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Background: Epigenetic mechanisms, including methylation, can contribute to childhood asthma. Identifying DNA methylation profiles in asthmatic patients can inform disease pathogenesis. Objective: We sought to identify differential DNA methylation in newborns and children related to childhood asthma. Methods: Within the Pregnancy And Childhood Epigenetics consortium, we performed epigenome-wide meta-analyses of school-age asthma in relation to CpG methylation (Illumina450K) in blood measured either in newborns, in prospective analyses, or cross-sectionally in school-aged children. We also identified differentially methylated regions. Results: In newborns (8 cohorts, 668 cases), 9 CpGs (and 35 regions) were differentially methylated (epigenome-wide significance, false discovery rate < 0.05) in relation to asthma development. In a cross-sectional meta-analysis of asthma and methylation in children (9 cohorts, 631 cases), we identified 179 CpGs (false discovery rate < 0.05) and 36 differentially methylated regions. In replication studies of methylation in other tissues, most of the 179 CpGs discovered in blood replicated, despite smaller sample sizes, in studies of nasal respiratory epithelium or eosinophils. Pathway analyses highlighted enrichment for asthma-relevant immune processes and overlap in pathways enriched both in newborns and children. Gene expression correlated with methylation at most loci. Functional annotation supports a regulatory effect on gene expression at many asthma-associated CpGs. Several implicated genes are targets for approved or experimental drugs, including IL5RA and KCNH2. Conclusion: Novel loci differentially methylated in newborns represent potential biomarkers of risk of asthma by school age. Cross-sectional associations in children can reflect both risk for and effects of disease. Asthma-related differential methylation in blood in children was substantially replicated in eosinophils and respiratory epithelium. more...

© 2018 The Author(s). Atopic asthma is a persistent disease characterized by intermittent wheeze and progressive loss of lung function. The disease is thought to be driven primarily by chronic aeroallergen-induced type 2-associated inflammation. However, the vast majority of atopics do not develop asthma despite ongoing aeroallergen exposure, suggesting additional mechanisms operate in conjunction with type 2 immunity to drive asthma pathogenesis. We employed RNA-Seq profiling of sputum-derived cells to identify gene networks operative at baseline in house dust mite-sensitized (HDM S ) subjects with/without wheezing history that are characteristic of the ongoing asthmatic state. The expression of type 2 effectors (IL-5, IL-13) was equivalent in both cohorts of subjects. However, in HDM S -wheezers they were associated with upregulation of two coexpression modules comprising multiple type 2- and epithelial-associated genes. The first module was interlinked by the hubs EGFR, ERBB2, CDH1 and IL-13. The second module was associated with CDHR3 and mucociliary clearance genes. Our findings provide new insight into the molecular mechanisms operative at baseline in the airway mucosa in atopic asthmatics undergoing natural aeroallergen exposure, and suggest that susceptibility to asthma amongst these subjects involves complex interactions between type 2- and epithelial-associated gene networks, which are not operative in equivalently sensitized/exposed atopic non-asthmatics. more...

Background: Vitamin D (25(OH)D) deficiency has been implicated as a possible risk factor for asthma development, but studies at selected time points measuring 25(OH)D levels during childhood have yielded conflicting findings. Prospective studies tracking 25(OH)D levels during the initiation phase of asthma in early childhood have not been reported. Objective: We sought to elucidate relationships between 25(OH)D levels from birth to age 10 years and susceptibility to allergic sensitization, respiratory tract infections, and asthma. Methods: Asthma-, allergy-, and respiratory tract infection-associated phenotypes (including pathogen identification) were characterized in a high-risk birth cohort. Plasma 25(OH)D concentrations were quantified at birth and at clinical follow-ups at the ages of 0.5, 1, 2, 3, 4, 5, and 10 years, and relationships with clinical outcomes were examined. Results: Cross-sectional analyses demonstrated inverse associations between 25(OH)D concentrations and the risk for concurrent sensitization at age 0.5, 2, and 3 years, and mixed-effects regression demonstrated inverse longitudinal associations of 25(OH)D levels with both sensitization and eczema. Multivariate regression modeling suggested that the number of 25(OH)D-deficient follow-ups was positively associated with risk for asthma/wheeze, eczema, and sensitization at 10 years; adjustment for sensitization (particularly by 2 years) in the asthma/wheeze models reduced 25(OH)D associations with these latter outcomes. 25(OH)D levels were also inversely associated with early nasopharyngeal colonization with . Streptococcus species and age of first febrile lower respiratory illness, both of which are known asthma risk factors. Conclusion: 25(OH)D deficiency in early childhood is associated with increased risk for persistent asthma, potentially through modulating susceptibility to early allergic sensitization, upper respiratory tract colonization with bacterial pathogens, or both. These relationshi more...

Background and objective: Mannitol challenge testing is an established tool for clinical asthma diagnosis, and can be performed outside of specialized respiratory laboratories. Despite applicability in both clinical and non-clinical populations, with different pre-test asthma probabilities, differences in diagnostic properties have not been well explored. This study aimed to quantify the diagnostic utility of mannitol challenge testing for asthma in a community cohort and a symptomatic wheezing subset of this cohort. Methods: During the 22-year follow-up of the Western Australian Pregnancy (Raine) Cohort, 772 participants (384 males) completed mannitol challenge and skin prick testing and respiratory health questionnaires, of whom 148 reporting wheeze in the past 12 months were included in a wheezing subset. Results: Responsiveness to mannitol had low sensitivity (19%) and high specificity (97%) to identify current asthma in the complete cohort, with positive and negative predictive values (PPV and NPV) of 45% and 92%, respectively. Within the wheezing subset, sensitivity (19%) and specificity (94%) remained similar, but PPV increased to 79%, and NPV decreased to 52%. Conclusion: Our findings support previously reported high specificity and good PPV for mannitol challenge testing in symptomatic wheezing populations, and highlight the need for caution when interpreting mannitol test results in non-clinical populations. more...

The nasopharynx (NP) is a reservoir for microbes associated with acute respiratory infections (ARIs). Lung inflammation resulting from ARIs during infancy is linked to asthma development. We examined the NP microbiome during the critical first year of life in a prospective cohort of 234 children, capturing both the viral and bacterial communities and documenting all incidents of ARIs. Most infants were initially colonized with Staphylococcus or Corynebacterium before stable colonization with Alloiococcus or Moraxella. Transient incursions of Streptococcus, Moraxella, or Haemophilus marked virus-associated ARIs. Our data identify the NP microbiome as a determinant for infection spread to the lower airways, severity of accompanying inflammatory symptoms, and risk for future asthma development. Early asymptomatic colonization with Streptococcus was a strong asthma predictor, and antibiotic usage disrupted asymptomatic colonization patterns. In the absence of effective anti-viral therapies, targeting pathogenic bacteria within the NP microbiome could represent a prophylactic approach to asthma. more...

Background: Young adulthood is a critical life period for health and health behaviours. Related measurements collected before and after birth, and during childhood and adolescence can provide a life-course analysis of important factors that contribute to health and behaviour in young adulthood. The Western Australian Pregnancy Cohort (Raine) Study has collected a large number of such measurements during the fetal, perinatal, infancy, childhood and adolescence periods and plans to relate them to common health issues and behaviours in young adults, including spinal pain, asthma, sleep disorders, physical activity and sedentary behaviour and, work absenteeism and presenteeism. The aim of this paper is to describe the rationale, design and methods of the 22 year follow-up of the Raine Study cohort. Methods/Design: The Raine Study is a prospective cohort study. Participants still active in the cohort (n = 2,086) were contacted around the time of their 22nd birthday and invited to participate in the 22 year follow-up. Each was asked to complete a questionnaire, attend a research facility for physical assessment and an overnight sleep study, wear activity monitors for a week, and to maintain a sleep and activity diary over this week. The questionnaire was broad and included questions related to sociodemographics, medical history, quality of life, psychological factors, lifestyle factors, spinal pain, respiratory, sleep, activity and work factors. Physical assessments included anthropometry, blood pressure, back muscle endurance, tissue sensitivity, lung function, airway reactivity, allergic status, 3D facial photographs, cognitive function, and overnight polysomnography. Discussion: Describing the prevalence of these health issues and behaviours in young adulthood will enable better recognition of the issues and planning of health care resources. Providing a detailed description of the phenotype of these issues will provide valuable information to help educate health profe more...

Vitamin D has been linked in some studies with atopy- and asthma-associated phenotypes in children with established disease, but its role in disease inception at the community level is less clear. The aim of the present study was to investigate associations between vitamin D status and biological signatures indicative of allergy and asthma development in children aged 6 and 14 years in Perth, WA, Australia (latitude 32° S). Serum vitamin D was assayed in 989 6-yr-olds and 1,380 14-yr-olds from an unselected community birth cohort; 689 subjects were assessed at both ages. Vitamin D levels were assessed as a risk modifier for respiratory and allergic outcomes at both ages, using previously ascertained phenotypic data. The predictive value of vitamin D levels at age 6 yrs for development of clinical phenotypes at age 14 yrs was also examined. Serum vitamin D levels in children of both ages were negatively associated with concurrent allergic phenotypes; sex stratification revealed that this association was restricted mainly to males. Furthermore, vitamin D levels at age 6 yrs were significant predictors of subsequent atopy/asthma-associated phenotypes at age 14 yrs. In an unselected community setting, children (particularly males) with inadequate vitamin D are at increased risk of developing atopy, and subsequently bronchial hyperresponsiveness (BHR) and asthma. In a large unselected cohort, males with inadequate vitamin D at 6 and 14 yrs of age had increased atopy and BHR. Low vitamin D at age 6 yrs was a predictor of atopy and asthma at 14 yrs of age. Copyright©ERS 2011. more...

Background: Atopy and asthma are commonly initiated during early life, and there is increasing interest in the development of preventive treatments for at-risk children. However, effective methods for assessing the level of risk in individual children are lacking. Objective: We sought to identify clinical and laboratory biomarkers in 2-year-olds that are predictive of the risk for persistent atopy and wheeze at age 5 years. Methods: We prospectively studied 198 atopic family history-positive children to age 5 years. Clinical and laboratory assessments related to asthma history and atopy status were undertaken annually; episodes of acute respiratory illness were assessed and classified throughout and graded by severity. Results: Aeroallergen-specific IgE titers cycled continuously within the low range in nonatopic subjects. Atopic subjects displayed similar cycling in infancy but eventually locked into a stable pattern of upwardly trending antibody production and TH2-polarized cellular immunity. The latter was associated with stable expression of IL-4 receptor in allergen-specific TH2 memory responses, which was absent from responses during infancy. Risk for persistent wheeze was strongly linked to early sensitization and in turn to early infection. Integration of these data by means of logistic regression revealed that attaining mite-specific IgE titers of greater than 0.20 kU/L by age 2 years was associated with a 12.7% risk of persistent wheeze, increasing progressively to an 87.2% risk with increasing numbers of severe lower respiratory tract illnesses experienced. Conclusion: The risk for development of persistent wheeze in children can be quantified by means of integration of measures related to early sensitization and early infections. Follow-up studies along similar lines in larger unselected populations to refine this approach are warranted. © 2010 American Academy of Allergy, Asthma & Immunology. more...

Background: Current treatment strategies for asthma in teenagers derive primarily from information on chronic disease in adults. More detailed understanding of risk factors related to teenage asthma might aid in the development of improved preventive and treatment strategies for this age group. Objective: We sought to identify biomarkers associated with asthma phenotypes in teenagers, particularly atopic asthma, and to identify markers that aid in discriminating between atopic subjects at high versus low risk of asthma. Methods: We studied 1380 unselected 14-year-olds and collected data on clinical history, allergic sensitization, and respiratory and immunoinflammatory function. The latter comprised measurements of circulating inflammatory markers and in vitro innate and adaptive immune functions, including house dust mite T-cell responses. We integrated the data into regression models to identify variables most strongly associated with asthma risk and severity among atopic subjects. Results: Eight hundred twenty-seven subjects were atopic, 140 subjects were asthmatic, and 81% of asthmatic subjects were also atopic. We identified asthma risk variables related to atopy intensity, including specific IgE and eosinophil levels, plus an additional series external to the TH2 cascade but that modified risk only in atopic subjects, including IFN-?, IL-10, and IL-12 responses and neutrophil numbers in blood. Moreover, bronchial hyperresponsiveness was associated strongly with atopic but not nonatopic asthma, and the bronchial hyperresponsiveness risk profile was itself dominated by atopy-associated variables. Conclusions: Asthma in teenagers is predominantly driven by atopy acting in concert with a second tier of TH2-independent immunoinflammatory mechanisms, which contribute to pathogenesis only against the background of pre-existing inhalant allergy. © 2009 American Academy of Allergy, Asthma & Immunology. more...

Interferon- (IFN) gene expression is tightly regulated in early life, and exaggerated negative control of IFN production in CD4+ T cells has been associated with risk for subsequent development of atopy. Recent studies have demonstrated hypermethylation of CpG sites in the IFN promoter in neonates, a mechanism which in mice leads to strong suppression of IFN gene transcription. In the present study, the methylation status of six CpG sites in the proximal promoter of the human IFN gene was determined by bisulphite sequencing. Cell populations studied were Th1 or Th2 polarized cell lines derived from neonatal and adult CD4+/CD45RA+ T cells, CD4+ and CD8+ naive T cells from cord blood of children followed to outcome age 2 for assessment of atopy status, and CD4+ and CD8+ naive T cells from 6 yr old and adult atopics and controls. We demonstrate that in vitro differentiation of CD4+ T cells down the Th1 pathway (but not the Th2 pathway) is accompanied by progressive demethylation of CpG sites in the IFN promoter, which is most marked in neonatal cells. Atopy development by age 2 was not associated with variations in methylation patterns in cord blood T cells. However, IFN promoter methylation was reduced in CD8+ T cells from atopic children in the age range in which hyperproduction of IFN as recently been identified as a common feature of the atopic phenotype. The findings demonstrate the potency of IFN promoter methylation as a mechanism for control of human IFN gene expression, particularly during early life. Differential regulation of IFN promoter methylation in T cells may be an important contributory factor in atopy development in childhood, and this possibility warrants further detailed investigation. more...