Combinations of genetic variants and/or environmental factors in relation to allergy and asthma in PARSIFAL.

<p>Odds ratios are shown for the top-hits rules identified for (<b>A</b>) allergic eczema; affected^1-10 and unaffected^58-67 (<b>B</b>) asthma; affected^1-10 and unaffected^44-53 and (<b>C</b>) atopic sensitization; affected^1-10 and unaffected^37-46. The odds ratios were calculated for children that fulfill all conditions in the rule using all other children as reference.</p

Visualization of co-occurring factors in rules for allergic eczema, asthma and atopic sensitization in PARSIFAL.

<p>Rule networks for (<b>A</b>-<b>B</b>) allergic eczema, (<b>C</b>-<b>D</b>) asthma and (<b>E</b>-<b>F</b>) atopic sensitization; affected and unaffected, respectively. Conditions that occur in the rules are on the outer ring, and co-occurrences of conditions in the rules are illustrated by ribbons across the circle connecting the conditions. The ribbon color indicates high (red) to low (grey) scores. The width of the edges is proportional to the number of correctly classified children.</p

SNP combinations with relevance for current asthma and wheeze in BAMSE and PARSIFAL.

<div><p>The combination of specific genetic variants in (<b>A</b>) <i>ORMDL3-RORA</i> increases the risk for current asthma¹, and in (<b>B</b>) <i>ORMDL3-RORA-COL29A1</i> increase the risk for wheeze². The risk for current asthma and wheeze increased with the number of risk genotypes described by corresponding rule (<b>C</b>-<b>D</b>). ORs and 95% confidence interval are shown. The major allele count is indicated for each gene below i.e. describing 0, 1 or 2 copies of the major allele. The reference category includes children who do not fulfill the rule.</p> <p>¹ IF ORMDL3_rs2305480=2[GG] AND RORA_rs17270362=1[AG] THEN current asthma.</p> <p>² IF COL29A1_rs11917356=2[AA] AND ORMDL3_rs7216389=0[TT] AND RORA_rs17270362=1[AG] THEN wheeze.</p></div

Analysis methodology for factors related to childhood allergy in the epidemiologic studies BAMSE and PARSIFAL.

<p>Allergy phenotypes were modeled based on genetic and exposure data to identify (<b>A</b>) rules using gene and (<b>B</b>) gene and environment data. MCFS selected significant predictors of a phenotype, which was used to generate rules by ROSETTA. First model used 110 SNPs in BAMSE and PARSIFAL, while the second model included both genetic and exposure data in PARSIFAL, using BAMSE for validation when applicable.</p

Allelic association tests between <i>RORA</i> SNPs and asthma.

<p>SNPs are represented by dots in relation to their genomic position. Grey-colored dots have a degree of linkage disequilibrium (r²) between 0.40 and 0.60 with the lead SNP (diamond). Recombination rate is indicated as a continuous line. Dashed line represents the significance threshold. (<b>A</b>) Asthma ever up to 4 years in BAMSE; (<b>B</b>) Asthma ever up to 8 years in BAMSE; (<b>C</b>) Physician-diagnosed asthma ever between 5–13 years in PARSIFAL; (<b>D</b>) Physician-diagnosed asthma in the combined dataset.</p

Overview of the genomic location and correlations of the <i>RORA</i> SNPs analyzed in this study.

<p>(<b>A</b>) Schematic representation of the exon intron distribution of the longest isoform of <i>RORA</i> (NM_134231) located in chromosome 15q22.2 and the position of the GWAS SNP rs11071559. An expansion of the 116.5 kb region surrounding the rs11071559 reveals the 35 SNPs analyzed in this study according to their positions along the intron 1. (<b>B</b>) The LD structure of the 116.5 kb region of RORA as defined by the solid spine algorithm in the combined dataset (n = 3153). Numbers in each box correspond to the pair-wise linkage disequilibrium coefficients (D’) between the respective SNPs. A similar LD structure was observed in BAMSE and PARSIFAL as separated populations. Additional information on the 35 <i>RORA</i> SNPs is presented in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060111#pone.0060111.s003" target="_blank">Table S3</a></b>.</p

Expression of <i>Rora</i> in the lung tissue of wildtype (WT) and <i>Npsr1</i> deficient (<i>Npsr1</i>^−/−) mice.

<p>(<b>A</b>) Lung tissue was collected from WT and <i>Npsr1</i>^−/− mice at 4 h intervals, and diurnal mRNA expression of <i>Rora</i> was measured with real-time PCR (n = 3–4/group/timepoint). The black bar represents the dark period. (<b>B</b>) Expression of <i>Rora</i> in the lung tissue of wildtype and <i>Npsr1</i>^−/− mice during the light period (8–12 am) (n = 6). Data are expressed as relative units indicating a fold change in <i>Rora</i> mRNA expression that is normalized to an endogenous reference gene (18S ribosomal RNA) and is relative to the non-template control calibrator (mean ± SEM). Similar data were obtained in two separate experiments. *p<0.05.</p

Cross-talk of RORA and NPSR1 in cell models.

<p>(<b>A</b>) Real-time PCR analysis of circadian clock genes in human SH-SY5Y neuroblastoma cell line over-expressing NPSR1 stimulated with 100 nM NPS for 0–24 h in the presence or absence of 3 µM SHA 68 (<i>N</i>-[(4-fluorophenyl)methyl]tetrahydro-3-oxo-1,1-diphenyl-3<i>H</i>-oxazolo[3,4-<i>a</i>]pyrazine-7(1<i>H</i>)-carboxamide), a selective antagonist of NPSR1. (<b>B</b>) Real-time PCR analysis of <i>RORA</i>, <i>NR1D1</i>, <i>NPAS2</i>, <i>CLOCK</i>, <i>ARNTL</i>, <i>PER1</i>, <i>CRY1</i>, and <i>DBP</i> mRNA expression in human embryonic kidney epithelial HEK-293H cell line over-expressing NPSR1 6 h after NPS (0.1–5 µM) stimulation. The results are presented as fold-changes in comparison to the unstimulated cells. GAPDH was used as the endogenous reference, and data are expressed as mean of triplicate samples ±95% confidence intervals. In all experiments, results were calculated with the comparative ΔΔ<i>C_t</i> method. (<b>C</b>) Schema of the <i>NPSR1</i> promoter and the location of the putative 6-bp AT-rich sequence preceding the half-core motif PuGGTCA (RORE). (<b>D</b>) NPSR1 driven luciferase in relative luminescence units after transfection with either RORA-1 encoding plasmid or an empty vector. Ten biological replicates per group. <i>Mann-Whitney</i> U test ***p<0.0001.</p

The <i>RORA</i> SNPs associated with asthma and their effects under additive and dominant models.

*<p>Exact Armitage P value.</p><p>B: Bamse; P: Parsifal, C: combined;</p>†<p>Rare homozygotes are in bold.</p><p>EM: Expectation-Maximization **From the lead SNP rs7164773.</p><p>Further information on the genotype distributions is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060111#pone.0060111.s007" target="_blank">File S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060111#pone.0060111.s008" target="_blank">File S4</a>.</p><p>ns: non-significant.</p

Gene-gene interactions between <i>RORA</i> and <i>NPSR1.</i>

<p>(A) Heatmap of the p-values for interaction between 35 <i>RORA</i> SNPs (<i>y</i>-axis) and 8 <i>NPSR1</i> SNPs (<i>x</i>-axis). A schema of the <i>NPSR1</i> gene is presented at the bottom of the figure with the relative positions of the SNPs. The alleles comprising the CAC haplotype are marked in red. (B) Effects of one (TC) or two copies (CC) of the <i>RORA</i> SNP rs7164773 on asthma risk according to the genotypes on the <i>NPSR1</i> SNP rs6972158 (Gln344Arg). Bars represent 95% confidence intervals. (C) Effects of <i>RORA</i> rs8024629 and <i>RORA</i> rs9302215 on asthma risk according to the functional <i>NPSR1</i> haplotype CAC (rs2530547<b>C</b>/rs324981<b>A</b>/rs727162<b>C</b>). All the results for gene-gene interactions analysis are presented after adjustment by age and country of origin.</p