In this issue of <i>PLOS Genetics</i>, Hancock et al. address gene–environment interaction effects based on smoking status, GWAS data, and lung function outcomes in over 50,000 adults.

<p>This Perspective highlights the main findings in Hancock et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003174#pgen.1003174-Hancock1" target="_blank">[11]</a> and discusses why interaction effects are so difficult to identify even in large, well-characterized data sets.</p

A schematic model on the functional crosstalk between NPS and NPSR1.

<p><b>(A)</b> The non-synonymous NPS-Leu(6) substitution affects NPS bioactivity. Depending on the NPSR1-Asn(107) or NPSR1-Ile(107) genotype, binding of NPS can lead to strong or minimal signaling as well as differential expression of downstream target genes such as the transcription factors FOS and NR4A1. <b>(B)</b> Two hypothetical mechanisms in which the differential expression of FOS and NR4A1 can modulate the outcomes of the NPS/NPSR1 pathway. FOS and NR4A1 bind to their regulatory elements in <i>NPSR1</i> creating a feed-back loop that affect cell surface expression of NPSR1 and availability, and also, affect a number of other downstream genes that modulate neuroinflammation and ultimately asthma risk.</p

Gene-gene interactions between NPSR1 and NPS.

<p>Location of six <i>NPSR1</i> SNPs with significant interactions with <i>NPS</i> rs10830123 in the multiplicative model for epistasis in connection with their p-value for interaction and linkage disequilibrium. The most significant interaction signals in BAMSE (green) and MAGIC/ISAAC (orange) are marked in bold. Statistics on LD between the functional rs324981 and other interacting SNPs are provided as D’ and r².</p

Haplotype association of NPS polymorphisms with asthma at 8 years.

<p>Haplotype association of NPS polymorphisms with asthma at 8 years.</p

Location and block structure of three genotyped SNPs in the <i>NPS</i> gene.

<p>(A) The scale represents the relative positions of rs1931704 (G>A) in the upstream regulatory region of <i>NPS</i>, followed by rs10830123 (G>C) in the intronic region and rs4751440 (G>C) in the coding region. Allele changes are indicated by > and given on the positive chromosomal strand of the Human Reference Assembly. The arrow indicates <i>NPS</i> transcription from the positive strand. (B) Effects of <i>NPS</i> SNPs on the risk of physician-diagnosed asthma in carriers of either one or two copies of the alternative allele. Bars represent 95% confidence intervals (CI). OR: Odds Ratio. Given the allele and genotype frequencies, the dominant model which best fits the data is shown.</p

Real time RT-PCR analysis of NPSR1 downstream target genes.

<p>(A) Time-dependent mRNA expression of <i>NR4A1</i>, <i>FOS</i>, <i>CXCL2</i>, and <i>CCL20</i> in human embryonic kidney epithelial (HEK293) cells transiently transfected with either NPSR1-Ile(107) or NPSR1-Asn(107) coding variants and stimulated with either NPS-Val(6) or NPS-Leu(6) NPS (100 nM) for 1, 3, 6, and 24 h. (B) Dose-response curves of <i>NR4A1</i>, <i>FOS</i>, <i>IER3</i>, and <i>EGR1</i> in human SH-SY5Y stable cell line over-expressing NPSR1-Ile(107) stimulated with either NPS-Leu(6) or NPS V6 (0.0001–1 μM) for 3 h. The results are presented as fold-changes in comparison to the unstimulated cells. <i>GAPDH</i> was used as the endogenous reference, and data are expressed as mean of triplicate samples. The error bars represent 95% confidence intervals. In all experiments, results were calculated with the comparative <i>C</i>_<i>t</i> method.</p

Electric Mobility Shift Assay (EMSA) for four sites in the promoter for Neuropeptide S Receptor 1 gene (<i>NPSR1</i>).

<p>The experiment was performed three separate times using nuclear protein extracts from two different cell types (Colo205 and HEK293). Data presented here is a representative gel using nuclear extract from Colo205. Data was similar for nuclear cell extracts from both cell lines. The sites studied included CpG site 2 coinciding with rs2168890 in a predicted HMX2 binding site, CpG site 3 in a predicted STAT1 binding site, CpG site 8 coinciding with rs2530547 in a predicted binding site for MYB, and CpG site 9 coinciding with rs887020 in a predicted binding site for AP1. Arrows indicates sites showing differential binding.</p

DNA methylation status compared between asthma cases and controls in the BAMSE cohort (n = 546) using adjusted linear regression models.

*<p>Only homozygous carriers of the allele (rs2168891) creating a CpG site were included in the analyses of CpG site 1 (Asthma ever n _{cases/controls} = 236/234, Allergic asthma n _{cases/controls} = 120/234, Non-allergic asthma n _{cases/controls} = 116/234, Current asthma n _{cases/controls} = 103/234).</p>**<p>Only homozygous carriers of the allele (rs2168890) creating a CpG site were included in the analyses of CpG site 2 (Asthma ever n _{cases/controls} = 196/201, Allergic asthma n _{cases/controls} = 94/201, Non-allergic asthma n _{cases/controls} = 201/201, Current asthma n _{cases/controls} = 89/201).</p>†<p>p-value for crude linear regression model.</p>††<p>p-value for linear regression model adjusted for gender, age at blood sampling and plate used for sodium-bisulfite treatment.</p

The promoter region of the Neuropeptide S Receptor 1 gene (<i>NPSR1</i>).

<p>Polymorphisms are marked by triangles, transcription factor binding sites are underlined and CpG sites are shaded in gray. Green color of the transcription factor indicates neurologically associated factors, blue indicates immunologically associated factors, and black indicates general factors.</p

Characteristics of the BIOAIR study population (n = 171).

<p>COPD - Chronic Obstructive Pulmonary Disease, BMI – Body Mass Index, FEV₁ – Forced Expiratory Volume in 1 second, FVC – Forced Vital Capacity. Data are presented as mean ± standard deviation. P-values were calculated using ANOVA for continuous variables and chi square test for distributions.</p>1<p>one individual with missing data,</p>2<p>five individuals with missing data,</p>3<p>two individuals with missing data,</p>4<p>seven individuals with missing data,</p>5<p>three individuals with missing data.</p