Assessing the Validity of Asthma Associations for Eight Candidate Genes and Age at Diagnosis Effects

<div><p>Background</p><p>Before the advent of genome-wide association studies (GWAS), <i>ADAM33, ADRB2, CD14, MS4A2</i> (alias <i>FCER1B</i>), <i>IL13, IL4, IL4R</i>, and <i>TNF</i> constituted the most replicated non-<i>HLA</i> candidate genes with asthma and related traits. However, except for the <i>IL13-IL4</i> region, none of these genes have been found in close proximity of genome-wide significant hits among GWAS for asthma or related traits. Here we aimed to assess the reproducibility of these asthma associations and to test if associations were more evident considering the effect of age at diagnosis.</p><p>Methodology/Principal Findings</p><p>We systematically evaluated 286 common single nucleotide polymorphisms (SNPs) of these 8 genes in a sample of 1,865 unrelated Spanish individuals (606 asthmatics and 1,259 controls). We found that variants at <i>MS4A2</i>, <i>IL4R</i> and <i>ADAM33</i> genes demonstrated varying association effects with the age at diagnosis of asthma, with 10 SNPs showing study-wise significance after the multiple comparison adjustment. In addition, <i>in silico</i> replication with GWAS data supported the association of <i>IL4R</i>.</p><p>Conclusions/Significance</p><p>Our results support the important role of <i>MS4A2</i>, <i>IL4R</i> and <i>ADAM33</i> genes in asthma and/or atopy susceptibility. However, additional studies in larger samples sets are needed to firmly implicate these genes in asthma susceptibility, and also to identify the causal variation underlying the associations found.</p></div

<i>P</i>-values of association by chromosome position with A) asthma ≤14years for <i>ADAM33</i>, B) asthma ≤39 years for <i>MS4A2</i> and, C) asthma ≤39 years for <i>IL4R</i>.

<p><b><i>P</i>-values are expressed in –log₁₀ scale.</b> The SNP number shown on the plot denotes the result for the most significant SNP for each gene and the results for the remaining were color coded to reflect their LD with this SNP based on pairwise <i>r</i>² values from the 1KGP. Estimated recombination rates (from 1KGP) were also plotted on the right axis to reflect the local LD structure.</p

Association summary of the 10 SNPs that resulted significantly associated with asthma after adjustments for the multiple comparisons.

<p>tSNPs are underlined.</p>a<p>According to NCBI build 36.3.</p>b<p>Computed for allele 1.</p>c<p>SNPs associated in previous studies.</p

Clinical data of patients included in this study.

<p>Clinical data of patients included in this study.</p

Lipid transfer proteins (LTP) included in the homemade array were separated by SDS-PAGE and stained with Coomassie Blue.

<p>Replicas were electrotransferred and incubated with polyclonal antibodies produced against peach LTP (dilution 1∶500). The name of the proteins corresponds to Table I.</p

Purified proteins included in the LTP microarray.

<p>r recombinant protein;</p>*<p>non-LTPs or control proteins included in the array are marked with an asterisk.</p

Frequency of LTP sensitization.

<p><b>A</b>. Recognition frequencies of food and pollen LTPs comparing fruit allergic patients with and without pollen sensitization (Pollen Fruit Allergy and Fruit Allergy, respectively). The recognition frequencies, shown as percentage of positive response (%), were obtained incubating the LTP microarray with single sera from allergic patients. <b>B</b>. Analysis of LTP crossreactivities by inhibition assays using the LTP microarray as solid phase and Pru p 3, Art v 3 and Cas s 8 (5, 1, 0.1, 0.01 µg/mL) as inhibitors. The inhibition percentage of the IgE binding capacity is indicated. Means (n = 3) and SDs (bars) are represented. All tests were performed in triplicate.</p

Co-sensitization graph of LTP allergens.

<p>Each node represents one allergen (LTP, white ovals non-LTP allergens, blue squares) and the links represent co-sensitization of one or more sera for the linked allergens. The weight of each link, between 0 and 1, measures the degree of co-sensitization. For the sake of clarity, only the 35 links of weights greater than 0.55 of the total 190 existing links are plotted.</p

Average weight of allergens included in the co-sensitization graph.

<p>Average weight of allergens included in the co-sensitization graph.</p

Recognition frequencies of food and pollen LTPs by geographical area.

<p>The recognition frequencies, shown as percentage of positive response (%), were obtained incubating the LTP microarray with single sera from allergic patients. Only LTPs with a positive response of more than 20% (taking all patients into account) are represented. Percentage positive responses and significant differences (p<0.05) are indicated.</p