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DNA methylation (DNAm) is a dynamic, age-dependent epigenetic modification that can be used to study interactions between genetic and environmental factors. Environmental exposures during critical periods of growth and development may alter DNAm patterns, leading to increased susceptibility to diseases such as asthma and allergies. One method to study the role of DNAm is the epigenetic clock-an algorithm that uses DNAm levels at select age-informative Cytosine-phosphate-Guanine (CpG) dinucleotides to predict epigenetic age (EA). The difference between EA and calendar age (CA) is termed epigenetic age acceleration (EAA) and reveals information about the biological capacity of an individual. Associations between EAA and disease susceptibility have been demonstrated for a variety of age-related conditions and, more recently, phenotypes such as asthma and allergic diseases, which often begin in childhood and progress throughout the lifespan. In this review, we explore different epigenetic clocks and how they have been applied, particularly as related to childhood asthma. We delve into how in utero and early life exposures (e.g., smoking, air pollution, maternal BMI) result in methylation changes. Furthermore, we explore the potential for EAA to be used as a biomarker for asthma and allergic diseases and identify areas for further study.

We examined common variation in asthma risk by conducting a meta-analysis of worldwide asthma genome-wide association studies (23,948 asthma cases, 118,538 controls) of individuals from ethnically diverse populations. We identified five new asthma loci, found two new associations at two known asthma loci, established asthma associations at two loci previously implicated in the comorbidity of asthma plus hay fever, and confirmed nine known loci. Investigation of pleiotropy showed large overlaps in genetic variants with autoimmune and inflammatory diseases. The enrichment in enhancer marks at asthma risk loci, especially in immune cells, suggested a major role of these loci in the regulation of immunologically related mechanisms.

Purpose of Review: Understanding the mechanisms involved in the development of asthma and allergic diseases is expanding, due in part to sequencing advances that have led to the identification of new viral strains such as human rhinovirus strain C (HRV-C) and the human microbiome project., Recent Findings: Recent studies have identified new ways in which viral and microbial exposures in early life interact with host genetic background/variants to modify the risk for developing asthma and allergic diseases. Recent research suggests that HRV-C is the main pathogenic agent associated with infant wheeze, hospitalizations and likely the subsequent development of asthma. Pulmonary He MRI suggests that HRV infection in early childhood and subsequent immune responses initiate airway remodeling. Numerous studies of the microbiome indicate that intestinal and airway microbiome diversity and composition contribute to the cause of asthma and allergic diseases., Summary: Susceptibility to asthma and allergic diseases is complex and involves genetic variants and environmental exposures (bacteria, viruses, smoking, and pet ownership), alteration of our microbiome and potentially large-scale manipulation of the environment over the past century.

Respiratory diseases are the most frequent chronic illnesses in babies and children. Although a vigorous innate immune system is critical for maintaining lung health, a balanced response is essential to minimize damaging inflammation. We investigated the functional and clinical impact of human genetic variants in the promoter of NFKBIA, which encodes IκBα, the major negative regulator of NF-κB. In this study, we quantified the functional impact of NFKBIA promoter polymorphisms (rs3138053, rs2233406, and rs2233409) on promoter-driven protein expression, allele-specific and total NFKBIA mRNA expression, IκBα protein expression, and TLR responsiveness; mapped innate immune regulatory networks active during respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia; and genotyped and analyzed independent cohorts of children with respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia. Genetic variants in the promoter of NFKBIA influenced NFKBIA gene expression, IκBα protein expression, and TLR-mediated inflammatory responses. Using a systems biology approach, we demonstrated that NFKBIA/IκBα is a central hub in transcriptional responses of prevalent childhood lung diseases, including respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia. Finally, by examining independent pediatric lung disease cohorts, we established that this immunologically relevant genetic variation in the promoter of NFKBIA is associated with differential susceptibility to severe bronchiolitis following infection with respiratory syncytial virus, airway hyperresponsiveness, and severe bronchopulmonary dysplasia. These data highlight the importance of negative innate immune regulators, such as NFKBIA, in pediatric lung disease and begin to unravel common aspects in the genetic predisposition to bronchopulmonary dysplasia, bronchiolitis, and childhood asthma.

Background: IgE is both a marker and mediator of allergic inflammation. Despite reported differences in serum total IgE levels by race-ethnicity, African American and Latino subjects have not been well represented in genetic studies of total IgE., Objective: We sought to identify the genetic predictors of serum total IgE levels., Methods: We used genome-wide association data from 4292 subjects (2469 African Americans, 1564 European Americans, and 259 Latinos) in the EVE Asthma Genetics Consortium. Tests for association were performed within each cohort by race-ethnic group (ie, African American, Latino, and European American) and asthma status. The resulting P values were meta-analyzed, accounting for sample size and direction of effect. Top single nucleotide polymorphism associations from the meta-analysis were reassessed in 6 additional cohorts comprising 5767 subjects., Results: We identified 10 unique regions in which the combined association statistic was associated with total serum IgE levels (P<5.0×10(-6)) and the minor allele frequency was 5% or greater in 2 or more population groups. Variant rs9469220, corresponding to HLA-DQB1, was the single nucleotide polymorphism most significantly associated with serum total IgE levels when assessed in both the replication cohorts and the discovery and replication sets combined (P=.007 and 2.45×10(-7), respectively). In addition, findings from earlier genome-wide association studies were also validated in the current meta-analysis., Conclusion: This meta-analysis independently identified a variant near HLA-DQB1 as a predictor of total serum IgE levels in multiple race-ethnic groups. This study also extends and confirms the findings of earlier genome-wide association analyses in African American and Latino subjects., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: The innate immune system is essential for host survival because of its ability to recognize invading pathogens and mount defensive responses., Objectives: We sought to identify genetic associations of innate immunity genes with atopy and asthma and interactions with early viral infections (first 12 months of life) in a high-risk birth cohort., Methods: Three Canadian family-based studies and 1 Australian population-based case-control study (n = 5565) were used to investigate associations of 321 single nucleotide polymorphisms (SNPs) in 26 innate immunity genes with atopy, asthma, atopic asthma, and airway hyperresponsiveness. Interactions between innate immunity genes and early viral exposure to 3 common viruses (parainfluenza, respiratory syncytial virus, and picornavirus) were examined in the Canadian Asthma Primary Prevention Study by using both an affected-only family-based transmission disequilibrium test and case-control methods., Results: In a joint analysis of all 4 cohorts, IL-1 receptor 2 (IL1R2) and Toll-like receptor 1 (TLR1) SNPs were associated with atopy after correction for multiple comparisons. In addition, an NFKBIA SNP was associated with atopic asthma. Six SNPs (rs1519309 [TLR3], rs740044 [ILIR2], rs4543123 [TLR1], rs5741812 [LBP], rs917998 [IL18RAP], and rs3136641 [NFKBIB]) were significant (P < .05, confirmed with 30,000 permutations) in both the combined analysis of main genetic effects and SNP-virus interaction analyses in both case-control and family-based methods. The TLR1 variant (rs4543123) was associated with both multiple viruses (respiratory syncytial virus and parainfluenza virus) and multiple phenotypes., Conclusion: We have identified novel susceptibility genes for asthma and related traits and interactions between these genes and early-life viral infections., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Genome-wide association studies (GWAS) have identified loci reproducibly associated with pulmonary diseases; however, the molecular mechanism underlying these associations are largely unknown. The objectives of this study were to discover genetic variants affecting gene expression in human lung tissue, to refine susceptibility loci for asthma identified in GWAS studies, and to use the genetics of gene expression and network analyses to find key molecular drivers of asthma. We performed a genome-wide search for expression quantitative trait loci (eQTL) in 1,111 human lung samples. The lung eQTL dataset was then used to inform asthma genetic studies reported in the literature. The top ranked lung eQTLs were integrated with the GWAS on asthma reported by the GABRIEL consortium to generate a Bayesian gene expression network for discovery of novel molecular pathways underpinning asthma. We detected 17,178 cis- and 593 trans- lung eQTLs, which can be used to explore the functional consequences of loci associated with lung diseases and traits. Some strong eQTLs are also asthma susceptibility loci. For example, rs3859192 on chr17q21 is robustly associated with the mRNA levels of GSDMA (P = 3.55 × 10(-151)). The genetic-gene expression network identified the SOCS3 pathway as one of the key drivers of asthma. The eQTLs and gene networks identified in this study are powerful tools for elucidating the causal mechanisms underlying pulmonary disease. This data resource offers much-needed support to pinpoint the causal genes and characterize the molecular function of gene variants associated with lung diseases., Competing Interests: The authors have declared that no competing interests exist.

Background: Genetic variants at the vitamin D receptor (VDR) locus are associated with asthma and atopy. We hypothesized that polymorphisms in other genes of the vitamin D pathway are associated with asthma or atopy., Methods: Eleven candidate genes were chosen for this study, five of which code for proteins in the vitamin D metabolism pathway (CYP27A1, CYP27B1, CYP2R1, CYP24A1, GC) and six that are known to be transcriptionally regulated by vitamin D (IL10, IL1RL1, CD28, CD86, IL8, SKIIP). For each gene, we selected a maximally informative set of common SNPs (tagSNPs) using the European-derived (CEU) HapMap dataset. A total of 87 SNPs were genotyped in a French-Canadian family sample ascertained through asthmatic probands (388 nuclear families, 1064 individuals) and evaluated using the Family Based Association Test (FBAT) program. We then sought to replicate the positive findings in four independent samples: two from Western Canada, one from Australia and one from the USA (CAMP)., Results: A number of SNPs in the IL10, CYP24A1, CYP2R1, IL1RL1 and CD86 genes were modestly associated with asthma and atopy (p < 0.05). Two-gene models testing for both main effects and the interaction were then performed using conditional logistic regression. Two-gene models implicating functional variants in the IL10 and VDR genes as well as in the IL10 and IL1RL1 genes were associated with asthma (p < 0.0002). In the replicate samples, SNPs in the IL10 and CYP24A1 genes were again modestly associated with asthma and atopy (p < 0.05). However, the SNPs or the orientation of the risk alleles were different between populations. A two-gene model involving IL10 and VDR was replicated in CAMP, but not in the other populations., Conclusion: A number of genes involved in the vitamin D pathway demonstrate modest levels of association with asthma and atopy. Multilocus models testing genes in the same pathway are potentially more effective to evaluate the risk of asthma, but the effects are not uniform across populations.

Background: The epithelial cell-derived protein thymic stromal lymphopoietin stimulates dendritic and mast cells to promote proallergic T(H)2 responses. Studies of transgenic expression of thymic stromal lymphopoietin and its receptor knockout mice have emphasized its critical role in the development of allergic inflammation. Association of genetic variation in thymic stromal lymphopoietin with IgE levels has been reported for human subjects., Objective: The aim of this study was to evaluate the relationship between variants of thymic stromal lymphopoietin and asthma and related phenotypes., Methods: We selected 6 single nucleotide polymorphisms in thymic stromal lymphopoietin and genotyped 5565 individuals from 4 independent asthma studies and tested for association with asthma, atopy, atopic asthma, and airway hyperresponsiveness by using a general allelic likelihood ratio test. P values were corrected for the effective number of independent single nucleotide polymorphisms and phenotypes., Results: The A allele of rs1837253, which is 5.7 kb upstream of the transcription start site of the gene, was associated with protection from asthma, atopic asthma, and airway hyperresponsiveness, with the odds ratios and corrected P values for each being 0.79 and 0.0058; 0.75 and 0.0074; and 0.76 and 0.0094, respectively. Associations between thymic stromal lymphopoietin and asthma-related phenotypes were the most statistically significant observations in our study, which has to date examined 98 candidate genes. Full results are available online at http://genapha.icapture.ubc.ca/., Conclusions: A genetic variant in the region of the thymic stromal lymphopoietin gene is associated with the phenotypes of asthma and airway hyperresponsiveness.

Background: T-cell immunoglobulin mucin-3 (TIM3) is a TH1-specific type 1 membrane protein that regulates TH1 proliferation and the development of immunological tolerance. TIM3 and its genetic variants have been suggested to play a role in regulating allergic diseases. Polymorphisms in the TIM3 promoter region have been reported to be associated with allergic phenotypes in several populations. The aims of this study were to examine whether genetic variation in the promoter region of TIM3 influenced transcription of the gene and risk for allergic phenotypes., Methods: We performed 5' rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction. We screened for polymorphisms in the promoter region. Deletion analysis was used to localize the promoter region of TIM3. Genotyping was performed by TaqMan assays in three asthma/allergy population samples., Results: We found two regions with promoter activity in TIM3. One region was from -214 bp to +58 bp and the other from -1.6 kb to -914 bp relative to the transcription start site. None of the single nucleotide polymorphisms (SNPs) or haplotypes affected the transcriptional activity in reporter gene assays. No association between the SNPs and any phenotype was observed in the study cohorts., Conclusion: Our findings indicate that SNPs and haplotypes in the TIM3 promoter region do not have a functional effect in vitro and are not associated with allergic diseases. These data suggest that polymorphisms in the TIM3 promoter region are unlikely to play an important role in susceptibility to allergic diseases.

Asthma, atopy, and related phenotypes are heterogeneous complex traits, with both genetic and environmental risk factors. Extensive research has been conducted and over hundred genes have been associated with asthma and atopy phenotypes, but many of these findings have failed to replicate in subsequent studies. To separate true associations from false positives, candidate genes need to be examined in large well-characterized samples, using standardized designs (genotyping, phenotyping and analysis). In an attempt to replicate previous associations we amalgamated the power and resources of four studies and genotyped 5,565 individuals to conduct a genetic association study of 93 previously associated candidate genes for asthma and related phenotypes using the same set of 861 single-nucleotide polymorphisms (SNPs), a common genotyping platform, and relatively harmonized phenotypes. We tested for association between SNPs and the dichotomous outcomes of asthma, atopy, atopic asthma, and airway hyperresponsiveness using a general allelic likelihood ratio test. No SNP in any gene reached significance levels that survived correction for all tested SNPs, phenotypes, and genes. Even after relaxing the usual stringent multiple testing corrections by performing a gene-based analysis (one gene at a time as if no other genes were typed) and by stratifying SNPs based on their prior evidence of association, no genes gave strong evidence of replication. There was weak evidence to implicate the following: IL13, IFNGR2, EDN1, and VDR in asthma; IL18, TBXA2R, IFNGR2, and VDR in atopy; TLR9, TBXA2R, VDR, NOD2, and STAT6 in airway hyperresponsiveness; TLR10, IFNGR2, STAT6, VDR, and NPSR1 in atopic asthma. Additionally we found an excess of SNPs with small effect sizes (OR < 1.4). The low rate of replication may be due to small effect size, differences in phenotypic definition, differential environmental effects, and/or genetic heterogeneity. To aid in future replication studies of asthma genes a comprehensive database was compiled and is available to the scientific community at http://genapha.icapture.ubc.ca/.

Background: Eight genes in the immune signaling pathway shown to be differentially expressed in asthmatic lung biopsy specimens in a previous microarray experiment were selected as candidate genes for asthma susceptibility., Objective: We sought to perform an association study with these genes and asthma-related phenotypes in 3 independent Canadian familial asthma collections and 1 Australian asthma case-control study., Methods: Tagging single nucleotide polymorphisms were selected by using the HapMap public database (r(2) > 0.8; minor allele frequency >0.10) and genotyped with the Illumina platform. Family-based association and trend tests for asthma, atopy, airway hyperresponsiveness, and allergic asthma phenotypes were done in each sample, correcting for multiple testing., Results: Uncorrected associations with polymorphisms within 7 genes were detected with 1 or more of the phenotypes in 1 or more of the 4 populations (.001

Rationale: Urokinase plasminogen activator (uPA) interacts with its receptor on inflammatory and migrating cells to regulate extracellular matrix degradation, cell adhesion, and inflammatory cell activation. It is necessary for the development of an appropriate immune response and is involved in tissue remodeling. The PLAU gene codes for this enzyme, and is located on 10q24. This region has demonstrated evidence for linkage in a genome scan for asthma in a sample from northeastern Quebec. Here, we hypothesized that uPA may function as a regulator of asthma susceptibility., Objectives: To test for association between asthma and genetic variants of PLAU., Methods: We sequenced PLAU and tested for genetic association between identified variants and asthma-related traits in a French-Canadian familial collection (231 families, 1,139 subjects). Additional association studies were performed in two other family-based Canadian cohorts (Canadian Asthma Primary Prevention Study [CAPPS], 238 trios; and Study of Asthma Genes and the Environment [SAGE], 237 trios)., Measurements and Main Results: In the original sample, under the dominant model, the common alleles, rs2227564C (P141) and rs2227566T, were associated with asthma (p = 0.011 and 0.045, respectively) and with airway hyperresponsiveness (AHR) (p = 0.026 and 0.038, respectively). Analysis of the linkage disequilibrium pattern also revealed association of the common haplotype for asthma, atopy, and AHR (p = 0.031, 0.043, and 0.006, respectively). Whereas no significant association was detected for PLAU single-nucleotide polymorphisms in the CAPPS cohort, association was observed in the SAGE cohort between the rs4065C allele and atopy under additive (p = 0.005) and dominant (p = 0.0001) genetic models., Conclusions: This suggests a role for the uPA pathway in the pathogenesis of the disease.