Volume 118, Issue 1 , Pages 277-279, July 2006
Lack of association between Toll-like receptor 2 and Toll-like receptor 4 polymorphisms and atopic eczema
Article Outline
To the Editor:
The etiology of atopic diseases (asthma, rhinoconjunctivitis, atopic eczema) is complex and multifactorial, involving both genetic and environmental factors. To explain the recent increase of their prevalence in Western societies, based on the hygiene hypothesis it has been speculated that an altered stimulation of the innate immune system might contribute to a dysbalance on the level of TH2 and TH1 immune responses. The innate immune system comprises a variety of pattern recognition receptors expressed on the cell surface, such as the Toll-like receptors (TLRs), or in intracellular compartments, like nucleotide-binding oligomerization domain protein 1 and 2.1 TLRs are known to function as mediators between innate and adaptive immunity via induction of dendritic cell maturation and shifting the adaptive immune response toward TH1.2 They are activated by a wide range of pathogen-associated and endogenous stimuli including agents associated with the development of allergic diseases.3 TLR4 is the principal receptor for bacterial endotoxin, the exposure to which has been suggested to protect from the development of asthma and atopy.4 TLR2 recognizes peptidoglycan, which predominates the cell wall of Staphylococcus aureus,1 a pathogen with important implications for atopic eczema. Recently, it has been shown that peptidoglycan activates nuclear factor-κB and induces abundant IL-8 production from keratinocytes via functionally TLR2.5 In humans, polymorphisms in TLRs have been shown to be associated with a variety of diseases.6 In previous studies, associations of the TLR2 polymorphisms rs4696480 with asthma7 and rs5743708 with severe atopic eczema8 as well as the TLR4 polymorphisms rs4986790 with asthma9 and rs4986791 with a modified response to endotoxin10 have been reported.
To study the candidacy of TLR2 and TLR4 as atopic eczema susceptibility genes, we performed an association study using all haplotype-tagging single nucleotide polymorphisms (htSNPs; information retrieved from the Innate Immunity PGA, NHLBI Program in Genomic Applications: http://innateimmunity.net/data/homology, June 2005) as well as SNPs previously associated with atopic traits (Fig 1) in a large cohort of 275 German white parent-offspring trios who had been recruited in Munich and Bonn, Germany. Children and their parents were administered a standardized questionnaire based on the European Community Respiratory Health Survey on respiratory health. In addition, all probands were interviewed in a standardized manner to report on symptoms of atopic diseases, basic family data, and parental history of atopic diseases. In all subjects, total as well as specific IgE antibodies against common environmental allergens (grass, birch, rye, mugwort pollen, Alternaria, Cladosporium, cat, dog, and Dermatophagoides pteronyssinus) were measured (enzyme-immuno assay, CAP-FEIA; Pharmacia, Uppsala, Sweden). Subjects were classified as having asthma or allergic rhinoconjunctivitis when they reported a physician's diagnosis. Atopic eczema was diagnosed on the basis of a skin examination by experienced senior dermatologists using the UK diagnostic criteria. The clinical severity of atopic eczema was determined according to the SCORAD index. Specific sensitization was defined to be present if at least one of the specific IgE antibodies was positive (CAP class ≥ 1, corresponding to ≥ 0.35 kU/L). Total serum IgE levels were considered dichotomously with the 50th percentile (246 kU/L), 66th percentile (466 kU/L), and 90th percentile (2000 kU/L) as cutoff levels.
Fig 1.
Gene structure of the TLR2 locus (A) and the TLR4 locus (B), according to ensembl v36 database (ENST00000339096/ENST00000260010). Location of genotyped SNPs is indicated by arrows above the line. Round-tailed arrows indicate exonic SNPs.
Assays for SNP genotyping were developed for all TLR2 and TLR4 SNPs, and genotyping was performed by using the MassARRAY system (Sequenom, San Diego, Calif). The Hardy-Weinberg equilibrium for genotype frequency distributions was tested using the χ2 goodness-of-fit test. Pairwise linkage disequilibrium between each pair of SNP loci was also evaluated using the χ2 test.
Evidence of association with the binary traits atopic eczema, moderate to severe atopic eczema (cutoff median SCORAD score, 44.6 points), asthma, allergic rhinoconjunctivitis, total serum IgE, and allergic sensitization was evaluated using the classical transmission/disequilibrium test implemented in Haploview 3.2 (J. Barrett and J. Maller, Whitehead Institute, Cambridge, Mass). Haplotype frequencies were estimated from htSNP genotype data using the expectation maximization (EM) algorithm implemented in Haploview, and haplotypes were evaluated for transmission distortion. Power analysis showed that our sample of 275 families has a power of >80% to detect a deviation of 20% in the allel transmission rate to offspring with atopic eczema on a Bonferroni-corrected significance level.
Parental genotype frequencies were found to be consistent with Hardy-Weinberg equilibrium. There were no significant deviations from expected transmission rates in our family-based study for any of the SNPs under consideration (Table I). The SNPs within TLR2 and TLR4 were in strong linkage disequilibrium with each other. The 4 TLR2 SNPs and 8 TLR4 SNPs defined 5 haplotypes with frequencies >5% for each gene, which accounted for 96.4% and 94.5% of all observed haplotypes. None of the individual SNPs showed associations with atopic eczema or severity of atopic eczema in our population of German white families (Table II), and there was no evidence of haplotype transmission disequilibrium. In particular, we could not replicate the reported association of the TLR2 polymorphism rs5743708 with severe atopic eczema, which was likely to be false-positive because of a low number of cases and an unbalanced study design.9 However, because there is substantial variation in frequencies of both genetic and nongenetic risk factors among different populations, our results cannot be generalized to other populations. Analysis of the subgroups of children with asthma, rhinitis, or allergic sensitization likewise did not reveal evidence of association with TLR2 or TLR4 polymorphisms. However, the number of children with these traits was lower, and the children were selected on the basis of atopic eczema. Therefore, the current sample might lack statistical power to replicate associations with asthma.
Table I. Phenotype characteristics of all individuals (parents and affected) included in analyses
| Trios (n = 275) | ||
|---|---|---|
| Parents | Offspring | |
| Male sex, % | 50.4 | 44.0 |
| Mean (±SD) age, y | 55.1 (±9.2) | 19.5 (±11.1) |
| Asthma, % | 9.9 | 30.3 |
| Atopic eczema, % | 11.6 | 100 |
| Extrinsic atopic eczema, % | 3.7 | 63.4 |
| Mean SCORAD | NA | 44.6 |
| Allergic rhinoconjunctivitis, % | 28.6 | 50.9 |
| Mean (±SD) ln(total serum IgE) | 3.16 (±4.11) | 4.93 (±4.01) |
Table II. Association analysis of TLR2 and TLR4 SNPs in the trio cohort∗
| Atopic eczema | Asthma | Rhinoconjunctivitis | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Gene | dbSNP rs# | Alleles | MAF (%) | T:U | P value | T:U | P value | T:U | P value |
| TLR2 | rs4696480 | T → A | 48.2 | 141:134 | .6729 | 47:47 | 1.0000 | 73:68† | .6737 |
| TLR2 | rs3804099 | T → C | 42.6 | 137:129 | .6238 | 51:47† | .6862 | 73:61† | .2999 |
| TLR2 | rs3804100 | T → C | 7.8 | 46:36 | .2695 | 12:08 | .3711 | 21:17† | .5164 |
| TLR2 | rs5743708 | G → A | 3.8 | 24:18 | .3545 | 07:04† | .3657 | 13:07† | .1797 |
| TLR4 | rs2770150 | T → C | 27.8 | 71:65 | .6069 | 40:32 | .3458 | 57:53 | .7029 |
| TLR4 | rs6478317 | A → G | 32.8 | 73:71 | .8676 | 42:40 | .8252 | 61:56† | .6439 |
| TLR4 | rs1927911 | C → T | 26.9 | 64:57 | .5245 | 40:36 | .6464 | 52:49 | .7653 |
| TLR4 | rs2149356 | C → A | 32.0 | 74:72 | .8685 | 41:40 | .9115 | 60:58† | .8539 |
| TLR4 | rs4986790 | A → G | 6.1 | 25:21 | .5553 | 11:11 | 1.0000 | 20:16 | .5050 |
| TLR4 | rs4986791 | C → T | 6.6 | 26:23 | .6682 | 12:12 | 1.0000 | 21:17 | .5164 |
| TLR4 | rs7873784 | G → C | 15.7 | 43:42 | .9136 | 25:22† | .6617 | 35:31† | .6225 |
| TLR4 | rs1927906 | A→ G | 9.8 | 36:30 | .4602 | 16:14† | .7150 | 29:23 | .4054 |
∗Alleles are listed in chromosomal order, as genotyped in the family collection Alleles are coded as major allele → minor allele. T:U ratio refers to the ratio of transmitted and untransmitted alleles of the allele underlined except for †(opposite allele overtransmitted). |
In conclusion, we have performed a comprehensive evaluation of the TLR2 and TLR4 locus in a large family-based study, which eliminates the possibility of spurious associations on the basis of population stratification. We have found no evidence that common TLR2 or TLR4 variants or haplotypes are associated with atopic eczema in our study population. Our data indicate that TLR2 and TLR4 do not represent major atopic eczema susceptibility loci in German white subjects.
We thank Caroline Bussmann, Department of Dermatology, University of Bonn, for continuous help on the recruitment of patients with AD and Caroline Stahl, Institute for Medical Statistics and Epidemiology (IMSE), Technical University Munich, for her excellent support in electronic data management.
References
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Supported by grants from the German Ministry of Education and Research (BMBF)/National Genome Research Network (NGFN), project NUW-S31T05, and the Deutsche Forschungsgemeinschaft (DFG NO454/1-4 and DFG NO454/2-3). N.N. is supported by a Heisenberg-Fellowship of the DFG NO454/3-1. S.W. is supported by research grant KKF-07/04 of the University Hospital “Rechts der Isar,” Technical University Munich.Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.
PII: S0091-6749(06)00927-4
doi:10.1016/j.jaci.2006.04.034
© 2006 American Academy of Allergy, Asthma and Immunology. Published by Elsevier Inc. All rights reserved.
Volume 118, Issue 1 , Pages 277-279, July 2006
