Association between polymorphisms in caspase recruitment domain containing protein 15 and allergy in two German populations

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1 Association between polymorphisms in caspase recruitment domain containing protein 15 and allergy in two German populations Michael Kabesch, MD, a Wilfried Peters, PhD, a David Carr, BSc, a Wolfgang Leupold, MD, b Stephan K. Weiland, MD, c and Erika von Mutius, MD a Munich, Dresden, and Ulm, Germany Background: Early exposure to microbial matter such as LPS may influence the development of asthma and allergies by activation of innate immunity pathways as indicated by studies in farming environments. Recently, polymorphisms in caspase recruitment domain containing protein 15 (CARD15), an intracellular LPS receptor protein, have been associated with Crohn s disease. Because these polymorphisms lead to changes in LPS recognition, they may affect the development of asthma and allergies. Objective: We genotyped a large population of German schoolchildren (N = 1872) from East and West Germany for 3 functional relevant CARD15 polymorphisms for their role in the development of asthma and allergy. Methods: By use of parental questionnaires, skin prick testing, pulmonary function tests, bronchial challenge tests, and measurements of serum IgE levels, children were phenotyped for the presence of atopic diseases. Genotyping was performed with PCR-based restriction enzyme assays. To assess associations between atopic phenotypes and genotypes standard statistical procedures were applied. Results: Children with the polymorphic allele C2722 had a more than 3-fold risk to develop allergic rhinitis (P <.001) and an almost 2-fold risk for atopic dermatitis (P <.05). Furthermore, the T2104 allele was associated with an almost 2-fold risk for allergic rhinitis (P <.05). When a C insertion at position 3020 was present, the risk of atopy increased by 50% (P <.05) and serum IgE levels were elevated (P <.01). Conclusion: The shared genetic background between Crohn s disease and atopy may indicate that an impaired recognition of microbial exposures results in an insufficient downregulation of excessive immune responses, giving rise to either T H 2 dominated allergies or T H 1 related Crohn s disease. (J Allergy Clin Immunol 2003;111:813-7.) From a University Children s Hospital, Munich, b University Children s Hospital, Dresden, and c Department of Epidemiology, University of Ulm. Supported by German Ministry of Education and Research (BMBF)/National Genome Research Network (NGFN): Research grant BMBF 01GS 0122 (NGFN). Received for publication September 4, 2002; revised December 8, 2002; accepted for publication December 17, Reprint requests: Michael Kabesch, MD, Children s University Hospital, Ludwig Maximilians University Munich, Lindwurmstrasse 4, D München, Germany Mosby, Inc. All rights reserved /2003 $ doi: /mai Key words: Asthma, atopic dermatitis, allergic rhinitis, atopy, childhood, IgE, CARD15, NOD2, polymorphism, SNP The struggle of the human organism with microbial matter is a thriving force of genetic evolution. Human genes involved in the innate immune response against microbial matter play a key role in the interaction with the environment. Thus, polymorphisms in these genes may result in profound functional changes and lead to the development of various diseases. Previous studies have indicated that single nucleotide polymorphisms (SNPs) in innate immunity genes such as the CD14 receptor 1,2 and Toll-like receptors (TLRs) 3 may be associated with the development and severity of atopic diseases and airway reactivity. CD14 and TLRs are involved in the extracellular recognition of bacterial matter such as LPS, an essential component of bacteria that represents a strong stimulus for the activation of T H 1 associated host defense mechanisms. According to the hygiene hypothesis, early infections or the exposure against microbial matter early in life are protective against the development of T H 2 driven atopic diseases. 4 Thus, variations in genes involved in the recognition of microbial matter may alter the balance between T H 1 and T H 2 driven immune responses and may therefore change the susceptibility to develop asthma and allergy. A number of genes other than CD14 and TLRs are associated with the recognition of microbial matter and LPS. One of these innate immunity genes recently discovered is the caspase recruitment domain containing protein 15 (CARD15) gene (previously known as nucleotide-binding oligomerization domain [NOD] 2), a member of the NOD1/apoptotic protease activating factor 1 (APAF1) gene family. Stimulation with LPS triggers CARD15 molecules that are primarily expressed in monocytes and act as an intracellular LPS receptor to activate nuclear factor kappa B (NF-κB), thereby initiating apoptosis and other immunoregulatory effects. How LPS gets internalized and interacts with CARD15 is not yet known. The CARD15 gene is located in the pericentromeric region of chromosome 16 and consists of 11 exons. In total, 13 polymorphisms have been identified within the gene. CARD15 contains 2 caspase recruitment domains (CARD 1 and 2), a nucleotide-binding domain (NBD), and ten 27-amino- 813

2 814 Kabesch et al J ALLERGY CLIN IMMUNOL APRIL 2003 Abbreviations used APAF1: Apoptotic protease activating factor 1 CARD: Caspase recruitment domain CARD15: Caspase recruitment domain containing protein 15 ISAAC: International Study of Childhood Asthma and Allergy LRR: Leucine-rich repeat NBD: Nucleotide-binding domain NF-κB: Nuclear factor kappa B NOD: Nucleotide-binding oligomerization domain OR: Odds ratio SNP: Single nucleotide polymorphism TLR: Toll-like receptor acid, leucine-rich repeats (LRRs) that represent the receptor domains for components of microbial pathogens. Recent reports have shown that 3 single nucleotide polymorphisms (SNPs) in the CARD15 gene that are not in linkage disequilibrium may be associated with the susceptibility to develop Crohn s disease, 5-7 a mostly T H 1 driven inflammatory bowel disease. Because there is evidence that early exposure to microbial matter 4 and the activation of monocyte and NF-κB dependent pathways 8 also play a role in the development of asthma and allergy, CARD15 polymorphisms may influence the genetic susceptibility to develop not only Crohn s disease but also atopic diseases. To test this hypothesis we genotyped 2 cross-sectional populations from West and East Germany (total N = 1872) for the 3 polymorphisms in the CARD15 gene previously reported to be related to Crohn s disease (3020insC, C2104T, and G2722C), and the association between these polymorphisms and asthma and allergy related traits was studied. METHODS Subjects Between 1995 and 1996, a cross-sectional study was conducted in Munich, West Germany, and Dresden, East Germany, to assess the prevalence of asthma and allergies in schoolchildren aged 9 to 11 years. 9 Parental questionnaires for self-completion were sent through the schools to the families. Children underwent skin prick testing, pulmonary function testing, and bronchial challenge with hyperosmolar saline (4.5%) at their schools. Blood was collected for serum IgE measurements and DNA extraction. Total serum IgE was measured in a 50% random sample of all children who had given blood in Dresden and all children with blood available in Munich. In this study only children of German origin who had both DNA and IgE data available were included in the analysis (total N = 1872, Munich n = 1161, Dresden n = 711). The study methods for phenotyping have been described in detail elsewhere. 9 Therefore, only methods pertaining to this analysis are given below. Informed written consent was obtained from all parents, and all study methods were approved by the ethics committee of the University of Münster (Coordination and Data Centre). Questionnaire Self-administered questionnaires included the International Study of Childhood Asthma and Allergy (ISAAC) core questions on symptoms of asthma, allergic rhinitis, and atopic eczema. 9 Children whose parents reported a doctor s diagnosis of asthma, recurrent spastic or recurrent asthmatic bronchitis were classified as having asthma. Current wheeze was defined as wheezing in the last 12 months. Nocturnal asthma was defined as waking up because of wheezing or coughing at least once a week. The definition of allergic rhinoconjunctivitis or atopic eczema was based on a parental report of a doctor s diagnosis of hay fever and atopic eczema, respectively. Skin prick test In Munich and Dresden, the sensitivity to 6 common aeroallergens (Dermatophagoides pteronyssinus, Dermatophagoides farinae, Alternaria tenuis, cat dander, and mixed grass and tree pollen) was assessed by using highly standardized extracts and lancets (ALK, Horsholm, Denmark) according to the ISAAC II protocol. A child was considered sensitized if a wheal reaction of more than 3 mm occurred to at least 1 specific allergen after subtraction of the negative control. Performance and reproducibility of this method were proved to be adequate and evaluation results were reported elsewhere. 10 Total serum IgE measurements Total serum IgE levels were measured by using the Insulite system (DPC Biermann, Bad Nauheim, Germany) and are summarized descriptively with geometric means and respective 95% CIs. Genotyping PCR reactions were carried out in a volume of 15 µl containing 20 ng of genomic DNA by using standard thermocyclers (Eppendorf, Cologne, Germany). Primers and restriction enzymes ( were GCT CTT GGC CTC ACC CGG, CAC CAG CTT TGC TCAG ACA CC and MspI for C2104T, ATA AAG TTC AAA GAC CTT CAG AAC TGG CGC, ATA AAG TTC AAA GAC CTT CAG AAC TGG CGC and CfoI for G2722C, and CCT CTC TCC CGT CAC CCC, GTG AAT GGA AGA GAG ACG GTT AC and NlaIV for 3020iC. Enzyme-digested PCR products were separated by size on agarose gels and subsequently visualized with ethidium bromide staining and ultraviolet illumination. For quality control a subsample of 10% of all samples was genotyped in duplicate to assess the consistency of applied genotyping methods, and Hardy Weinberg equations were calculated for all genotypes. The reasons for the selection of the 3 polymorphisms 3020insC, C2104T, and G2722C for genotyping in our study population were the following: these polymorphisms (1) were associated with Crohn s disease, (2) were not in linkage disequilibrium with each other, (3) had an allele frequency reasonable for analysis in our sample size, and (4) had been associated with functional alterations in the ability to react with LPS. Data analysis Standard ANOVA, chi-square tests, and logistic regression were used to compare means and proportions between groups. All calculations were done with the SAS software package 8.2 (SAS Institute, Inc, Cary, NC). First, multivariate logistic regression models with the 3 polymorphisms under review as independent factors enabled the estimation of the individual odds ratios (ORs) for the presence or otherwise of the separate polymorphisms for each of the outcome variables. Second, a multivariate logistic regression model with dummy variables for the number of the 3 CARD15 polymorphisms present enabled the assessment of the associated risks for varying numbers of the polymorphisms under consideration. To assess linkage disequilibrium, only homozygous subjects for the referenced polymorphisms were used in which haplotypes could be determined unambiguously. Levin s δ was used to calculate linkage disequilibrium between the reference polymorphism (3020iC) and all other polymorphisms because this measure is assumed to be most appropriate for case-control studies. 11

3 J ALLERGY CLIN IMMUNOL VOLUME 111, NUMBER 4 Kabesch et al 815 RESULTS All available blood samples in Munich (N = 1161) and a random sample of 50% of all available blood samples in Dresden (N = 711) were genotyped for the 3 SNPs in the CARD15 gene previously reported to be associated with Crohn s disease. 5-7 No selection bias could be detected between children genotyped for this study and those who were not (Tables I and II). Furthermore, no significant difference was present between children genotyped for CARD15 polymorphisms in Munich and Dresden (Tables I and II). Allele frequencies (for the less frequent allele) were for the C insertion at position 3020, for C2104T, and for G2722C. These frequencies were similar to the previously reported frequencies in white populations. Allele frequencies did not differ significantly between the Eastern and Western study populations. Furthermore, no significant linkage disequilibrium was observed between the 3 SNPs genotyped in the CARD15 gene. When the study populations from Dresden and Munich were analyzed together, children with the C allele at position 2722 had a more than 3-fold risk to develop allergic rhinitis and an almost 2-fold risk to develop atopic dermatitis (Table III). Furthermore, the T2104 allele was also associated with an almost 2-fold risk for allergic rhinitis. The risk to develop atopy increased by 50% when a C insertion at position 3020 was present. When the populations from Dresden and Munich were analyzed separately, trends were similar in both populations for all the observed associations. However, because allele frequencies are low, some of the associations only reached borderline significance in some of the separate analyses. In the Munich population, polymorphism 3020iC showed the strongest effect with atopy, which was defined by a positive skin prick test (37.2% vs 24.6%; OR 1.84; 95% CI, 1.14 to 2.97; P <.05). Polymorphism C2722 was also associated with atopic dermatitis (29.4% vs 19.1%; OR 1.78; 95% CI, 0.84 to 3.79) and atopic rhinitis (17.6% vs 7.7%; OR 2.44; 95% CI, 0.98 to 6.07; P <.10) in the Munich sample. In Dresden, the effect of polymorphism C2722 on the presence of atopic dermatitis (29.7% vs 17.6%; OR 2.00; 95% CI, 0.96 to 4.18; P <.010) and atopic rhinitis (25.0% vs 7.6%; OR 4.05; 95% CI, 1.79 to 9.17; P <.001) was even stronger than in Munich, whereas polymorphism T2104 was also associated with atopic rhinitis to a lesser degree (16.9% vs 7.6%; OR 2.43; 95% CI, 1.24 to 4.78; P <.05). Geometric means for total serum IgE levels were significantly elevated in atopic children with a C insertion at position 3020 (312.1 IU/mL [95% CI, to 448.6] vs IU/mL [95% CI, to 210.3]; P =.009), indicating that the severity of the atopy status may be enhanced by this polymorphism. However, no effect by any CARD15 polymorphism was observed on the increase of serum IgE levels in nonatopic children. Furthermore, the presence of any 2 of the 3 SNPs was strongly related to atopy (OR, 3.06; 95% CI, 1.20 to 7.78; P <.05) and allergic rhinitis (OR, 4.64; 95% CI, 1.62 to 13.26; P <.01). When the number of positive skin FIG 1. Depiction of the putative domain structure of the CARD15 gene corresponding to the amino acid residues. The protein NOD2 contains 2 caspase recruitment domains (CARD 1 & 2), an NBD, and ten 27-amino-acid LRRs. CARD15 variants alter the structure of the NBD, the LRR domain of the protein, or the adjacent region. prick test results was analyzed, children with polymorphisms in the CARD15 gene had a higher number of positive skin prick test results. Geometric means for the number of positive skin prick test results were 0.47 (95% CI, 0.46 to 0.48) in children without a CARD15 polymorphism compared to 0.53 (95% CI, 0.48 to 0.58) when the C insertion at position 3020 was present (P =.001), 0.54 (95% CI, 0.47 to 0.62) when the polymorphism C2722 was present (P =.001), and 0.49 (95% CI, 0.46 to 0.53) when polymorphism T2104 was identified. No significant association between asthma and any single polymorphism in the CARD15 gene was observed, not in the 2 populations separately or in the combined population. DISCUSSION In this study we showed that 3 polymorphisms in the CARD15 gene previously related to the development of Crohn s disease are also associated with atopic diseases. This observation was consistent in 2 population samples from East and West Germany. The strongest association was observed with allergic rhinitis, in which 2 CARD15 polymorphisms independently increased the risk to develop the disease by 2- to 3-fold. From these results we conclude that even though the frequency of CARD15 polymorphisms in the general population is low, CARD15 SNPs are significantly associated with the susceptibility to develop different forms of atopic diseases during childhood. Furthermore, CARD15 polymorphisms may

4 816 Kabesch et al J ALLERGY CLIN IMMUNOL APRIL 2003 TABLE I. Comparison between children included and not included in the Munich study population Total study population* Questionnaire data, no Questionnaire data, Genotyped Munich (N = 2612) blood available (n = 1311) blood available (n = 1300) (n = 1161) Male gender 49.3% 49.9% 48.8% 48.9% Mean age (y) Asthma 10.3 ( ) 9.6 ( ) 10.9 ( ) 11.3 ( ) Atopic dermatitis 17.5 ( ) 16.3 ( ) 18.8 ( ) 19.0 ( ) Atopic rhinitis 9.3 ( ) 10.0 ( ) 8.6 ( ) 8.6 ( ) Skin prick test reactivity 23.5 ( ) 21.7 ( ) 24.2 ( ) 24.8 ( ) 95% CI indicated for prevalence rates. *Children of German origin in whom phenotyping data were available. More details on phenotypes are given in reference 8. Children with complete phenotyping data and DNA available. TABLE II. Comparison between children included and not included in the Dresden study population Total study population* Questionnaire data, no Questionnaire data, Genotyped Dresden (N = 3016) blood available (n = 971) blood available (n = 2045) (n = 711) Male sex 51.9% 53.5% 51.2% 51.6% Mean age (y) Asthma 7.9 ( ) 8.3 ( ) 7.7 ( ) 9.0 ( ) Atopic dermatitis 16.6 ( ) 15.4 ( ) 17.2 ( ) 18.0 ( ) Atopic rhinitis 9.8 ( ) 10.1 ( ) 9.6 ( ) 9.2 ( ) Skin prick test reactivity 25.7 ( ) 23.0 ( ) 26.1 ( ) 26.1 ( ) 95% confidence interval indicated for prevalence rates. *Children of German origin in whom phenotyping data were available. More details on phenotypes are given in reference 8. 50% random sample of all children with complete phenotyping data and DNA available. TABLE III. Association between CARD15 polymorphisms and atopic diseases Atopy Atopic dermatitis Atopic rhinitis Asthma Polymorphism (n = 456/1789) (n = 330/1747) (n = 154/1765) (n = 185/1794) C2104T (Arg675Trp) 26.9% vs 24.6% 16.2% vs 18.5% 12.9% vs 7.7% 11.9% vs 10.1% (n = 202/1805) NS NS 1.73 ( )* NS G2722C (Gly881Arg) 31.5% vs 24.6% 29.6% vs 18.5% 21.4% vs 7.7% 8.3 vs 10.1% (n = 73/1805) NS 1.85 ( )* 3.16 ( ) NS 3020iC 32.8% vs 24.6% 23.0% vs 18.5% 9.4% vs 7.7% 13.1% vs 10.1% (n = 132/1805) 1.49 ( )* NS NS NS Prevalence rates and ORs (95% CIs) for atopic phenotypes by polymorphisms in the CARD15 gene assessed in 1873 German schoolchildren. Prevalence rates were compared between children with the polymorphism (first prevalence rate shown) and those without any CARD15 polymorphism. NS, Not significant. *P <.05. P <.001. increase the severity of atopic diseases; this is indicated by their association with elevated serum IgE levels in atopic patients and the increase in the number of positive skin prick test results. 2 No significant association between asthma and any single polymorphism in the CARD15 gene was observed. However, because the prevalence of asthma in this cross-sectional population sample was approximately 10.5% and the allele frequency of CARD15 is low, the number of asthmatic subjects investigated in this study may not have been large enough to rule out any role of CARD15 polymorphisms in the development of childhood asthma. Furthermore, the effect of multiple polymorphisms present in one individual on the risk to develop atopic diseases could not be studied because of the low gene frequency of the polymorphisms. CARD15 represents an intracellular receptor for LPS requiring the presence of a functional LRR domain for its function. This domain is similar to the LPS responsive LLR domain of the TLR4, which is also involved in LPS signaling. Thus, it seems that LRR domains represent a basic receptor structure redundantly used by LPS interaction proteins. Although 2 of the polymorphisms described here (G2722C and 3020iC) are located within the LRR domain, the third polymorphism is located between the LRR domain and the NBD of the CARD15 molecule (Fig 1). For the 3020iC polymorphism the functional implications have been demonstrated. Polymorphism 3020iC leads to a frameshift mutation at amino acid position 980/981 of the protein and the truncation of the LRR domain. Notably, the absence of the last of 10 tandem LRRs severely impedes the capability of the CARD15 protein to interact with LPS and to activate the NF-κB pathway in response to bacterial stimuli. 7 Polymorphisms G2722C and C2104T both result in putative amino acid variations. Polymorphism G2722C leads to a change from arginine at position 675 to tryptophan; polymorphism C2104T results in an amino acid change from glycine to arginine at position 881. It has been shown recently that both these polymorphisms alter the CARD15 protein in a way that the originating variants are significantly less active than the wild-type protein in conferring responsiveness to bacterial LPS (Gabriel Nunez, personal communication, June 2002).

5 J ALLERGY CLIN IMMUNOL VOLUME 111, NUMBER 4 Kabesch et al 817 CARD15 activates the NF-κB pathway by interacting with serine-threonine kinase RICK via a homophilic CARD-CARD interaction. 12 Interestingly, the expression of a CARD15 mutant completely lacking the LRR domain resulted in enhanced NF-κB activation, whereas mutants containing the LRR or NBD domains alone did not lead to NF-κB activation. 7 Thus, it can be concluded that LRR domains may represent response elements for LPS but are not per se involved in the NF-κB activation. The response to LPS as mediated by NOD1 and CARD15 appears to be independent from TLR4 because it is unaffected by dominant interfering mutants of MyD88 and TRAF6, two essential molecules in the TLR4 signaling pathway. 13 NF-κB activation induced by LPS was inhibited by dominant forms of NOD1 but not by TRAF. Cell culture experiments showed that even when the LRR domain is truncated by polymorphism 3020iC, the basal capability to activate the NF-κB pathway by unspecific stimuli is not significantly affected. 7 However, NF-κB activation by LPS mediated through CARD15 is almost absent in the presence of polymorphism 3020iC and significantly reduced in the case of polymorphisms G2722C or C2104T. Therefore, naturally occurring polymorphisms in the LPS-responsive element of the CARD15 gene seem to specifically influence the capability of the organism to react toward bacterial stimuli with NF-κB activation. In Crohn s disease this may lead to an impaired defense against microbial matter in the bowel and may thus allow bacteria to progress beyond the level of the CARD15 defense system. As a consequence, this may cause greater local damage and a more severe immunologic response at a more profound level of the intestinal immunodefense. On the other hand, it may be speculated that the impairment of LPS recognition facilitated by CARD15 polymorphisms reduces the general capability of the innate immunity to interact with bacterial matter early in life and to develop a robust regulatory T-cell reservoir. 14 Repeated early stimulation of the regulatory T-cell system seems necessary to induce a negative immunoregulatory feedback loop suppressing unwanted and exaggerated responses against pathogens. If such early stimulation is absent or this interaction is impaired by genetic predisposition (eg, CARD15 polymorphisms), autoimmune as well as atopic diseases may emerge. 14 Therefore, the protective effect against the development of atopic diseases that is conferred by early contact with microbial matter may be diminished. 4 The hypothesis seems tempting that CARD15 polymorphisms influence the development of atopic diseases by changes in the capability to mount an adequate NF-κB response after bacterial challenges (as may be suggested by our results). However, it was also shown that CARD15 enhances apoptosis via the augmentation of the apoptosis induction by caspase-9 expression. 15 In this context it is important that mechanisms involved in the regulation of the survival and apoptosis of inflammatory cells may play an important role in the persistent inflammatory process characterizing asthma and atopy. At this point it is not known whether the 3 polymorphisms that we investigated in CARD15 and that lead to alterations in the LPS responsiveness also influence the capability of CARD15 to induce apoptosis and how this could relate to the development of atopic diseases and Crohn s disease at the same time. Furthermore, it cannot be ruled out that CARD15 has further functional properties that have not been investigated so far that are also affected by gene polymorphisms. On the population level, the prevalence of both atopic and autoimmune diseases has increased concomitantly during the last decades in Western societies. 16 This concomitant rise in prevalence may challenge the paradigm of a dichotomy opposing allergic illnesses as the prototype of a T H 2-like immune response to autoimmune disorders such as Crohn s disease as the antagonistic T H 1 driven disease. Instead, an additional regulatory element suppressing excessive immune responses to self-antigens or allergens may be impaired. 14 CARD15 polymorphisms may play a role in this. The shared genetic background between atopy and Crohn s disease supports this notion, suggesting that it is a malfunctioned recognition of microbial exposures that contributes to an excessive response of both types of immune responses. REFERENCES 1. Baldini M, Lohman IC, Halonen M, Erickson RP, Holt PG, Martinez FD. A polymorphism* in the 5 flanking region of the CD14 gene is associated with circulating soluble CD14 levels and with total serum immunoglobulin E. Am J Respir Cell Mol Biol 1999;20: Koppelman GH, Reijmerink NE, Colin Stine O, Howard TD, Whittaker PA, Meyers DA, et al. Association of a promoter polymorphism of the CD14 gene and atopy. Am J Respir Crit Care Med 2001;163: Arbour NC, Lorenz E, Schutte BC, Zabner J, Kline JN, Jones M, et al. 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