Demographic information for the clinical cohorts evaluated in this study.

<p>Definition of abbreviations: N = number of subjects providing DNA samples evaluated in this study; SD = standard deviation; FEV₁ = forced expiratory volume in 1 second (mL); LOCCS = Leukotriene Modifier Or Corticosteroid or Corticosteroid-Salmeterol Trial; LODO = Effectiveness of Low Dose Theophylline as Add On Therapy for the Treatment of Asthma; CLIC = Characterizing the Response to a LT Receptor Antagonist and Inhaled Corticosteroid trial; PACT = Pediatric Asthma Controller Trial.</p><p>Demographic information for the clinical cohorts evaluated in this study.</p

Improvement in lung function related to montelukast treatment, by rs6475448 genotype.

<p>The least-squares (LS) means (adjusted for study, race and gender) and 95% confidence intervals for ΔFEV₁ related to montelukast treatment were generated using R (<a href="http://cran.r-project.org/web/packages/lsmeans/lsmeans.pdf" target="_blank">http://cran.r-project.org/web/packages/lsmeans/lsmeans.pdf</a>), and plotted for each study (panels), by rs6475448 genotypes: homozygous reference (“GG”: LOCCS = 32; LODO = 38; CLIC = 25; PACT = 65), heterozygous (“GA”: LOCCS = 28; LODO = 21; CLIC = 30; PACT = 75) and homozygous variant (“AA”: LOCCS = 9; LODO = 5; CLIC = 5; PACT = 5).</p

Replicated^* GWAS SNPs.

<p>Definition of abbreviations: “SNP” = single nucleotide polymorphism; “Chr.” = chromosome (1–22); “Chr. Location.” = chromosomal position of listed SNP; “β” = effect size estimates (ΔFEV₁, (mL)) for the minor allele.</p><p><b>*</b>Table lists GWA results adjusted for baseline FEV₁, age, race and gender as covariates (additive genetic model), for the SNPs that met criteria for replication in all cohorts (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129385#sec006" target="_blank">Methods</a>) and remained significant after correction for multiple testing. Minor allele frequencies for all SNPs in all cohorts is >5%.</p><p>^<b>‡</b>Combined P value for all cohorts.</p><p>Replicated^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129385#t002fn002" target="_blank">*</a>} GWAS SNPs.</p

Results of the discovery GWAS.

<p>Manhattan plots (<b>A</b> and <b>B</b>) contain −log P values (y-axis) associated with 8-week change in FEV₁ after montelukast treatment, for 532,264 genotyped SNPs organized by chromosome (x-axis), for LOCCS (<b>A</b>) and LODO (<b>B</b>). The threshold for genome-wide significance and suggestive genome-wide significance are indicated as blue and red lines, respectively, in the Manhattan plots. Q-Q plots (<b>C</b> and <b>D</b>) demonstrate the observed −log P values vs. expected −log P values, for SNPs from LOCCS (<b>C</b>) and LODO (<b>D</b>) populations. In all plots, individual SNPs are represented as filled circles.</p

Summary of BDR by genotype of the SNP (rs295137) near <i>SPATS2L</i> with lowest P-value among all subjects in the primary populations.

<p>The TT genotype was associated with higher BDR (median 16.0; inter-quartile range (IQR) = [6.2, 32.4]), than the TC genotype (median 11.2; IQR = [5.2, 22.6]) or the CC genotype (median 10.3; IQR = [4.1, 21.7]).</p

Primary GWAS Top Results (SNPs with Combined P-values <1e-05).

<p>CLLS = CAMP/LOCCS/LODO/Sepracor. CLLS used genotyped SNP data. CARE and ACRN used HapMap Phase 2 imputed data with Mach ratio of empirically observed dosage variance to the expected dosage variance (Rsq) values indicated.</p

Effect of siRNA-mediated <i>SPATS2L</i> knockdown on β₂-adrenergic receptor levels in HASM cells.

<p>(A) Knockdown efficiency of two <i>SPATS2L</i> siRNAs. Quantitative real-time PCR was done on RNAs extracted from HASM cells transfected with control non-targeting (NT) or <i>SPATS2L</i>-specific siRNAs. (B) Western blot analysis of β₂AR protein in <i>SPATS2L</i> knockdown HASM cells. Three independent experiments (siRNA transfection and Western blot analysis) were done in HASM cells. The upper panel is a representative of the three Western blots. Quantification of the β₂AR protein amount (normalized against the control β-actin protein) from three Western blots is shown in the lower panel.</p

Characteristics of GWAS subjects.

<p>Pre-BD = Pre-bronchodilator. SD = Standard deviation. For continuous variables, Mean (SD) and [Range] are shown.</p>*<p>Subjects were allowed to use rescue medication during wash-out period unless otherwise indicated.</p>**<p>550Kv3, 610, 317K, and 370K refer to Illumina BeadChips.</p>§<p>CARE trials included: CLIC, PACT, MARS.</p>¶<p>ACRN trials included: BAGS, DICE, IMPACT, PRICE.</p

Study overview.

<p>(A) Primary GWAS was conducted using subjects from CAMP, LOCCS, LODO, Sepracor, ACRN, and CARE cohort. Samples genotyped on Illumina platforms (i.e. CAMP/LOCCS/LODO/Sepracor) were pooled and analyzed first. Results from samples genotyped on Affymetrix platforms were analyzed separately and then combined to obtain the primary GWAS results. 1000GP imputed data was utilized to expand the primary GWAS association results. (B) Replication of the top (i.e. P-value<1E-04) SNPs from the primary GWAS were attempted in two independent populations: SARP and DAG. (C) The <i>SPATS2L</i> gene was selected for functional validation based on nominal replication of association results in SARP and analysis of publicly available resources.</p

Region of association near <i>SPATS2L</i> SNPs to BDR.

<p>The x-axis denotes position along Chromosome 2. The y-axis denotes −Log₁₀(P) corresponding to 1000GP imputed data P-values. LD between the SNP with the lowest P-value (rs295137) to each SNP in the plot is denoted in colors and was computed according to 1000GP June 2010 CEU data. Plot was created using LocusZoom <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002824#pgen.1002824-Pruim1" target="_blank">[44]</a>.</p