3214A and C3473T. These authors described a strong allelic association between bronchial asthma in childhood and two of the SNP sites, T3214A (Cys1072Ser) and C3473T (Pro1158Leu); P=0.000004 for T3214A and P=0.0009 for C3473T. T3214A, unlike C3473T, was also associated with bronchial asthma in adult patients (P=0.000002). Moreover, a strong association was also found between bronchial asthma in childhood and a specific haplotype, TGAAAT, P=0.00004. As bronchial asthma in most childhood patients had been diagnosed as atopic, these results suggest an etiological role of IKAP especially in atopic bronchial asthma.

The aim of our study was to test the possible role of five selected SNPs in the coding region of the IKAP gene, T819C (Leu273Leu), G2295A (Gly765Gly), A2490G (Ile830Met), T3214A (Cys1072Ser) and C3473T (Pro1158Leu), in atopic asthma or other atopic phenotypes in the highly homogenous Czech population, and to compare the results with those received previously in Japanese population.

Subjects and methods

Subjects

Caucasian subjects of exclusively Czech nationality (n=682) were included in this case-control study. All subjects were selected using a detailed questionnaire modified from the American Thoracic Society respiratory questionnaire (American Thoracic Society 1988) regarding lifetime symptoms suggestive of asthma, rhinitis and dermatitis, with additional questions on symptoms and therapy, as well as on other diseases. Phenotype status was assigned without previous knowledge of genotypes by two investigators independently.

Unrelated, random-sampled controls included 309 subjects (158 men and 151 women), aged 35.3±15.6 years (mean±SD) without symptoms of any atopic, lung and skin diseases and with no familial history of asthma/atopy as well. They were recruited from general practitioners' surgeries. A total of 373 patients with clinically manifested atopic asthma, allergic rhinitis, atopic dermatitis or their combinations, 208 men and 165 women, aged 25.5±12.6 years were studied. Out of 373 patients, 27 individuals had only atopic asthma, 77 had both asthma and rhinitis, 16 had asthma and dermatitis, 149 had only rhinitis, 29 had both rhinitis and dermatitis, 11 had only dermatitis and 65 had all three diseases. A subgroup of 184 atopic patients, 112 men and 72 women, aged 25.1±12.6 years, with diagnosis of bronchial asthma alone or its combination with other atopic diseases was made in order to compare our results with previous findings. The patients were recruited from the Department of Clinical Immunology and Allergology, St. Anne's Teaching Hospital, Brno, from the Faculty Children Hospital, Brno, and from allergologists' surgeries. The patients were interviewed and the diagnoses of atopic diseases were defined as described previously (Buckova et al. 2002).

All the subjects, or their parents in case of children, gave written informed consent to participation in the study. The study was approved by the Committee for the Ethics of Medical Experiment on Human Subjects, Medical Faculty, Masaryk University, Brno.

Genotype identification

The genomic DNA was isolated from peripheral blood leukocytes by a standard method, using proteinase K digestion of cells, according to Sambrook et al. (1989). Five SNPs in the coding region of the IKAP gene were studied. DNA fragments containing C819T, A2490G and C3473T sites were amplified using PCR conditions and primers described by Takeoka et al. (2001). To amplify DNA fragments containing T3214A and G2295A sites, we used PCR with primers of 5′-aggaattgagtttacctggggac-3′ and 5′-agtcaactgctgcttattgtctc-3′ for T3214A and 5′-ctgtttaatgaaggtttccagatt-3′ and 5′-ccctaaggtaactttctaagctg-3′ for G2295A. Reactions were carried out in a final volume of 25 μl containing DNA, 2.5 mM MgCl2, 0,75 U Taq polymerase (Fermentas), 0.4 μM of each primer, 200 μM dNTPs mixed (Fermentas) and 1×PCR buffer (Fermentas). Amplification conditions were 95 °C for 2 min, followed by 30 cycles of 94 °C for 20 s, 60 °C for 20 s and 72 °C for 20 s with a final extension for 5 min at 72 °C. PCR products were digested with the HinfI restriction enzyme at 37 °C for 4 h, except C3473T, where KpnI at 37 °C for 4 h was used. The digested fragments were separated by electrophoresis in 2% agarose gel and visualised with ethidium bromide. Restriction fragment sizes are shown in Table 1.

Table 1. Restriction analysis of the IKAP gene variants
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Statistics

Allelic frequencies were calculated from the observed numbers of genotypes. Fisher's exact test and the χ2test were used to determine the significance of differences in frequencies of alleles and genotypes between two groups. Additionally, χ2 analysis was used to test for deviations of genotype distributions from Hardy-Weinberg equilibrium. Pairwise linkage disequilibrium coefficients were calculated and expressed as the D′=D/Dmax, according to Thompson et al. (1988). The statistical analysis was performed using the programme package Statistica v3.0 (StatSoft, Tulsa, USA). Detection power calculation was performed using EpiInfo 6 software (WHO, Geneva, Switzerland/Centers for Disease Control, Atlanta, Ga.).

Results

We genotyped 373 patients and 309 healthy control individuals for five of six SNPs in the coding region of the IKAP gene described by Takeoka et al. (2001). In order to compare our results with those presented by Takeoka and co-workers, data of a subgroup of 184 atopic patients with diagnosis of bronchial asthma alone or its combination with other atopic phenotypes are also shown. We found no polymorphism in position 2295, where only allele A was detected in both patient and control groups. The genotype distributions for the other four polymorphisms (Table 2) were consistent with the Hardy-Weinberg equilibrium in both groups and also in the subgroup of asthmatic patients. We found no significant differences in the genotype and allele distributions for any SNPs in the IKAP gene in atopic patients or the subgroup of patients with atopic asthma compared with healthy controls. All the polymorphisms were in a complete or quasi-complete pair-wise linkage disequilibrium with each other (Table 3). When more detailed analysis was carried out, no association was found between variants of the IKAP gene and the total or specific IgE levels or the skin prick test reactivity (data not shown).

Table 2. Genotype distribution of the IKAP gene variants in patients with atopic diseases, the subgroup of patients with atopic asthma and controls. No significant differences were observed between the tested groups
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Table 3. Pairwise linkage disequilibrium coefficients (D′) between four SNPs in the IKAP gene in patients with atopic diseases, the subgroup of patients with atopic asthma and controls. D′=D/D max, according to Thompson et al. (1988)
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Discussion

IKK-complex-associated protein (IKAP) was originally referred to as a scaffold protein and a regulator for three different kinases involved in pro-inflammatory cytokine signalling when purified as a 150-kDa protein component of the interleukin-1-inducible IKK complexes that contain NIK, IKK-α, IKK-β, IκB-α, NF-κB/RelA (Cohen et al. 1998). In a later report, this specific role of IKAP in NF-κB-mediated signalling was not confirmed by Krappmann et al. (2000), who instead suggested its possible action on the level of general transcription. Indeed, IKAP was later identified as a subunit of the human elongator complex as a part of its three-subunit core form. Its role in transcription elongation may be to support the elongator complex assembly (Hawkes et al. 2002). However, recently a novel role for IKAP in the regulation of activation of the mammalian stress response via the c-Jun N-terminal kinase (JNK)-signalling pathway has been demonstrated (Holmberg et al. 2002). Although all the mechanisms mentioned above could be involved in the pathophysiology of bronchial asthma and other phenotypes typical for atopy, the role of the IKAP protein in these diseases is still unclear and it appears to be complex.

Besides bronchial asthma, which is a common multifactorial disease with polygenic inheritance pattern, variants of the IKAP gene on the chromosome 9q31 were also studied in the autosomal recessive disease called Familial Dysautonomia. Slaugenhaupt et al. (2001) and Anderson et al. (2001), independently, showed two mutations in the IKAP gene to be responsible for this sensory neuropathy characterised by widespread sensory and variable autonomic dysfunction.

In the only paper regarding this gene and bronchial asthma or related traits, so far published, Takeoka et al. (2001) showed a strong allelic association between two of the SNP sites in the IKAP gene, T3214A (Cys1072Ser) and C3473T (Pro1158Leu) and bronchial asthma in a Japanese population. Regarding the 3214A allele, this was expressed in both adults (P=0.000002) and children (P=0.000004), whereas for the 3473T allele the result was significant only in children (P=0.0009). The other four polymorphisms (C819T, G2295A, A2446C and A2490G) had no association with asthma in this study.

We performed an association study of the polymorphisms in the IKAP gene and atopic diseases in the Czech (Caucasian) population. Consistently with the Japanese study, we found no significant association between T819C, G2295A and A2490G SNPs in the IKAP gene and atopic asthma and/or other atopic phenotypes. On the other hand, we failed to replicate an association between 3214A and 3473T and atopic asthma. In the Japanese study, when we consider asthma in children, which was diagnosed as atopic asthma in almost all cases, odds ratios calculated for the 3214A and 3473T alleles were 1.72 (95% CI, 1.23–2.23) and 1.51 (95% CI, 1.19–1.93), respectively. Although these alleles were less frequent in the Czech population and the sample size of our asthma case-control study was smaller than that of Takeoka et al. (2001), the power to detect the association in our study was 0.92 for the T3214A polymorphism, assuming the odds ratio to be 1.72, and for the C3473T polymorphism, the power was 0.72, assuming the odds ratio to be 1.51, the alpha level being set to 0.05 in both cases. Concerning any of the five studied polymorphisms, allele frequencies in our population differed from those reported in the Japanese population. Although the C allele in the position 819 prevailed in the Japanese individuals, it was rare in the Czech ones. In the position 2295, only the allele A was observed in the Czech population, which is consistent with the nucleotide mRNA sequences available on NCBI database (AF 044195, AF 153419, NM 003640, BC 033094), but it is different from Japanese finding, where the G allele appeared more frequently than A. However, these nucleotide differences don't lead to amino acid differences, as both these substitutions are silent. In the other three studied SNPs, which lead to alterations in the protein product (A2490G-Ile830Met, T3214A-Cys1072Ser and C3473T-Pro1158Leu), the same alleles (2490A, 3214T and 3473C) prevailed in both Czech and Japanese populations, but the frequency of these alleles was significantly higher in our study. For example, while the frequency of the 3214T allele was 63% in Japanese children with bronchial asthma, it was 82.1% in our atopic asthma patients, which was even higher than in Japanese controls (74.5%). Additionally, pairwise linkage disequilibrium between any of four polymorphic sites, when measured by standardized coefficient D', was expressed stronger in the Czech population.

In summary, although the results of the Japanese study indicated that specific variants of the IKAP gene might be associated with mechanisms responsible for early-onset bronchial asthma which was mostly diagnosed as atopic, the results of our study suggest that the polymorphisms in the coding region of the IKAP gene are unlikely to contribute to atopic disease risk in the Czech population.

This inconsistency contributes to the phenomenon that results of candidate gene studies vary frequently and associations found in some populations are often not replicated in others. However, this is not completely unexpected for genetic studies in multifactorial diseases where particular gene is studied on the different genetic and environmental "background" in various populations.

Thus, more functional and genetic studies are needed to reveal the role of the IKAP gene and IKAP protein in the pathogenesis of diseases related to atopy.