Epigenome-wide association study identifies DNA methylation markers for asthma remission in whole blood and nasal epithelium.

BACKGROUND: Asthma is a chronic respiratory disease which is not curable, yet some patients experience spontaneous remission. We hypothesized that epigenetic mechanisms may be involved in asthma remission. METHODS: Clinical remission (ClinR) was defined as the absence of asthma symptoms and medication for at least 12 months, and complete remission (ComR) was defined as ClinR with normal lung function and absence of airway hyperresponsiveness. We analyzed differential DNA methylation of ClinR and ComR comparing to persistent asthma (PersA) in whole blood samples (n = 72) and nasal brushing samples (n = 97) in a longitudinal cohort of well characterized asthma patients. Significant findings of whole blood DNA methylation were tested for replication in two independent cohorts, Lifelines and Epidemiological study on the Genetics and Environment of Asthma (EGEA). RESULTS: We identified differentially methylated CpG sites associated with ClinR (7 CpG sites) and ComR (129 CpG sites) in whole blood. One CpG (cg13378519, Chr1) associated with ClinR was replicated and annotated to PEX11 (Peroxisomal Biogenesis Factor 11 Beta). The whole blood DNA methylation levels of this CpG were also different between ClinR and healthy subjects. One ComR-associated CpG (cg24788483, Chr10) that annotated to TCF7L2 (Transcription Factor 7 Like 2) was replicated and associated with expression of TCF7L2 gene. One out of seven ClinR-associated CpG sites and 8 out of 129 ComR-associated CpG sites identified from whole blood samples showed nominal significance (P < 0.05) and the same direction of effect in nasal brushes. CONCLUSION: We identified DNA methylation markers possibly associated with clinical and complete asthma remission in nasal brushes and whole blood, and two CpG sites identified from whole blood can be replicated in independent cohorts and may play a role in peroxisome proliferation and Wnt signaling pathway.

Nuclear Receptor Nur77 Attenuates Airway Inflammation in Mice by Suppressing NF-κB Activity in Lung Epithelial Cells.

Allergic asthma is characterized by persistent chronic airway inflammation, which leads to mucus hypersecretion and airway hyperresponsiveness. Nuclear receptor Nur77 plays a pivotal role in distinct immune and inflammatory cells and is expressed in eosinophils and lung epithelium. However, the role of Nur77 in allergic airway inflammation has not been studied so far. In the present study, we determined the role of Nur77 in airway inflammation using a murine model of OVA-induced allergic airway inflammation. We found that OVA-challenged Nur77 knockout (KO) mice show significantly enhanced infiltration of inflammatory cells, including eosinophils and lymphocytes, and aggravated mucus production. The infiltration of macrophages is limited in this model and was similar in wild-type and Nur77 KO mice. Higher levels of Th2 cytokines were found in bronchoalveolar lavage fluid and draining lymph node cells of Nur77-KO mice, as well as increased serum IgG1 and IgG2a levels. Knockdown of Nur77 in human lung epithelial cells resulted in a marked increase in IκBα phosphorylation, corresponding with elevated NF-κB activity, whereas Nur77 overexpression decreased NF-κB activity. Consistently, Nur77 significantly decreased mRNA levels of inflammatory cytokines and Muc5ac expression and also attenuated mucus production in lung epithelial cells. To further corroborate these findings, we searched for association of single nucleotide polymorphisms in Nur77 gene with asthma and with the severity of bronchial hyperresponsiveness. We identified three Nur77 single nucleotide polymorphisms showing association with severity of bronchial hyperresponsiveness in asthma patients. Collectively, these findings support a protective role of Nur77 in OVA-induced airway inflammation and identify Nur77 as a novel therapeutic target for airway inflammation.

Genome-wide protein QTL mapping identifies human plasma kallikrein as a post-translational regulator of serum uPAR levels.

The soluble cleaved urokinase plasminogen activator receptor (scuPAR) is a circulating protein detected in multiple diseases, including various cancers, cardiovascular disease, and kidney disease, where elevated levels of scuPAR have been associated with worsening prognosis and increased disease aggressiveness. We aimed to identify novel genetic and biomolecular mechanisms regulating scuPAR levels. Elevated serum scuPAR levels were identified in asthma (n=514) and chronic obstructive pulmonary disease (COPD; n=219) cohorts when compared to controls (n=96). In these cohorts, a genome-wide association study of serum scuPAR levels identified a human plasma kallikrein gene (KLKB1) promoter polymorphism (rs4253238) associated with serum scuPAR levels in a control/asthma population (P=1.17 × 10(-7)), which was also observed in a COPD population (combined P=5.04 × 10(-12)). Using a fluorescent assay, we demonstrated that serum KLKB1 enzymatic activity was driven by rs4253238 and is inverse to scuPAR levels. Biochemical analysis identified that KLKB1 cleaves scuPAR and negates scuPAR's effects on primary human bronchial epithelial cells (HBECs) in vitro. Chymotrypsin was used as a proproteolytic control, while basal HBECs were used as a control to define scuPAR-driven effects. In summary, we reveal a novel post-translational regulatory mechanism for scuPAR using a hypothesis-free approach with implications for multiple human diseases.

ITGB5 and AGFG1 variants are associated with severity of airway responsiveness.

BACKGROUND: Airway hyperresponsiveness (AHR), a primary characteristic of asthma, involves increased airway smooth muscle contractility in response to certain exposures. We sought to determine whether common genetic variants were associated with AHR severity. METHODS: A genome-wide association study (GWAS) of AHR, quantified as the natural log of the dosage of methacholine causing a 20% drop in FEV1, was performed with 994 non-Hispanic white asthmatic subjects from three drug clinical trials: CAMP, CARE, and ACRN. Genotyping was performed on Affymetrix 6.0 arrays, and imputed data based on HapMap Phase 2, was used to measure the association of SNPs with AHR using a linear regression model. Replication of primary findings was attempted in 650 white subjects from DAG, and 3,354 white subjects from LHS. Evidence that the top SNPs were eQTL of their respective genes was sought using expression data available for 419 white CAMP subjects. RESULTS: The top primary GWAS associations were in rs848788 (P-value 7.2E-07) and rs6731443 (P-value 2.5E-06), located within the ITGB5 and AGFG1 genes, respectively. The AGFG1 result replicated at a nominally significant level in one independent population (LHS P-value 0.012), and the SNP had a nominally significant unadjusted P-value (0.0067) for being an eQTL of AGFG1. CONCLUSIONS: Based on current knowledge of ITGB5 and AGFG1, our results suggest that variants within these genes may be involved in modulating AHR. Future functional studies are required to confirm that our associations represent true biologically significant findings.

Integration of mouse and human genome-wide association data identifies KCNIP4 as an asthma gene.

Asthma is a common chronic respiratory disease characterized by airway hyperresponsiveness (AHR). The genetics of asthma have been widely studied in mouse and human, and homologous genomic regions have been associated with mouse AHR and human asthma-related phenotypes. Our goal was to identify asthma-related genes by integrating AHR associations in mouse with human genome-wide association study (GWAS) data. We used Efficient Mixed Model Association (EMMA) analysis to conduct a GWAS of baseline AHR measures from males and females of 31 mouse strains. Genes near or containing SNPs with EMMA p-values <0.001 were selected for further study in human GWAS. The results of the previously reported EVE consortium asthma GWAS meta-analysis consisting of 12,958 diverse North American subjects from 9 study centers were used to select a subset of homologous genes with evidence of association with asthma in humans. Following validation attempts in three human asthma GWAS (i.e., Sepracor/LOCCS/LODO/Illumina, GABRIEL, DAG) and two human AHR GWAS (i.e., SHARP, DAG), the Kv channel interacting protein 4 (KCNIP4) gene was identified as nominally associated with both asthma and AHR at a gene- and SNP-level. In EVE, the smallest KCNIP4 association was at rs6833065 (P-value 2.9e-04), while the strongest associations for Sepracor/LOCCS/LODO/Illumina, GABRIEL, DAG were 1.5e-03, 1.0e-03, 3.1e-03 at rs7664617, rs4697177, rs4696975, respectively. At a SNP level, the strongest association across all asthma GWAS was at rs4697177 (P-value 1.1e-04). The smallest P-values for association with AHR were 2.3e-03 at rs11947661 in SHARP and 2.1e-03 at rs402802 in DAG. Functional studies are required to validate the potential involvement of KCNIP4 in modulating asthma susceptibility and/or AHR. Our results suggest that a useful approach to identify genes associated with human asthma is to leverage mouse AHR association data.

Genome-wide association analysis in asthma subjects identifies SPATS2L as a novel bronchodilator response gene.

Bronchodilator response (BDR) is an important asthma phenotype that measures reversibility of airway obstruction by comparing lung function (i.e. FEV(1)) before and after the administration of a short-acting ß(2)-agonist, the most common rescue medications used for the treatment of asthma. BDR also serves as a test of ß(2)-agonist efficacy. BDR is a complex trait that is partly under genetic control. A genome-wide association study (GWAS) of BDR, quantified as percent change in baseline FEV(1) after administration of a ß(2)-agonist, was performed with 1,644 non-Hispanic white asthmatic subjects from six drug clinical trials: CAMP, LOCCS, LODO, a medication trial conducted by Sepracor, CARE, and ACRN. Data for 469,884 single-nucleotide polymorphisms (SNPs) were used to measure the association of SNPs with BDR using a linear regression model, while adjusting for age, sex, and height. Replication of primary P-values was attempted in 501 white subjects from SARP and 550 white subjects from DAG. Experimental evidence supporting the top gene was obtained via siRNA knockdown and Western blotting analyses. The lowest overall combined P-value was 9.7E-07 for SNP rs295137, near the SPATS2L gene. Among subjects in the primary analysis, those with rs295137 TT genotype had a median BDR of 16.0 (IQR = [6.2, 32.4]), while those with CC or TC genotypes had a median BDR of 10.9 (IQR = [5.0, 22.2]). SPATS2L mRNA knockdown resulted in increased ß(2)-adrenergic receptor levels. Our results suggest that SPATS2L may be an important regulator of ß(2)-adrenergic receptor down-regulation and that there is promise in gaining a better understanding of the biological mechanisms of differential response to ß(2)-agonists through GWAS.