metaProbiotics: a tool for mining probiotic from metagenomic binning data based on a language model.

Beneficial bacteria remain largely unexplored. Lacking systematic methods, understanding probiotic community traits becomes challenging, leading to various conclusions about their probiotic effects among different publications. We developed language model-based metaProbiotics to rapidly detect probiotic bins from metagenomes, demonstrating superior performance in simulated benchmark datasets. Testing on gut metagenomes from probiotic-treated individuals, it revealed the probioticity of intervention strains-derived bins and other probiotic-associated bins beyond the training data, such as a plasmid-like bin. Analyses of these bins revealed various probiotic mechanisms and bai operon as probiotic Ruminococcaceae's potential marker. In different health-disease cohorts, these bins were more common in healthy individuals, signifying their probiotic role, but relevant health predictions based on the abundance profiles of these bins faced cross-disease challenges. To better understand the heterogeneous nature of probiotics, we used metaProbiotics to construct a comprehensive probiotic genome set from global gut metagenomic data. Module analysis of this set shows that diseased individuals often lack certain probiotic gene modules, with significant variation of the missing modules across different diseases. Additionally, different gene modules on the same probiotic have heterogeneous effects on various diseases. We thus believe that gene function integrity of the probiotic community is more crucial in maintaining gut homeostasis than merely increasing specific gene abundance, and adding probiotics indiscriminately might not boost health. We expect that the innovative language model-based metaProbiotics tool will promote novel probiotic discovery using large-scale metagenomic data and facilitate systematic research on bacterial probiotic effects. The metaProbiotics program can be freely downloaded at https://github.com/zhenchengfang/metaProbiotics.

Association between leisure sedentary behaviors and hypertension risk: A prospective cohort study and two-sample Mendelian randomization analysis in Europeans.

OBJECTIVE: This study aimed to investigate the potential causal relationship between domain-specific sedentary behaviors (including television watching, computer use, and driving) and hypertension risk in European populations. METHODS: Initially, we conducted a multivariable Cox regression analysis to evaluate the associations between domain-specific sedentary behaviors and the risk of developing hypertension using data from 261,829 hypertension-free participants in the UK Biobank. To validate the findings of observational analysis, we employed two-sample univariable mendelian randomization (UVMR) analysis utilizing summary statistics from genome-wide association study conducted on European populations. We then performed multivariable mendelian randomization (MVMR) analysis to account for the influence of the risk factors for hypertension. RESULTS: In this prospective observational analysis, individuals who spent >3 h per day watching television had significantly higher risk of developing hypertension (HR = 1.24, 95% CI: 1.20-1.29, P < 0.001) compared to those who watched television for 0-1 h per day. The mendelian randomization analysis provided consistent evidence for a causal relationship between prolonged television watching time and hypertension risk (OR = 1.45, 95% CI: 1.25-1.69, P < 0.001; all PMVMR < 0.05) in both UVMR and MVMR results. No significant associations were found between computer use, driving behaviors and the risk of hypertension in either the observational or UVMR/MVMR analyses. CONCLUSIONS: These findings provide evidence for a causal effect specifically linking higher television watching time to an increased risk of hypertension and indicate the potential effectiveness of reducing television viewing time as a preventive measure to mitigate the risk of hypertension.

Common and distinct metabolomic markers related to immune aging in Western European and East African populations.

In old age, impaired immunity causes high susceptibility to infections and cancer, higher morbidity and mortality, and poorer vaccination efficiency. Many factors, such as genetics, diet, and lifestyle, impact aging. This study aimed to investigate how immune responses change with age in healthy Dutch and Tanzanian individuals and identify common metabolites associated with an aged immune profile. We performed untargeted metabolomics from plasma to identify age-associated metabolites, and we correlated their concentrations with ex-vivo cytokine production by immune cells, DNA methylation-based epigenetic aging, and telomere length. Innate immune responses were impacted differently by age in Dutch and Tanzanian cohorts. Age-related decline in steroid hormone precursors common in both populations was associated with higher systemic inflammation and lower cytokine responses. Hippurate and 2-phenylacetamide, commonly more abundant in older individuals, were negatively correlated with cytokine responses and telomere length and positively correlated with epigenetic aging. Lastly, we identified several metabolites that might contribute to the stronger decline in innate immunity with age in Tanzanians. The shared metabolomic signatures of the two cohorts suggest common mechanisms of immune aging, revealing metabolites with potential contributions. These findings also reflect genetic or environmental effects on circulating metabolites that modulate immune responses.

Species-level resolution for the vaginal microbiota with short amplicons.

Specific bacterial species have been found to play important roles in human vagina. Achieving high species-level resolution is vital for analyzing vaginal microbiota data. However, contradictory conclusions were yielded from different methodological studies. More comprehensive evaluation is needed for determining an optimal pipeline for vaginal microbiota. Based on the sequences of vaginal bacterial species downloaded from NCBI, we conducted simulated amplification with various primer sets targeting different 16S regions as well as taxonomic classification on the amplicons applying different combinations of algorithms (BLAST+, VSEARCH, and Sklearn) and reference databases (Greengenes2, SILVA, and RDP). Vaginal swabs were collected from participants with different vaginal microecology to construct 16S full-length sequenced mock communities. Both computational and experimental amplifications were performed on the mock samples. Classification accuracy of each pipeline was determined. Microbial profiles were compared between the full-length and partial 16S sequencing samples. The optimal pipeline was further validated in a multicenter cohort against the PCR results of common STI pathogens. Pipeline V1-V3_Sklearn_Combined had the highest accuracy for classifying the amplicons generated from both the NCBI downloaded data (84.20% ± 2.39%) and the full-length sequencing data (95.65% ± 3.04%). Vaginal samples amplified and sequenced targeting the V1-V3 region but merely employing the forward reads (223 bp) and classified using the optimal pipeline, resembled the mock communities the most. The pipeline demonstrated high F1-scores for detecting STI pathogens within the validation cohort. We have determined an optimal pipeline to achieve high species-level resolution for vaginal microbiota with short amplicons, which will facilitate future studies.IMPORTANCEFor vaginal microbiota studies, diverse 16S rRNA gene regions were applied for amplification and sequencing, which affect the comparability between different studies as well as the species-level resolution of taxonomic classification. We conducted comprehensive evaluation on the methods which influence the accuracy for the taxonomic classification and established an optimal pipeline to achieve high species-level resolution for vaginal microbiota with short amplicons, which will facilitate future studies.

European and multi-ancestry genome-wide association meta-analysis of atopic dermatitis highlights importance of systemic immune regulation.

Atopic dermatitis (AD) is a common inflammatory skin condition and prior genome-wide association studies (GWAS) have identified 71 associated loci. In the current study we conducted the largest AD GWAS to date (discovery N = 1,086,394, replication N = 3,604,027), combining previously reported cohorts with additional available data. We identified 81 loci (29 novel) in the European-only analysis (which all replicated in a separate European analysis) and 10 additional loci in the multi-ancestry analysis (3 novel). Eight variants from the multi-ancestry analysis replicated in at least one of the populations tested (European, Latino or African), while two may be specific to individuals of Japanese ancestry. AD loci showed enrichment for DNAse I hypersensitivity and eQTL associations in blood. At each locus we prioritised candidate genes by integrating multi-omic data. The implicated genes are predominantly in immune pathways of relevance to atopic inflammation and some offer drug repurposing opportunities.

Serum Anti-Aging Protein α-Klotho Mediates the Association between Diet Quality and Kidney Function.

Adherence to healthy dietary patterns is associated with a reduced risk of kidney dysfunction. Nevertheless, the age-related mechanisms that underpin the relationship between diet and kidney function remain undetermined. This study aimed to investigate the mediating role of serum α-Klotho, an anti-aging protein, in the link between a healthy diet and kidney function. A cross-sectional study was conducted on a cohort of 12,817 individuals aged between 40 and 79 years who participated in the National Health and Nutrition Examination Survey (NHANES) from 2007 to 2016. For each participant, the Healthy Eating Index 2015 (HEI-2015) score was calculated as a measure of a healthy dietary pattern. Creatinine-based estimated glomerular filtration rate (eGFR) was used to assess kidney function. Multivariable regression models were used to analyze the association between the standardized HEI-2015 score and eGFR after adjusting for potential confounders. Causal mediation analysis was performed to assess whether serum α-Klotho influenced this association. The mean (±SD) eGFR of all individuals was 86.8 ± 19.8 mL/min per 1.73 m2. A high standardized HEI-2015 score was associated with a high eGFR (ß [95% CI], 0.94 [0.64-1.23]; p < 0.001). The mediation analysis revealed that serum α-Klotho accounted for 5.6-10.5% of the association of standardized overall HEI-2015 score, total fruits, whole fruits, greens and beans, and whole grain with eGFR in the NHANES. According to the results from the subgroup analysis, serum α-Klotho exerted a mediating effect in the participants aged 60-79 years and in males. A healthy diet may promote kidney function by up-regulating serum anti-aging α-Klotho. This novel pathway suggests important implications for dietary recommendations and kidney health.

Oxidative stress gene expression, DNA methylation, and gut microbiota interaction trigger Crohn's disease: a multi-omics Mendelian randomization study.

BACKGROUND: Oxidative stress (OS) is a key pathophysiological mechanism in Crohn's disease (CD). OS-related genes can be affected by environmental factors, intestinal inflammation, gut microbiota, and epigenetic changes. However, the role of OS as a potential CD etiological factor or triggering factor is unknown, as differentially expressed OS genes in CD can be either a cause or a subsequent change of intestinal inflammation. Herein, we used a multi-omics summary data-based Mendelian randomization (SMR) approach to identify putative causal effects and underlying mechanisms of OS genes in CD. METHODS: OS-related genes were extracted from the GeneCards database. Intestinal transcriptome datasets were collected from the Gene Expression Omnibus (GEO) database and meta-analyzed to identify differentially expressed genes (DEGs) related to OS in CD. Integration analyses of the largest CD genome-wide association study (GWAS) summaries with expression quantitative trait loci (eQTLs) and DNA methylation QTLs (mQTLs) from the blood were performed using SMR methods to prioritize putative blood OS genes and their regulatory elements associated with CD risk. Up-to-date intestinal eQTLs and fecal microbial QTLs (mbQTLs) were integrated to uncover potential interactions between host OS gene expression and gut microbiota through SMR and colocalization analysis. Two additional Mendelian randomization (MR) methods were used as sensitivity analyses. Putative results were validated in an independent multi-omics cohort from the First Affiliated Hospital of Sun Yat-sen University (FAH-SYS). RESULTS: A meta-analysis from six datasets identified 438 OS-related DEGs enriched in intestinal enterocytes in CD from 817 OS-related genes. Five genes from blood tissue were prioritized as candidate CD-causal genes using three-step SMR methods: BAD, SHC1, STAT3, MUC1, and GPX3. Furthermore, SMR analysis also identified five putative intestinal genes, three of which were involved in gene-microbiota interactions through colocalization analysis: MUC1, CD40, and PRKAB1. Validation results showed that 88.79% of DEGs were replicated in the FAH-SYS cohort. Associations between pairs of MUC1-Bacillus aciditolerans and PRKAB1-Escherichia coli in the FAH-SYS cohort were consistent with eQTL-mbQTL colocalization. CONCLUSIONS: This multi-omics integration study highlighted that OS genes causal to CD are regulated by DNA methylation and host-microbiota interactions. This provides evidence for future targeted functional research aimed at developing suitable therapeutic interventions and disease prevention.

Nasal DNA methylation at three CpG sites predicts childhood allergic disease.

Childhood allergic diseases, including asthma, rhinitis and eczema, are prevalent conditions that share strong genetic and environmental components. Diagnosis relies on clinical history and measurements of allergen-specific IgE. We hypothesize that a multi-omics model could accurately diagnose childhood allergic disease. We show that nasal DNA methylation has the strongest predictive power to diagnose childhood allergy, surpassing blood DNA methylation, genetic risk scores, and environmental factors. DNA methylation at only three nasal CpG sites classifies allergic disease in Dutch children aged 16 years well, with an area under the curve (AUC) of 0.86. This is replicated in Puerto Rican children aged 9-20 years (AUC 0.82). DNA methylation at these CpGs additionally detects allergic multimorbidity and symptomatic IgE sensitization. Using nasal single-cell RNA-sequencing data, these three CpGs associate with influx of T cells and macrophages that contribute to allergic inflammation. Our study suggests the potential of methylation-based allergy diagnosis.

Plasma metabolome predicts trained immunity responses after antituberculosis BCG vaccination.

The antituberculosis vaccine Bacillus Calmette-Guérin (BCG) induces nonspecific protection against heterologous infections, at least partly through induction of innate immune memory (trained immunity). The amplitude of the response to BCG is variable, but the factors that influence this response are poorly understood. Metabolites, either released by cells or absorbed from the gut, are known to influence immune responses, but whether they impact BCG responses is not known. We vaccinated 325 healthy individuals with BCG, and collected blood before, 2 weeks and 3 months after vaccination, to assess the influence of circulating metabolites on the immune responses induced by BCG. Circulating metabolite concentrations after BCG vaccination were found to have a more pronounced impact on trained immunity responses, such as the increase in IL-1ß and TNF-α production upon Staphylococcus aureus stimulation, than on specific adaptive immune memory, assessed as IFN-γ production in response to Mycobacterium tuberculosis. Circulating metabolites at baseline were able to predict trained immunity responses at 3 months after vaccination and enrichment analysis based on the metabolites positively associated with trained immunity revealed enrichment of the tricarboxylic acid (TCA) cycle and glutamine metabolism, both of which were previously found to be important for trained immunity. Several new metabolic pathways that influence trained immunity were identified, among which taurine metabolism associated with BCG-induced trained immunity, a finding validated in functional experiments. In conclusion, circulating metabolites are important factors influencing BCG-induced trained immunity in humans. Modulation of metabolic pathways may be a novel strategy to improve vaccine and trained immunity responses.

Pulmonary Function and Blood DNA Methylation: A Multiancestry Epigenome-Wide Association Meta-analysis.

Rationale: Methylation integrates factors present at birth and modifiable across the lifespan that can influence pulmonary function. Studies are limited in scope and replication. Objectives: To conduct large-scale epigenome-wide meta-analyses of blood DNA methylation and pulmonary function. Methods: Twelve cohorts analyzed associations of methylation at cytosine-phosphate-guanine probes (CpGs), using Illumina 450K or EPIC/850K arrays, with FEV1, FVC, and FEV1/FVC. We performed multiancestry epigenome-wide meta-analyses (total of 17,503 individuals; 14,761 European, 2,549 African, and 193 Hispanic/Latino ancestries) and interpreted results using integrative epigenomics. Measurements and Main Results: We identified 1,267 CpGs (1,042 genes) differentially methylated (false discovery rate, <0.025) in relation to FEV1, FVC, or FEV1/FVC, including 1,240 novel and 73 also related to chronic obstructive pulmonary disease (1,787 cases). We found 294 CpGs unique to European or African ancestry and 395 CpGs unique to never or ever smokers. The majority of significant CpGs correlated with nearby gene expression in blood. Findings were enriched in key regulatory elements for gene function, including accessible chromatin elements, in both blood and lung. Sixty-nine implicated genes are targets of investigational or approved drugs. One example novel gene highlighted by integrative epigenomic and druggable target analysis is TNFRSF4. Mendelian randomization and colocalization analyses suggest that epigenome-wide association study signals capture causal regulatory genomic loci. Conclusions: We identified numerous novel loci differentially methylated in relation to pulmonary function; few were detected in large genome-wide association studies. Integrative analyses highlight functional relevance and potential therapeutic targets. This comprehensive discovery of potentially modifiable, novel lung function loci expands knowledge gained from genetic studies, providing insights into lung pathogenesis.

Multi-Omics Integration Reveals Only Minor Long-Term Molecular and Functional Sequelae in Immune Cells of Individuals Recovered From COVID-19.

The majority of COVID-19 patients experience mild to moderate disease course and recover within a few weeks. An increasing number of studies characterized the long-term changes in the specific anti-SARS-CoV-2 immune responses, but how COVID-19 shapes the innate and heterologous adaptive immune system after recovery is less well known. To comprehensively investigate the post-SARS-CoV-2 infection sequelae on the immune system, we performed a multi-omics study by integrating single-cell RNA-sequencing, single-cell ATAC-sequencing, genome-wide DNA methylation profiling, and functional validation experiments in 14 convalescent COVID-19 and 15 healthy individuals. We showed that immune responses generally recover without major sequelae after COVID-19. However, subtle differences persist at the transcriptomic level in monocytes, with downregulation of the interferon pathway, while DNA methylation also displays minor changes in convalescent COVID-19 individuals. However, these differences did not affect the cytokine production capacity of PBMCs upon different bacterial, viral, and fungal stimuli, although baseline release of IL-1Ra and IFN-γ was higher in convalescent individuals. In conclusion, we propose that despite minor differences in epigenetic and transcriptional programs, the immune system of convalescent COVID-19 patients largely recovers to the homeostatic level of healthy individuals.

Infant RSV immunoprophylaxis changes nasal epithelial DNA methylation at 6 years of age.

BACKGROUND: Respiratory syncytial virus (RSV) infection has been associated with childhood wheeze and asthma, and potential mechanisms include persistent epigenetic effects. METHODS: In the randomized, placebo-controlled MAKI trial, 429 preterm infants randomly received RSV immunoprophylaxis with palivizumab or placebo during their first RSV season. Children were followed until age 6 for asthma evaluation. DNA methylation in cells obtained by nasal brushes at age 6 was measured by Illumina MethylationEPIC array. RESULTS: RSV immunoprophylaxis in infancy had a significant impact on global methylation patterns in nasal cells at age 6. The first principal component (PC) related to the immunoprophylaxis intervention was enriched for the pathway "detection of chemical stimulus involved in sensory perception of smell" and "T cell differentiation." Subsequent analysis of these PCs indicated an effect of RSV immunoprophylaxis on cell type composition of nasal brushed cells. Three CpG sites, cg18040241, cg08243963, and cg19555973 which are annotated to genes GLB1L2, SC5D, and BPIFB1, were differentially methylated at genome-wide significance, but were not associated with asthma. CONCLUSION: The study provides the first proof of concept that RSV immunoprophylaxis during infancy has long-term effects on nasal epigenetic signatures at age 6, relating to host sensory perception, epidermal growth factor receptor signaling, and adaptive immune responses.

Epigenome-Wide DNA Methylation and Pesticide Use in the Agricultural Lung Health Study.

BACKGROUND: Pesticide exposure is associated with many long-term health outcomes; the potential underlying mechanisms are not well established for most associations. Epigenetic modifications, such as DNA methylation, may contribute. Individual pesticides may be associated with specific DNA methylation patterns but no epigenome-wide association study (EWAS) has evaluated methylation in relation to individual pesticides. OBJECTIVES: We conducted an EWAS of DNA methylation in relation to several pesticide active ingredients. METHODS: The Agricultural Lung Health Study is a case-control study of asthma, nested within the Agricultural Health Study. We analyzed blood DNA methylation measured using Illumina's EPIC array in 1,170 male farmers of European ancestry. For pesticides still on the market at blood collection (2009-2013), we evaluated nine active ingredients for which at least 30 participants reported past and current (within the last 12 months) use, as well as seven banned organochlorines with at least 30 participants reporting past use. We used robust linear regression to compare methylation at individual C-phosphate-G sites (CpGs) among users of a specific pesticide to never users. RESULTS: Using family-wise error rate (p<9×10-8) or false-discovery rate (FDR<0.05), we identified 162 differentially methylated CpGs across 8 of 9 currently marketed active ingredients (acetochlor, atrazine, dicamba, glyphosate, malathion, metolachlor, mesotrione, and picloram) and one banned organochlorine (heptachlor). Differentially methylated CpGs were unique to each active ingredient, and a dose-response relationship with lifetime days of use was observed for most. Significant CpGs were enriched for transcription motifs and 28% of CpGs were associated with whole blood cis-gene expression, supporting functional effects of findings. We corroborated a previously reported association between dichlorodiphenyltrichloroethane (banned in the United States in 1972) and epigenetic age acceleration. DISCUSSION: We identified differential methylation for several active ingredients in male farmers of European ancestry. These may serve as biomarkers of chronic exposure and could inform mechanisms of long-term health outcomes from pesticide exposure. https://doi.org/10.1289/EHP8928.

Residential PM2.5 exposure and the nasal methylome in children.

RATIONALE: PM2.5-induced adverse effects on respiratory health may be driven by epigenetic modifications in airway cells. The potential impact of exposure duration on epigenetic alterations in the airways is not yet known. OBJECTIVES: We aimed to study associations of fine particulate matter PM2.5 exposure with DNA methylation in nasal cells. METHODS: We conducted nasal epigenome-wide association analyses within 503 children from Project Viva (mean age 12.9 y), and examined various exposure durations (1-day, 1-week, 1-month, 3-months and 1-year) prior to nasal sampling. We used residential addresses to estimate average daily PM2.5 at 1 km resolution. We collected nasal swabs from the anterior nares and measured DNA methylation (DNAm) using the Illumina MethylationEPIC BeadChip. We tested 719,075 high quality autosomal CpGs using CpG-by-CpG and regional DNAm analyses controlling for multiple comparisons, and adjusted for maternal education, household smokers, child sex, race/ethnicity, BMI z-score, age, season at sample collection and cell-type heterogeneity. We further corrected for bias and genomic inflation. We tested for replication in a cohort from the Netherlands (PIAMA). RESULTS: In adjusted analyses, we found 362 CpGs associated with 1-year PM2.5 (FDR < 0.05), 20 CpGs passing Bonferroni correction (P < 7.0x10-8) and 10 Differentially Methylated Regions (DMRs). In 445 PIAMA participants (mean age 16.3 years) 11 of 203 available CpGs replicated at P < 0.05. We observed differential DNAm at/near genes implicated in cell cycle, immune and inflammatory responses. There were no CpGs or regions associated with PM2.5 levels at 1-day, 1-week, or 1-month prior to sample collection, although 2 CpGs were associated with past 3-month PM2.5. CONCLUSION: We observed wide-spread DNAm variability associated with average past year PM2.5 exposure but we did not detect associations with shorter-term exposure. Our results suggest that nasal DNAm marks reflect chronic air pollution exposure.

Exposure to violence, chronic stress, nasal DNA methylation, and atopic asthma in children.

BACKGROUND: Exposure to violence (ETV) or chronic stress may influence asthma through unclear mechanisms. METHODS: Epigenome-wide association study (EWAS) of ETV or chronic stress measures and DNA methylation in nasal epithelium from 487 Puerto Ricans aged 9-20 years who participated in the Epigenetic Variation and Childhood Asthma in Puerto Ricans study [EVA-PR]). We assessed four measures of ETV and chronic stress in children (ETV scale, gun violence, and perceived stress) and their mothers (perceived stress). Each EWAS was conducted using linear regression, with CpGs as dependent variables and the stress/violence measure as a predictor, adjusting for age, sex, the top five principal components, and SVA latent factors. We then selected the top 100 CpGs (by p value) associated with each stress/violence measure in EVA-PR and conducted a meta-analysis of the selected CpGs and atopic asthma using data from EVA-PR and two additional cohorts (Project Viva and PIAMA). RESULTS: Three CpGs (in SNN, PTPRN2, and LINC01164) were associated with maternal perceived stress or gun violence (p = 1.28-3.36 × 10-7 ), but not with atopic asthma, in EVA-PR. In a meta-analysis of three cohorts, which included the top CpGs associated with stress/violence measures in EVA-PR, 12 CpGs (in STARD3NL, SLC35F4, TSR3, CDC42SE2, KLHL25, PLCB1, BUD13, OR2B3, GALR1, TMEM196, TEAD4, and ANAPC13) were associated with atopic asthma at FDR-p < .05. CONCLUSIONS: Pending confirmation in longitudinal studies, our findings suggest that nasal epithelial methylation markers associated with measures of ETV and chronic stress may be linked to atopic asthma in children and adolescents.

Shared DNA methylation signatures in childhood allergy: The MeDALL study.

BACKGROUND: Differential DNA methylation associated with allergy might provide novel insights into the shared or unique etiology of asthma, rhinitis, and eczema. OBJECTIVE: We sought to identify DNA methylation profiles associated with childhood allergy. METHODS: Within the European Mechanisms of the Development of Allergy (MeDALL) consortium, we performed an epigenome-wide association study of whole blood DNA methylation by using a cross-sectional design. Allergy was defined as having symptoms from at least 1 allergic disease (asthma, rhinitis, or eczema) and positive serum-specific IgE to common aeroallergens. The discovery study included 219 case patients and 417 controls at age 4 years and 228 case patients and 593 controls at age 8 years from 3 birth cohorts, with replication analyses in 325 case patients and 1111 controls. We performed additional analyses on 21 replicated sites in 785 case patients and 2124 controls by allergic symptoms only from 8 cohorts, 3 of which were not previously included in analyses. RESULTS: We identified 80 differentially methylated CpG sites that showed a 1% to 3% methylation difference in the discovery phase, of which 21 (including 5 novel CpG sites) passed genome-wide significance after meta-analysis. All 21 CpG sites were also significantly differentially methylated with allergic symptoms and shared between asthma, rhinitis, and eczema. The 21 CpG sites mapped to relevant genes, including ACOT7, LMAN3, and CLDN23. All 21 CpG sties were differently methylated in asthma in isolated eosinophils, and 10 were replicated in respiratory epithelium. CONCLUSION: Reduced whole blood DNA methylation at 21 CpG sites was significantly associated with childhood allergy. The findings provide novel insights into the shared molecular mechanisms underlying asthma, rhinitis, and eczema.