DNA Methylation in the Neuropeptide S Receptor 1 (NPSR1) Promoter in Relation to Asthma and Environmental Factors

Peer reviewe

An integrative genomics approach identifies new asthma pathways related to air pollution exposure

The evidence on traffic-related air pollution exposure (TRAP) and incident childhood asthma is inconsistent, and may depend on genetic factors. We aimed to identify mechanisms of childhood asthma using genome-wide SNP data and individual TRAP exposure, and to evaluate the effect of susceptibility SNPs and TRAP on DNA-methylation and gene expression. We used LUR models to estimate individual outdoor NO2 levels at the birth address and performed a genome-wide interaction study for doctor's diagnosis of asthma up to 8 years in three European birth cohorts with replication in two North American cohorts (n=3,322 subjects). The top GWIS and replicated SNPs were assessed for methQTL effects in peripheral blood cells and eQTL effects in human lung specimens. Short- and long-term TRAP associations with methylation patterns and TRAP-induced differential gene expression in blood cells were also assessed. The novel loci MAGI1, B4GALT5, MOCOS and DLG2, and the previously lung disease linked locus ADCY2 showed strong evidence for interaction with TRAP (genome-wide significance or replication). The top replication SNP rs686237 was identified as an eQTL for B4GALT5 (p=1.18x10-17) and affected TRAP-induced gene expression (p=0.03). Differential methylation following TRAP exposure was seen for DLG2, ADCY2, MAGI1 and MOCOS. Identified genes belong to the guanylate kinase, sphingolipid and calcium signaling pathways, suggesting involvement in asthma pathogenesis. Our results indicate that gene-environment interactions are important for asthma development and that functional genomics analyses in conjunction with environmental exposures may give valuable insights about pathophysiologic mechanisms

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

Neuropeptide S Receptor 1 gene (<i>NPSR1</i>) DNA methylation levels in isolated cells from peripheral blood.

<p>Peripheral blood donations were obtained from six healthy adult male donors. Data were obtained using the Illumina Infinium 450K bead array and the M-value represents methylation degree <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053877#pone.0053877-Reinius1" target="_blank">[25]</a>. Dark grey bars represent lymphoid cells, light grey bars myeloid cells and black bar whole blood. TSS – Transcription start site.</p

The effect of respiratory phenotypes and lifestyle on the levels of DNA methylation in the <i>NPSR1</i> promoter region covering CpG site 1 to 12 in adults from the BIOAIR study (n = 171) using adjusted linear regression models.

<p>COPD – Chronic Obstructive Pulmonary Disease, BMI – Body Mass Index, FEV₁ – Forced Expiratory Volume in 1 second, FVC – Forced Vital Capacity. Mild asthma patients were used as reference in the comparison to severe asthma and COPD. P: p-value for linear regression model adjusted for age, gender and country of origin. Age and gender were also adjusted for asthma and other obstructive airway disease (mild asthma, severe asthma or COPD).</p>*<p>Data analyzed in homozygous carriers of the C allele only (n_{mild asthma/severe asthma/COPD} = 47/56/44),</p>**<p>Data analyzed in homozygous carriers of the C allele only (n_{mild asthma/severe asthma/COPD} = 38/45/33),</p>†<p>Data analyzed in homozygous carriers of the C allele only (n_{mild asthma/severe asthma/COPD} = 16/27/19),</p>††<p>Data analyzed in homozygous carriers of the G allele only (n_{mild asthma/severe asthma/COPD} = 10/20/10).</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