Prevention of allergies and infections by minimally processed milk in infants-The MARTHA feasibility and safety trial.

BACKGROUND: Consumption of raw cow's milk has repeatedly been shown to protect from asthma, allergies, and respiratory infections. As raw milk bears potential health hazards, it cannot be recommended for prevention. Therefore, we performed an intervention study with microbially safe but otherwise minimally processed cow's milk. Here we describe feasibility and safety of the trial. METHODS: The MARTHA trial (DRKS00014781) was set up as a double-blind randomized intervention in a population residing in Bavaria. Infants from 6 to 36 months of age consumed minimally processed cow's milk (intervention arm) or ultra-heat-treated (UHT) semi-skimmed milk (comparator arm). RESULTS: At the age of 6 to 12 months, 260 infants were enrolled, with 72% having a family history of atopy. The extensive screening system for milk consumption and symptoms suggestive of adverse events was well accepted with 22,988 completed weekly surveys and an average completion of 82% surveys sent out. The children consumed the study milk on average on 457 days (61% of intervention days). The intervention proved to be safe without any case of milk allergy or milk intolerance under the intervention in both arms. All 6 cases of serious adverse events were unrelated to milk. The most common reason was unscheduled hospitalization of more than 3 days. CONCLUSIONS: The intervention with minimally processed milk and the study instruments proved feasible. During the age of 6 to 36 months, there was no increased risk of milk allergy in a population with a substantial proportion of family history of atopy.

IgA+ memory B-cells are significantly increased in patients with asthma and small airway dysfunction.

BACKGROUND: Comprehensive studies investigated the role of T-cells in asthma which led to personalised treatment options targeting severe eosinophilic asthma. However, little is known about the contribution of B-cells to this chronic inflammatory disease. In this study we investigated the contribution of various B-cell populations to specific clinical features in asthma. METHODS: In the All Age Asthma Cohort (ALLIANCE), a subgroup of 154 adult asthma patients and 28 healthy controls were included for B-cell characterisation by flow cytometry. Questionnaires, lung function measurements, blood differential counts and allergy testing of participants were analysed together with comprehensive data on B-cells using association studies and multivariate linear models. RESULTS: Patients with severe asthma showed decreased immature B-cell populations while memory B-cells were significantly increased compared with both mild-moderate asthma patients and healthy controls. Furthermore, increased frequencies of IgA+ memory B-cells were associated with impaired lung function and specifically with parameters indicative for augmented resistance in the peripheral airways. Accordingly, asthma patients with small airway dysfunction (SAD) defined by impulse oscillometry showed increased frequencies of IgA+ memory B-cells, particularly in patients with mild-moderate asthma. Additionally, IgA+ memory B-cells significantly correlated with clinical features of SAD such as exacerbations. CONCLUSIONS: With this study we demonstrate for the first time a significant association of increased IgA+ memory B-cells with asthma and SAD, pointing towards future options for B-cell-directed strategies in preventing and treating asthma.

T2-high asthma phenotypes across lifespan.

RATIONALE: In adults, personalised asthma treatment targets patients with type 2 (T2)-high and eosinophilic asthma phenotypes. It is unclear whether such classification is achievable in children. OBJECTIVES: To define T2-high asthma with easily accessible biomarkers and compare resulting phenotypes across all ages. METHODS: In the multicentre clinical All Age Asthma Cohort (ALLIANCE), 1125 participants (n=776 asthmatics, n=349 controls) were recruited and followed for 2 years (1 year in adults). Extensive clinical characterisation (questionnaires, blood differential count, allergy testing, lung function and sputum induction (in adults)) was performed at baseline and follow-ups. Interleukin (IL)-4, IL-5 and IL-13 were measured after stimulation of whole blood with lipopolysaccharide (LPS) or anti-CD3/CD28. MEASUREMENTS AND MAIN RESULTS: Based on blood eosinophil counts and allergen-specific serum IgE antibodies, patients were categorised into four mutually exclusive phenotypes: "atopy-only", "eosinophils-only", "T2-high" (eosinophilia + atopy) and "T2-low" (neither eosinophilia nor atopy). The T2-high phenotype was found across all ages, even in very young children in whom it persisted to a large degree even after 2 years of follow-up. T2-high asthma in adults was associated with childhood onset, suggesting early origins of this asthma phenotype. In both children and adults, the T2-high phenotype was characterised by excessive production of specific IgE to allergens (p<0.0001) and, from school age onwards, by increased production of IL-5 after anti-CD3/CD28 stimulation of whole blood. CONCLUSIONS: Using easily accessible biomarkers, patients with T2-high asthma can be identified across all ages delineating a distinct phenotype. These patients may benefit from therapy with biologicals even at a younger age.

Identification of OCA2 as a novel locus for the co-morbidity of asthma-plus-eczema.

BACKGROUND: Numerous genes have been associated with the three most common allergic diseases (asthma, allergic rhinitis or eczema) but these genes explain only a part of the heritability. In the vast majority of genetic studies, complex phenotypes such as co-morbidity of two of these diseases, have not been considered. This may partly explain missing heritability. OBJECTIVE: To identify genetic variants specifically associated with the co-morbidity of asthma-plus-eczema. METHODS: We first conducted a meta-analysis of four GWAS (Genome-Wide Association Study) of the combined asthma-plus-eczema phenotype (total of 8807 European-ancestry subjects of whom 1208 subjects had both asthma and eczema). To assess whether the association with SNP(s) was specific to the co-morbidity, we also conducted a meta-analysis of homogeneity test of association according to disease status ("asthma-plus-eczema" vs. the presence of only one disease "asthma only or eczema only"). We then used a joint test by combining the two test statistics from the co-morbidity-SNP association and the phenotypic heterogeneity of SNP effect meta-analyses. RESULTS: Seven SNPs were detected for specific association to the asthma-plus-eczema co-morbidity, two with significant and five with suggestive evidence using the joint test after correction for multiple testing. The two significant SNPs are located in the OCA2 gene (Oculocutaneous Albinism II), a new locus never detected for significant evidence of association with any allergic disease. This gene is a promising candidate gene, because of its link to skin and lung diseases, and to epithelial barrier and immune mechanisms. CONCLUSION: Our study underlines the importance of studying sub-phenotypes as co-morbidities to detect new susceptibility genes.

Trajectories of asthma and allergy symptoms from childhood to adulthood.

BACKGROUND: Phenotypes of asthma and allergic diseases are mainly studied separately for children and adults. To explore the role of adolescence and young adulthood, we investigated symptom trajectories at the transition from childhood into adulthood. METHODS: Latent class analysis (LCA) was conducted in a population initially recruited for the German arm of Phase II of the International Study of Asthma and Allergies in Childhood and followed-up three times until their early 30s (N=2267). Indicators included in LCA were 12-month prevalences of symptoms of wheeze, rhinoconjunctivitis, and eczema. Latent classes were further characterised regarding important traits such as skin prick tests. Logistic regression models were used to investigate associations with environmental determinants such as smoking and occupational exposures. RESULTS: Six latent classes were identified: an asymptomatic one as well as three with single and two with co-occurring symptoms. All trajectories essentially established between baseline assessment at around 10 years and the first follow-up at around 17 years. Probabilities for symptoms increased from childhood to adolescence, especially for wheeze-related latent classes, while they remained constant in adulthood. Wheeze-related latent classes were also positively associated with exposures during adolescence (e.g. active smoking). CONCLUSION: Distinct trajectories of asthma and allergy symptoms establish from childhood through adolescence and stabilize during early adulthood. This pattern was most notable in wheeze-related latent classes which also showed the strongest positive associations with environmental exposures in adolescence/young adulthood. Therefore, not only childhood but also adolescence is relevant for disease development and offers considerable potential for prevention and health promotion.

Early priming of asthma and respiratory allergies: Future aspects of prevention: A statement by the European Forum for Education and Research in Allergy and Airway Disease (EUFOREA) and the EAACI-Clemens von Pirquet Foundation.

In order to summarize recent research on the prevention of allergies-particularly asthma-and stimulate new activities for future initiatives, a virtual workshop sponsored by the EAACI Clemens von Pirquet foundation and EUFOREA was held in October 2021. The determinants of the "allergic march" as well as the key messages from intervention studies were reviewed by an international faculty of experts. Several unmet needs were identified, and a number of priorities for future studies were proposed.

Asthma in farm children is more determined by genetic polymorphisms and in non-farm children by environmental factors.

BACKGROUND: The asthma syndrome is influenced by hereditary and environmental factors. With the example of farm exposure, we study whether genetic and environmental factors interact for asthma. METHODS: Statistical learning approaches based on penalized regression and decision trees were used to predict asthma in the GABRIELA study with 850 cases (9% farm children) and 857 controls (14% farm children). Single-nucleotide polymorphisms (SNPs) were selected from a genome-wide dataset based on a literature search or by statistical selection techniques. Prediction was assessed by receiver operating characteristics (ROC) curves and validated in the PASTURE cohort. RESULTS: Prediction by family history of asthma and atopy yielded an area under the ROC curve (AUC) of 0.62 [0.57-0.66] in the random forest machine learning approach. By adding information on demographics (sex and age) and 26 environmental exposure variables, the quality of prediction significantly improved (AUC = 0.65 [0.61-0.70]). In farm children, however, environmental variables did not improve prediction quality. Rather SNPs related to IL33 and RAD50 contributed significantly to the prediction of asthma (AUC = 0.70 [0.62-0.78]). CONCLUSIONS: Asthma in farm children is more likely predicted by other factors as compared to non-farm children though in both forms, family history may integrate environmental exposure, genotype and degree of penetrance.

Development of atopic sensitization in Finnish and Estonian children: A latent class analysis in a multicenter cohort.

BACKGROUND: The prevalence of atopy is associated with a Western lifestyle, as shown by studies comparing neighboring regions with different socioeconomic backgrounds. Atopy might reflect various conditions differing in their susceptibility to environmental factors. OBJECTIVE: We sought to define phenotypes of atopic sensitization in early childhood and examine their association with allergic diseases and hereditary background in Finland and Estonia. METHODS: The analysis included 1603 Finnish and 1657 Estonian children from the DIABIMMUNE multicenter young children cohort. Specific IgE levels were measured at age 3, 4, and 5 years, respectively, and categorized into 3 CAP classes. Latent class analysis was performed with the statistical software package poLCA in R software. RESULTS: Both populations differed in terms of socioeconomic status and environmental determinants, such as pet ownership, farm-related exposure, time spent playing outdoors, and prevalence of allergic diseases (all P < .001). Nevertheless, we found similar latent classes in both populations: an unsensitized class, a food class, 2 inhalant classes differentiating between seasonal and perennial aeroallergens, and a severe atopy class. The latter was characterized by high total and specific IgE levels and strongly associated with wheeze (odds ratio [OR], 5.64 [95% CI, 3.07-10.52] and 4.56 [95% CI, 2.35-8.52]), allergic rhinitis (OR, 22.4 [95% CI, 11.67-44.54] and 13.97 [95% CI, 7.33-26.4]), and atopic eczema (OR, 9.39 [95% CI, 4.9-19.3] and 9.5 [95% CI, 5.2-17.5] for Finland and Estonia, respectively). Environmental differences were reflected in the larger seasonal inhalant atopy class in Finland, although composition of classes was comparable between countries. CONCLUSION: Despite profound differences in environmental exposures, there might exist genuine patterns of atopic sensitization. The distribution of these patterns might determine the contribution of atopic sensitization to disease onset.

Genome-wide interaction study of early-life smoking exposure on time-to-asthma onset in childhood.

BACKGROUND: Asthma, a heterogeneous disease with variable age of onset, results from the interplay between genetic and environmental factors. Early-life tobacco smoke (ELTS) exposure is a major asthma risk factor. Only a few genetic loci have been reported to interact with ELTS exposure in asthma. OBJECTIVE: Our aim was to identify new loci interacting with ELTS exposure on time-to-asthma onset (TAO) in childhood. METHODS: We conducted genome-wide interaction analyses of ELTS exposure on time-to-asthma onset in childhood in five European-ancestry studies (totalling 8273 subjects) using Cox proportional-hazard model. The results of all five genome-wide analyses were meta-analysed. RESULTS: The 13q21 locus showed genome-wide significant interaction with ELTS exposure (P = 4.3 × 10-8 for rs7334050 within KLHL1 with consistent results across the five studies). Suggestive interactions (P < 5 × 10-6 ) were found at three other loci: 20p12 (rs13037508 within MACROD2; P = 4.9 × 10-7 ), 14q22 (rs7493885 near NIN; P = 2.9 × 10-6 ) and 2p22 (rs232542 near CYP1B1; P = 4.1 × 10-6 ). Functional annotations and the literature showed that the lead SNPs at these four loci influence DNA methylation in the blood and are located nearby CpG sites reported to be associated with exposure to tobacco smoke components, which strongly support our findings. CONCLUSIONS AND CLINICAL RELEVANCE: We identified novel candidate genes interacting with ELTS exposure on time-to-asthma onset in childhood. These genes have plausible biological relevance related to tobacco smoke exposure. Further epigenetic and functional studies are needed to confirm these findings and to shed light on the underlying mechanisms.

Parents know it best: Prediction of asthma and lung function by parental perception of early wheezing episodes.

BACKGROUND: Childhood asthma is often preceded by early wheeze. Usually, wheezing episodes are recorded retrospectively, which may induce recall bias. AIMS AND OBJECTIVES: The aim of this study was to investigate true-positive recall of parent-reported wheeze at 1 year of age, its determinants, and its implications for asthma and lung function at 6 years of age. METHODS: The PASTURE (Protection Against Allergy-Study in Rural Environments) study followed 880 children from rural areas in 5 European countries from birth to age 6 years. Wheeze symptoms in the first year were asked weekly. At age 6, parent-reported asthma diagnosis was ascertained and lung function measurements were conducted. Correct parental recall of wheeze episodes at the end of the first year was assessed for associations with lung function, asthma, and the asthma risk locus on chromosome 17q21. RESULTS: Parents correctly recalled wheeze after the first year in 54% of wheezers. This true-positive recall was determined by number of episodes, timing of the last wheeze episode, and parental asthma. Independently from these determinants, true-positive recall predicted asthma at age 6 years (odds ratio 4.54, 95% confidence interval (CI) [1.75-14.16]) and impaired lung function (ß = -0.62, 95% CI [-1.12; -0.13], P-value = .02). Associations were stronger in children with asthma risk SNPs on chromosome 17q21. CONCLUSION: Correct parental recall of wheezing episodes may reflect clinical relevance of early wheeze and its impact on subsequent asthma and lung function impairment. Questions tailored to parental perception of wheezing episodes may further enhance asthma prediction.

Correction to: Analysis of the Fungal Flora in Environmental Dust Samples by PCR-SSCP Method.

The original version of this article contained a mistake in the co-author's conflict of interest statement. Erika von Mutius wishes to add the following disclosures: "E. von Mutius is listed as an inventor on the following patents: publication number EP 1411977, composition containing bacterial antigens used for the prophylaxis and the treatment of allergic diseases, granted on 18 April 2007; publication number EP1637147, stable dust extract for allergy protection, granted on 10 December 2008; publication number EP 1964570, pharmaceutical compound to protect against allergies and inflammatory diseases, granted on 21 November 2012. E. von Mutius is listed as inventor and has received royalties on the following patent: publication number EP2361632, specific environmental bacteria for the protection from and/or the treatment of allergic, chronic inflammatory and/or autoimmune disorders, granted on 19 March 2014".

Latent class analysis reveals clinically relevant atopy phenotypes in 2 birth cohorts.

BACKGROUND: Phenotypes of childhood-onset asthma are characterized by distinct trajectories and functional features. For atopy, definition of phenotypes during childhood is less clear. OBJECTIVE: We sought to define phenotypes of atopic sensitization over the first 6 years of life using a latent class analysis (LCA) integrating 3 dimensions of atopy: allergen specificity, time course, and levels of specific IgE (sIgE). METHODS: Phenotypes were defined by means of LCA in 680 children of the Multizentrische Allergiestudie (MAS) and 766 children of the Protection against allergy: Study in Rural Environments (PASTURE) birth cohorts and compared with classical nondisjunctive definitions of seasonal, perennial, and food sensitization with respect to atopic diseases and lung function. Cytokine levels were measured in the PASTURE cohort. RESULTS: The LCA classified predominantly by type and multiplicity of sensitization (food vs inhalant), allergen combinations, and sIgE levels. Latent classes were related to atopic disease manifestations with higher sensitivity and specificity than the classical definitions. LCA detected consistently in both cohorts a distinct group of children with severe atopy characterized by high seasonal sIgE levels and a strong propensity for asthma; hay fever; eczema; and impaired lung function, also in children without an established asthma diagnosis. Severe atopy was associated with an increased IL-5/IFN-γ ratio. A path analysis among sensitized children revealed that among all features of severe atopy, only excessive sIgE production early in life affected asthma risk. CONCLUSIONS: LCA revealed a set of benign, symptomatic, and severe atopy phenotypes. The severe phenotype emerged as a latent condition with signs of a dysbalanced immune response. It determined high asthma risk through excessive sIgE production and directly affected impaired lung function.

Bacterial microbiota of the upper respiratory tract and childhood asthma.

BACKGROUND: Patients with asthma and healthy controls differ in bacterial colonization of the respiratory tract. The upper airways have been shown to reflect colonization of the lower airways, the actual site of inflammation in asthma, which is hardly accessible in population studies. OBJECTIVE: We sought to characterize the bacterial communities at 2 sites of the upper respiratory tract obtained from children from a rural area and to relate these to asthma. METHODS: The microbiota of 327 throat and 68 nasal samples from school-age farm and nonfarm children were analyzed by 454-pyrosequencing of the bacterial 16S ribosomal RNA gene. RESULTS: Alterations in nasal microbiota but not of throat microbiota were associated with asthma. Children with asthma had lower α- and ß-diversity of the nasal microbiota as compared with healthy control children. Furthermore, asthma presence was positively associated with a specific operational taxonomic unit from the genus Moraxella in children not exposed to farming, whereas in farm children Moraxella colonization was unrelated to asthma. In nonfarm children, Moraxella colonization explained the association between bacterial diversity and asthma to a large extent. CONCLUSIONS: Asthma was mainly associated with an altered nasal microbiota characterized by lower diversity and Moraxella abundance. Children living on farms might not be susceptible to the disadvantageous effect of Moraxella. Prospective studies may clarify whether Moraxella outgrowth is a cause or a consequence of loss in diversity.

Identification of fungal candidates for asthma protection in a large population-based study.

BACKGROUND: Exposure to molds has been related to asthma risk both positively and negatively, depending on the environmental setting. The pertinent results are based on generic markers or culturing methods although the majority of present fungi cannot be cultured under laboratory conditions. The aim of the present analysis was to assess environmental dust samples for asthma-protective fungal candidates with a comprehensive molecular technique covering also non-cultivable and non-viable fungi. METHODS: Mattress dust samples of 844 children from the GABRIELA study were analyzed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) of the fungus-specific internal transcribed spacer (ITS) region. Known asthma candidate species were tested for their associations with asthma, and further gel positions were sought to explain the above. As a second, data-driven, analysis, we tested the association of each individual gel position with asthma. RESULTS: In the hypothesis-driven approach, Penicillium chrysogenum emerged with an odds ratio of 0.80 (95% confidence interval 0.66-0.96; p = 0.020). The effect size was changed by 39% toward the null when adjusting for the two bands 683 (DNA of Metschnikowia sp., Aureobasidium spp.) and 978 (DNA of Epicoccum spp., Galactomyces spp., uncultured Penicillium). The data-driven approach yielded an additional band (containing DNA of Pseudotaeniolina globosa) with reduced risk of asthma (OR = 0.80 [0.66-0.96], p = 0.012). CONCLUSIONS: A large population-based study revealed several fungal taxa with inverse associations with childhood asthma. Molds produce a variety of bioactive compounds with detrimental but also beneficial immunoregulatory capacities, which renders them promising targets for further asthma research.

The Early Development of Wheeze. Environmental Determinants and Genetic Susceptibility at 17q21.

RATIONALE: Growing up on a farm protects from childhood asthma and early wheeze. Virus-triggered wheeze in infancy predicts asthma in individuals with a genetic asthma risk associated with chromosome 17q21. OBJECTIVES: To test environmental determinants of infections and wheeze in the first year of life, potential modifications of these associations by 17q21, and the implications for different trajectories of wheeze. METHODS: We followed 983 children in rural areas of Europe from birth until age 6 years. Symptoms of wheeze, rhinitis, fever, and environmental exposures were documented with weekly diaries during year 1. Asthma at age 6 was defined as ever having a reported doctor's diagnosis. Single-nucleotide polymorphisms related to ORMDL3 (rs8076131) and GSDMB (rs7216389, rs2290400) at 17q21 were genotyped. MEASUREMENTS AND MAIN RESULTS: Early wheeze was positively associated with presence of older siblings among carriers of known asthma risk alleles at 17q21 (e.g., rs8076131) (adjusted odds ratio [aOR], 1.53; 95% confidence interval [CI], 1.16-2.01). Exposure to farm animal sheds was inversely related to wheeze (aOR, 0.44; 95% CI, 0.33-0.60). Both effects were similarly observed in children with transient wheeze up to age 3 years without subsequent development of asthma (aOR, 1.71 [95% CI, 1.09-2.67]; and aOR, 0.48 [95% CI, 0.30-0.76], respectively). CONCLUSIONS: These findings suggest that the chromosome 17q21 locus relates to episodes of acute airway obstruction common to both transient wheeze and asthma. The previously identified asthma risk alleles are the ones susceptible to environmental influences. Thus, this gene-environment interaction reveals two faces of 17q21: The same genotype constitutes genetic risk and allows for environmental protection, thereby providing options for prospective prevention strategies.

ω-3 fatty acids contribute to the asthma-protective effect of unprocessed cow's milk.

BACKGROUND: Living on a farm has repeatedly been shown to protect children from asthma and allergies. A major factor involved in this effect is consumption of unprocessed cow's milk obtained directly from a farm. However, this phenomenon has never been shown in a longitudinal design, and the responsible milk components are still unknown. OBJECTIVES: We sought to assess the asthma-protective effect of unprocessed cow's milk consumption in a birth cohort and to determine whether the differences in the fatty acid (FA) composition of unprocessed farm milk and industrially processed milk contributed to this effect. METHODS: The Protection Against Allergy-Study in Rural Environments (PASTURE) study followed 1133 children living in rural areas in 5 European countries from birth to age 6 years. In 934 children milk consumption was assessed by using yearly questionnaires, and samples of the "usually" consumed milk and serum samples of the children were collected at age 4 years. Doctor-diagnosed asthma was parent reported at age 6 years. In a nested case-control study of 35 asthmatic and 49 nonasthmatic children, 42 FAs were quantified in milk samples. RESULTS: The risk of asthma at 6 years of age was reduced by previous consumption of unprocessed farm milk compared with shop milk (adjusted odds ratio for consumption at 4 years, 0.26; 95% CI, 0.10-0.67). Part of the effect was explained by the higher fat content of farm milk, particularly the higher levels of ω-3 polyunsaturated FAs (adjusted odds ratio, 0.29; 95% CI, 0.11-0.81). CONCLUSION: Continuous farm milk consumption in childhood protects against asthma at school age partially by means of higher intake of ω-3 polyunsaturated FAs, which are precursors of anti-inflammatory mediators.

Microbes and asthma: Opportunities for intervention.

The worldwide incidence and prevalence of asthma continues to increase. Asthma is now understood as an umbrella term for different phenotypes or endotypes, which arise through different pathophysiologic pathways. Understanding the many factors contributing to development of the disease is important for the identification of novel therapeutic targets for the treatment of certain asthma phenotypes. The hygiene hypothesis has been formulated to explain the increasing prevalence of allergic disease, including asthma. This hypothesis postulates that decreased exposure at a young age to certain infectious agents as a result of improved hygiene, increased antibiotic use and vaccination, and changes in lifestyle and dietary habits is associated with changes in the immune system, which predispose subjects to allergy. Many microbes, during their coevolution with human subjects, developed mechanisms to manipulate the human immune system and to increase their chances of survival. Improving models of asthma, as well as choosing adequate end points in clinical trials, will lead to a more complete understanding of the underlying mechanisms, thus providing an opportunity to devise primary and secondary interventions at the same time as identifying new molecular targets for treatment. This article reports the discussion and conclusion of a workshop under the auspices of the Netherlands Lung Foundation to extend our understanding of how modulation of the immune system by bacterial, parasitic, and viral infections might affect the development of asthma and to map out future lines of investigation.

Identification of a new locus at 16q12 associated with time to asthma onset.

BACKGROUND: Asthma is a heterogeneous disease in which age of onset plays an important role. OBJECTIVE: We sought to identify the genetic variants associated with time to asthma onset (TAO). METHODS: We conducted a large-scale meta-analysis of 9 genome-wide association studies of TAO (total of 5462 asthmatic patients with a broad range of age of asthma onset and 8424 control subjects of European ancestry) performed by using survival analysis techniques. RESULTS: We detected 5 regions associated with TAO at the genome-wide significant level (P < 5 × 10-8). We evidenced a new locus in the 16q12 region (near cylindromatosis turban tumor syndrome gene [CYLD]) and confirmed 4 asthma risk regions: 2q12 (IL-1 receptor-like 1 [IL1RL1]), 6p21 (HLA-DQA1), 9p24 (IL33), and 17q12-q21 (zona pellucida binding protein 2 [ZPBP2]-gasdermin A [GSDMA]). Conditional analyses identified 2 distinct signals at 9p24 (both upstream of IL33) and 17q12-q21 (near ZPBP2 and within GSDMA). Together, these 7 distinct loci explained 6.0% of the variance in TAO. In addition, we showed that genetic variants at 9p24 and 17q12-q21 were strongly associated with an earlier onset of childhood asthma (P ≤ .002), whereas the 16q12 single nucleotide polymorphism was associated with later asthma onset (P = .04). A high burden of disease risk alleles at these loci was associated with earlier age of asthma onset (4 vs 9-12 years, P = 10-4). CONCLUSION: The new susceptibility region for TAO at 16q12 harbors variants that correlate with the expression of CYLD and nucleotide-binding oligomerization domain 2 (NOD2), 2 strong candidates for asthma. This study demonstrates that incorporating the variability of age of asthma onset in asthma modeling is a helpful approach in the search for disease susceptibility genes.