A functional genomics pipeline to identify high-value asthma and allergy CpGs in the human methylome

BACKGROUND: DNA methylation of cytosines at cytosine-phosphate-guanine (CpG) dinucleotides (CpGs) is a widespread epigenetic mark, but genome-wide variation has been relatively unexplored due to the limited representation of variable CpGs on commercial high-throughput arrays. OBJECTIVES: To explore this hidden portion of the epigenome, this study combined whole-genome bisulfite sequencing with in silico evidence of gene regulatory regions to design a custom array of high-value CpGs. This study focused on airway epithelial cells from children with and without allergic asthma because these cells mediate the effects of inhaled microbes, pollution, and allergens on asthma and allergic disease risk. METHODS: This study identified differentially methylated regions from whole-genome bisulfite sequencing in nasal epithelial cell DNA from a total of 39 children with and without allergic asthma of both European and African ancestries. This study selected CpGs from differentially methylated regions, previous allergy or asthma epigenome-wide association studies (EWAS), or genome-wide association study loci, and overlapped them with functional annotations for inclusion on a custom Asthma&Allergy array. This study used both the custom and EPIC arrays to perform EWAS of allergic sensitization (AS) in nasal epithelial cell DNA from children in the URECA (Urban Environment and Childhood Asthma) birth cohort and using the custom array in the INSPIRE [Infant Susceptibility to Pulmonary Infections and Asthma Following RSV Exposure] birth cohort. Each CpG on the arrays was assigned to its nearest gene and its promotor capture Hi-C interacting gene and performed expression quantitative trait methylation (eQTM) studies for both sets of genes. RESULTS: Custom array CpGs were enriched for intermediate methylation levels compared to EPIC CpGs. Intermediate methylation CpGs were further enriched among those associated with AS and for eQTMs on both arrays. CONCLUSIONS: This study revealed signature features of high-value CpGs and evidence for epigenetic regulation of genes at AS EWAS loci that are robust to race/ethnicity, ascertainment, age, and geography

Gene-based association study of rare variants in children of diverse ancestries implicates TNFRSF21 in the development of allergic asthma

BACKGROUND: Most genetic studies of asthma and allergy have focused on common variation in individuals primarily of European ancestry. Studying the role of rare variation in quantitative phenotypes and in asthma phenotypes in populations of diverse ancestries can provide additional, important insights into the development of these traits. OBJECTIVE: We sought to examine the contribution of rare variants to different asthma- or allergy-associated quantitative traits in children with diverse ancestries and explore their role in asthma phenotypes. METHODS: We examined whole-genome sequencing data from children participants in longitudinal studies of asthma (n = 1035; parent-identified as 67% Black and 25% Hispanic) to identify rare variants (minor allele frequency \u3c 0.01). We assigned variants to genes and tested for associations using an omnibus variant-set test between each of 24,902 genes and 8 asthma-associated quantitative traits. On combining our results with external data on predicted gene expression in humans and mouse knockout studies, we identified 3 candidate genes. A burden of rare variants in each gene and in a combined 3-gene score was tested for its associations with clinical phenotypes of asthma. Finally, published single-cell gene expression data in lower airway mucosal cells after allergen challenge were used to assess transcriptional responses to allergen. RESULTS: Rare variants in USF1 were significantly associated with blood neutrophil count (P = 2.18 × 10 CONCLUSIONS: We report novel associations between rare variants in genes and allergic and inflammatory phenotypes in children with diverse ancestries, highlighting TNFRSF21 as contributing to the development of allergic asthma

Childhood Allergy and the NeOnatal Environment (CANOE) Research Protocol and Recruitment Redesign during the COVID-19 Pandemic

Rationale: Recruitment for research studies is a challenging endeavor that has been further complicated by the COVID-19 pandemic. While clinical research was temporarily halted due to the pandemic, it was hypothesized that study and recruitment restructuring would enable brisk enrollment when research resumed. Methods: A new NIH/ECHO-supported multi-center birth cohort, “Childhood Allergy and the NeOnatal Environment” (CANOE) was launched in January 2019 across four sites to determine how pre-, peri-, and post-natal factors influence development of recurrent wheezing and atopic dermatitis. Study recruitment was halted for nine months due to the COVID-19 pandemic, during which recruitment and study procedures were redesigned. Results: Recruitment strategies were modified to limit in-person contact, shifting toward alternative HIPAA-compliant methods like clinician referrals, institutional social media, and telemedicine consenting. Protocol changes included reducing frequency of in-person visits, leveraging clinical care visits to collect bio-samples, expanded self-collection of samples at home, and posting study materials online. Recruitment rates range from 3-12 families per month per site. In-clinic recruitment with modifications for social distancing has been successful across all sites. Other successful strategies have included targeted social media posts, mailed letters, and email. Rates of consent have been similar across recruitment strategies and the implementation of multiple recruitment strategies has yielded the highest rates of ongoing consent and enrollment of mother-infant dyads. Conclusions: Study procedures that prioritize health and safety measures such as social distancing, study participant convenience, and diversification of recruitment strategies enable continued birth cohort recruitment and data collection while adhering to public health restrictions during the pandemic

Examining virtual research recruitment and participant diversity in a multi-center birth cohort, Childhood Allergy and the NeOnatal Environment (CANOE)

Rationale: Recruitment for a NIH/ECHO-supported multi-center birth cohort, “Childhood Allergy and the NeOnatal Environment” (CANOE) stopped due to the COVID-19 pandemic. Redesign of study procedures emphasized virtual and socially distanced activities. We hypothesized that “virtual” recruitment methods (social media, websites, email) would surpass “traditional” methods (in-clinic, telephone, flyers/print materials) and increase enrollment of families from diverse backgrounds and communities. Methods: Pregnant women (n=439, target 500) were recruited from four academic medical centers in Detroit MI, Madison WI, Nashville TN, and St. Louis MO. We collected demographic and social information by questionnaires and examined race, ethnicity, age, parity, and employment status in relation to recruitment method using chi-square tests. Results: In-clinic and telephone recruitment comprised 55% of enrollment, followed by print materials (17%), and social media and email (15%). The cohort includes families self-identifying as Caucasian/White (63%), African American/Black (27%), Hispanic/Latino (3.3%), Asian (3.5%), and mixed races (1.2%). This reflects site demographics for White and Black patients, while other populations are not as well recruited into this cohort. Recruitment method success did not vary by race, ethnicity, maternal age, or employment status (p=ns for each comparison). Most (63%) multigravida mothers (9.1% of participants) were recruited in clinic, while primigravida participants were recruited more evenly via all methods. Conclusions: “Virtual” recruitment methods comprised a smaller proportion of cohort enrollment than hypothesized and study recruitment method did not vary by race/ethnicity; however, consideration of combined, varied, and novel recruitment methods may add to the development of best practices for more representative research study recruitment

Longitudinal Characterization of Atopic Dermatitis Phenotypes in The Children\u27s Respiratory and Environmental Workgroup (CREW) Birth Cohort Consortium

Rationale: Previously identified longitudinal patterns of atopic dermatitis (AD) may lack generalizability and precision due to small sample size and limited time points. We identify and describe longitudinal AD phenotypes in a large consortium study. Methods: Data from 11 birth cohorts across the United States from the CREW (Children’s Respiratory and Environmental Workgroup) consortium were harmonized to determine physician diagnosis of AD in each year of life from 0-7 years of age (N=7,900). AD phenotypes were identified using Longitudinal Latent Class Analysis, and relationships with demographic variables were determined using multinomial logistic regression with a 3-step procedure to account for uncertainty in class membership. Results: We identified 5 classes of AD expression, selected based on model fit, interpretability, and clinical utility: Persistent AD (15.4%), Early AD with Potential Reoccurrence (2.7%), Late-Onset AD (7.0%), Transient Early AD (3.0%), and Minimal/No AD (72.0%). Males had significantly higher odds of Persistent AD (OR [95% CI]=1.47 [1.22, 1.75]) and Early AD with Potential Reoccurrence (OR [95% CI]=1.89 [1.19, 2.94]). Relative to White children, Black children had higher odds of Persistent AD (OR [95% CI]=2.50 [2.05, 3.05]), Early AD with Potential Reoccurrence (OR [95% CI]=3.07 [1.94, 4.85]), and Transient Early AD (OR [95% CI]=4.12 [2.62, 6.48]). Conclusions: Five AD phenotypes exist in a diverse national sample of children. Black children and males are at increased risk of early and persistent AD. These findings illustrate potential risk factors to target AD prevention

Longitudinal assessment of Allergic Outcomes and Atopic Dermatitis Phenotypes in The Children\u27s Respiratory and Environmental Workgroup (CREW) Birth Cohort Consortium

Rationale: Atopic dermatitis (AD) is a heterogenous inflammatory skin disease often associated with other allergic diseases. We characterized AD phenotypes and associated allergic outcomes longitudinally across a multi-site consortium. Methods: AD expression in 11 U.S. birth cohorts from the CREW (Children’s Respiratory and Environmental Workgroup) consortium was assessed in each year of life from age 0-7 years (N=7,900). Longitudinal Latent Class Analysis was performed to identify AD phenotypes. Five classes of AD were identified: Persistent AD (15.4%), Early AD with Potential Reoccurrence (2.7%), Late-Onset AD (7.0%), Transient Early AD (3.0%), and Minimal/No AD (72.0%). Serum allergen sensitization patterns and allergic clinical disease were associated with AD phenotype using multinomial logistic regression with a 3-step procedure to account for uncertainty in class membership. Results: Children with Persistent AD, Early AD with Potential Reoccurrence, and Transient Early AD were more likely to have food allergy compared to those with Minimal/No AD (OR[95% CI]=2.73[2.15, 3.45], 2.69[1.63, 4.45], 2.54[1.55, 4.16], respectively). These groups had similarly higher odds of food sensitization. Persistent AD (OR[95% CI]=1.81[1.48, 2.21]) and Early AD with Potential Reoccurrence (OR[95% CI]=3.66[1.90, 7.05]) had significantly higher odds of ever asthma relative to Minimal/No AD. At both 2-4 years and 5-7 years, persistent AD (OR[95% CI]=1.35[1.04, 1.74], 1.25[1.01, 1.53]) and Late-Onset AD (OR[95% CI]=1.68[1.13, 2.50], 2.22[1.33, 3.70]) relative to Minimal/No AD had higher odds of allergic rhinitis. Conclusions: Longitudinal AD phenotypes had varying associations with allergic sensitization, food allergy, asthma and allergic rhinitis, demonstrating the heterogeneity of allergic comorbidity risk associated with AD

The Pediatric Asthma Risk Score: A New Gold Standard for Asthma Prediction

Rationale: Early prediction of asthma is critical to identify potential primary prevention strategies. The Pediatric Asthma Risk Score (PARS) is a continuous score to predict early-life asthma but was developed and validated in relatively homogenous populations. We compared PARS directly to the Asthma Predictive Index (API) and validated in 10 cohorts with varying race, ethnicity, sex, cohort type, missing data and birth decades, and perform a meta-analysis across all 10 cohorts. Methods: We utilized data from 5674 children participating in the Children’s Respiratory and Environmental Workgroup. We applied both PARS and the API in each cohort, as well as harmonized across all cohorts, and directly compared the ability of each tool to predict asthma development at ages 5-10. Results: The PARS area under the curve (AUC) was significantly higher than the AUC of the API in 9 cohorts (p-value range 0.01 - \u3c0.001). The PARS AUC did not differ by cohort type (high risk or general population), decade of enrollment, race, sex, ethnicity, missing PARS factors or polysensitization definition (skin prick test vs. specific IgE). The weights of the 6 PARS factors in the meta-analysis were very similar to the original weights, validating the original PARS scoring. Conclusions: This multi-cohort study makes the PARS the most validated model of asthma prediction in children to date, not only with respect to the number of cohorts used but also with regards to capturing the diversity of asthma in the United States. Future studies may consider PARS the new gold standard in pediatric asthma risk prediction

Distinct Airway Inflammatory Pathways Associated with Asthma Exacerbations are Modulated by Mepolizumab Therapy in Children

Rationale: Identification of specific airway inflammatory pathways can lead to effective personalized treatment with biologics in asthma and insights to mechanisms of action. Methods: 290 urban children with exacerbation-prone asthma and ≥150/mm3 blood eosinophils were randomized (1:1) to placebo or mepolizumab added to guideline-based care. Nasal lavage samples were collected at randomization and during treatment for RNA-sequencing, and analyzed by cell-deconvolution modular analysis to assess genome-wide expression patterns associated with exacerbation number and effect of treatment. Results: Mepolizumab significantly reduced the frequency of exacerbations compared to placebo. At randomization, there were no differences in expression between treatment groups; multiple modules were subsequently differentially expressed during mepolizumab but not placebo treatment. Furthermore, expression levels of multiple modules were associated with the exacerbation number during the study, with distinct relationships observed in the placebo and/or mepolizumab groups. Notably, higher expression at randomization of an eosinophil-associated module enriched for Type-2 genes including IL4, IL5, and IL13, was associated with increased exacerbations in placebo (β=0.19, p\u3c0.001), but not mepolizumab-treated children (interaction p\u3c0.01). Furthermore, mepolizumab treatment reduced expression of this module (Fold-change=0.62, p\u3c0.001). In contrast, higher expression at randomization of an eosinophil-associated module enriched for eosinophil activation (e.g. CD9) and mucus hypersecretion (e.g. MUC5AC) genes was associated with exacerbation number in both groups throughout the study (β=0.18, p\u3c0.01) and was unaltered by mepolizumab therapy. Conclusions: Multiple distinct airway inflammation patterns were identified associated with exacerbation frequency. These findings identify inflammatory endotypes and indicate likelihood and potential mechanisms of a beneficial clinical response to mepolizumab therapy to prevent exacerbations

The Effect of Subcutaneous German Cockroach Immunotherapy (SCIT) on Nasal Allergen Challenge (NAC) and Cockroach-specific Antibody Responses Among Urban Children and Adolescents

Rationale: Cockroach allergy contributes to asthma and rhinitis morbidity among many urban children. Treatment with cockroach SCIT could be beneficial. Methods: 8-17 year-old children with mild-moderate asthma from 11 urban sites participated in a randomized double-blind placebo-controlled SCIT trial using non-standardized, glycerinated German cockroach extract. Positive cockroach skin tests, cockroach-specific IgE, and nasal challenge response with total nasal symptom scores (TNSS) ≥6 or maximal sneeze scores of 3 during a graded NAC were required for enrollment. Following dose escalation, 0.4 ml of undiluted extract was targeted for maintenance dosing (∼7 mcg Bla g2/dose). The primary endpoint was change in NAC-induced mean TNSS from baseline to one year post randomization. Changes in cockroach-specific IgE (CRsIgE) and IgG4 (CRsIgG4) were also analyzed. Results: Mean TNSS did not significantly change from baseline in either group (placebo n=29, SCIT n=28). There was no significant difference in the change in mean TNSS between placebo and SCIT [−0.79±0.35 vs. −1.02±0.37, respectively, difference=0.2(−1.15, 0.70), p=0.63]. Baseline CRsIgE and CRsIgG4 didn’t differ between groups. Mean CRsIgE decreased in both groups following treatment: 3.6 to 2.3 kU/L (0.64 fold change), p=0.015 and 8.3 to 4.2 kU/L (0.51 fold change), p\u3c0.001 in placebo and SCIT respectively, but did not differ between groups [p=0.33]. Significant increases in CRsIgG4 post-treatment were observed among SCIT recipients only: 0.07 to 12.3 mg/L (176 fold change), p\u3c0.001. Conclusions: Cockroach SCIT increased CRsIgG4 levels but did not significantly alter NAC-induced TNSS responses. The extent to which NAC in these children may reflect clinical efficacy for rhinitis or asthma is uncertain

Phenotype-directed Therapy with Mepolizumab for Urban Children with Exacerbation-Prone Asthma

Rationale: Asthma exacerbations are common in urban children and have significant short- and long-term consequences. Elevated peripheral blood and airway eosinophils have been identified as risk factors for exacerbations, and therapies targeting these biomarkers reduce exacerbations in adults; however, data on anti-eosinophil treatment in children and adolescents are limited. The primary objective of this study is to determine if phenotype-directed use of mepolizumab reduces the rate of asthma exacerbations in urban children. Methods: Urban children 6-17 years of age (n=290) with exacerbation-prone asthma (2+ exacerbations in previous year) and blood eosinophils ≥150/mm3 were randomized 1:1 to mepolizumab (6-11 years: 40 mg; 12-17 years: 100 mg) or placebo every 4 weeks added to guideline-based care for 1 year. The primary outcome was the number of asthma exacerbations treated with systemic corticosteroids; a comparison of the two treatment groups was evaluated using a negative-binomial model. Results: Mepolizumab significantly reduced peripheral blood eosinophils (p\u3c0.01) and nasal eosinophils (p\u3c0.01). The rate of asthma exacerbations was significantly lower in mepolizumab (0.96 exacerbations/year) vs. placebo (1.30 exacerbations/year) treated participants [relative risk 0.73 (95% confidence interval 0.56-0.96), p=0.027]. There were no significant differences in secondary outcomes, including time to first exacerbation, lung function, quality of life, or composite asthma severity index (CASI). Post hoc, the time to second asthma exacerbation increased significantly with mepolizumab (p=0.02). Adverse events were similar between groups. Conclusions: Phenotype-directed therapy with mepolizumab in urban children and adolescents with exacerbation-prone eosinophilic asthma significantly reduced recurrent exacerbations and was well tolerated, but did not impact other asthma outcomes