Association between polymorphisms in caspase recruitment domain containing protein 15 and allergy in two German populations
Authors: Stephan K Weiland
Michael Kabesch
David Carr
Wilfried Peters
Erika von Mutius
Wolfgang Leupold
Journal: Journal of Allergy and Clinical Immunology (2003)
Discipline: IMMUNOLOGY
Keywords: microbial matter; LPS; asthma; allergies; CARD15 polymorphisms
Affiliations: University Children's Hospital, Ludwig Maximilians University Munich, München, Germany.

Abstract:  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 &lt;.001) and an almost 2-fold risk for atopic dermatitis (P &lt;.05). Furthermore, the T2104 allele was associated with an almost 2-fold risk for allergic rhinitis (P &lt;.05). When a C insertion at position 3020 was present, the risk of atopy increased by 50% (P &lt;.05) and serum IgE levels were elevated (P &lt;.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. 

Retracted: No

PubPeer comments:
 Approx 50 issues with this paper. Full account at https: www.wjst.de blog sciencesurf 2005 03 is-there-an-association-of-card15-variants-with-allergy which includes also the history + references. Journal responded but did never correct anything. Main points: Laboratory Methods. There is no information available about DNA extraction procedures, re-identification of samples, quality control, pre-amplification, details of PCR reaction, size of restriction fragments and scoring of genotypes. The SNP8 assay seems to include a MspI restriction site at the forward primer which could interfere with cutting the amplified fragment. The SNP12 assay has identical forward and backward primer which would not lead to any amplification. The assay for SNP13 has a poor design as it does not include any control site to ensure enzyme activity. Neither results of duplicate sample testing nor results of Hardy- Weinberg equilibrium are being reported. There is also no rationale given why only half of the Dresden samples are being genotyped. According to table I and II only 81% of genotyping attempts were successful while it is known that high genotyping failure rates is leading to false positive associations. Even if the noted errors in the primer sequence may be attributed to simple typing errors, this still raises doubts about the validity of the laboratory procedures. Analysis. A posthoc control group created by the absence of any genetic variant seems to inflate risks. It may even be argued that the calculation of relative risks would be more appropriate than that of odds ratios (resulting in considerable lower effect estimates). Without any detailed information of phenotypic and genotypic details of the control group, effects are hard to understand and might be also a reason why this definition of controls has been abandoned in further analysis of this population. The 1:2 matching of u201csupernormalu201d controls in the consecutive analysis is also questionable as selecting individuals with certain homogenous levels of confounders imposes restrictions of the analysis. With the absence of u201cnormalu201d controls even a more severe bias may be introduced. Results. The method section details the estimation of haplotypes but results are being omitted. Instead of haplotypes associations, the risk of co-occurrence of any two SNPs is reported which increase from 3.16 to 4.64. This result is considerably lower from what is being reported in the Crohn u0301s literature. Neither results of separate allelic and genotypic analysis are being reported and -as only significant risk estimates are given in table III- it is impossible to judge about any possible dose-response effect. The risks are also not adjusted for strong confounders like season and there is no assessment of goodness-of-fit of the models used which further undermines the validity of this study. The numbers in table III are confusing: The legend in table III refers to a total sample of 1873 children while introduction and methods reports 1872; row numbers in table III do not exceed 1805 and column numbers do not exceed 1765 individuals. Table I, II and III data do not match: For example 8,6% of 1161 genotyped children in table I have atopic rhinitis (may be rounded to N=100) plus 9,2% of 711 genotyped children in table II (may be rounded to N=65) which does not add to N=154 genotyped children with atopic rhinitis (table III). Similar restrictions apply to all other traits tested. A sample of 1872 children is reported in another paper to originate also from Leipzig and not only from Munich and Dresden. Other reports on the same collection report more than 3,000 samples. The results section of the current paper report that u201cIn Dresden, [u2026.] polymorphism T2104 was also associated with atopic rhinitis to a lesser degree [than in Munich] (16,9% vs 7.6%; OR 2,43; 95% CI 1,24 to 4,78; P .05)u201d. Results for Munich are not given, but the overall result for C2104T and atopic rhinitis in table III may indicate that the above sentence is wrong. In the results section the allele frequency of SNP8 is reported to be 5,6%, SNP12 of 2,0% and SNP13 of 3,8%. Percentages calculated from column 1 of table III give 11,2% for SNP8, 4,0% for SNP12 and 7,3% for SNP13. No significant linkage disequilibrium was observed between the examined SNPs while it is unclear why only homozygous subjects were included for this procedure. As linkage disequilibrium results seems to be unlikely in another paper from the same group this might refer to outstanding problems in the genetics module of SAS version 8.2 used by the authors (SAS notes SN-011039 and SN-008611). Linkage disequilibrium is not reported correctly if at least one haplotype frequency for a marker pair is estimated to be greater than the frequency of either allele. Another another error may be introduced if there are individuals with all missing alleles as already noted above. The moderate increase of total IgE probably does not indicate a u201chigher severity of atopyu201d. The difference between mean 186,6 IU ml and 312,1 IU ml reflect less than the transition between 70thand 80th percentile as may be assumed from another paper. IgE is a laboratory value that may be influenced also by other reasons and is otherwise not used as severity marker of atopy. Sources of bias. There is no information if the difference observed between the study centres is caused by population stratification (which could have been tested with anonymous marker and been completely avoided by using family-based samples). This is expected to be a particular problem as the authors reported a much lower prevalence of allergy of the Turkish minority in the Munich study center that have a considerable different genetic background. Was ancestry defined by passport or by self-reported affiliation? How were probands with mixed ancestry treated? Mild stratification might exist also in less admixed populations when looking for alleles with modest disease effect. Which steps have been taken to ensure that controls are non-cases? References. There are numerous referencing errors and misunderstandings: Fig. 1 locates SNP12 in the 6th LRR and SNP13 in the 9th LRR. According to Ogura SNP12 resides in the 7th LRR and SNP13 in the 10th LRR. Table I and II footnotes refers to reference 8 which belongs to another topic. Reference 10 cited in the methods is misleading as it relates to a different skin prick test device. Reference 14 is used to show that impaired LPS recognition by NOD2 polymorphism reduces the capability to interact with bacteria and to develop a Treg reservoir. Unfortunately this is not the content of their reference 14: u201cNODu201d denotes u201cnon-obese diabetic miceu201d and the review discusses helminth (but not bacterial) effects on T cells. CARD15 gene is also not located in the pericentromeric region of chromosome 16 (which would be q11.1) but on the cytogenetic band q12.1. There exists also a 12 exon isoform of CARD15 (and not only the reported 11 exon form). Even more important is the omission, that at the time of the study 67 (and not only the reported 13) polymorphism have been known. Linkage disequilibrium with untyped SNPs could therefore confound the current analysis. Interpretation. It is hard to understand why SNP8 and SNP12 impose the highest risk for atopic rhinitis while the risk for Crohn u0301s disease comes mainly with SNP13. Why is the excess risk for atopic rhinitis not found with the underlying biological traits? Although not being discussed in the paper, this could point towards chance effects introduced by multiple testing. The overall number of tests performed is not given in the paper. If we assume, however, 6 traits (total IgE, number of skin prick tests, atopy, atopic dermatitis, atopic rhinitis, asthma), tested in 3 groups (total and 2 subgroups), 4 series (as single SNP plus all combinations of 2 SNPs) and assume their u201cbestu201d p value from table III to be 0,001 (an exact estimate is not given), the Bonferoni corrected p value would be pcorr= 1-(1-0,001)6*3*4 = 0,0695 which is above conventional standards. ,