Control for Population Structure and Relatedness for Binary Traits in Genetic Association Studies via Logistic Mixed Models.

Linear mixed models (LMMs) are widely used in genome-wide association studies (GWASs) to account for population structure and relatedness, for both continuous and binary traits. Motivated by the failure of LMMs to control type I errors in a GWAS of asthma, a binary trait, we show that LMMs are generally inappropriate for analyzing binary traits when population stratification leads to violation of the LMM's constant-residual variance assumption. To overcome this problem, we develop a computationally efficient logistic mixed model approach for genome-wide analysis of binary traits, the generalized linear mixed model association test (GMMAT). This approach fits a logistic mixed model once per GWAS and performs score tests under the null hypothesis of no association between a binary trait and individual genetic variants. We show in simulation studies and real data analysis that GMMAT effectively controls for population structure and relatedness when analyzing binary traits in a wide variety of study designs.

Gun Violence, African Ancestry, and Asthma: A Case-Control Study in Puerto Rican Children.

BACKGROUND: Exposure to gun violence and African ancestry have been separately associated with increased risk of asthma in Puerto Rican children. OBJECTIVE: The objective of this study was to examine whether African ancestry and gun violence interact on asthma and total IgE in school-aged Puerto Rican children. METHODS: This is a case-control study of 747 Puerto Rican children aged 9 to 14 years living in San Juan, Puerto Rico (n = 472), and Hartford, Connecticut (n = 275). Exposure to gun violence was defined as the child's report of hearing gunshots more than once, and the percentage of African ancestry was estimated using genome-wide genotypic data. Asthma was defined as parental report of physician-diagnosed asthma and wheeze in the previous year. Serum total IgE (IU/mL) was measured in study participants. Multivariate logistic and linear regressions were used for the analysis of asthma and total IgE, respectively. RESULTS: In multivariate analyses, there was a significant interaction between exposure to gun violence and African ancestry on asthma (P = .001) and serum total IgE (P = .04). Among children exposed to gun violence, each quartile increase in the percentage of African ancestry was associated with approximately 45% higher odds of asthma (95% CI, 1.15-1.84; P = .002) and an approximately 19% increment in total IgE (95% , 0.60-40.65, P = .04). In contrast, there was no significant association between African ancestry and asthma or total IgE in children not exposed to gun violence. CONCLUSIONS: Our results suggest that exposure to gun violence modifies the estimated effect of African ancestry on asthma and atopy in Puerto Rican children.

Folate Deficiency, Atopy, and Severe Asthma Exacerbations in Puerto Rican Children.

BACKGROUND: Little is known about folate and atopy or severe asthma exacerbations. We examined whether folate deficiency is associated with number of positive skin tests to allergens or severe asthma exacerbations in a high-risk population and further assessed whether such association is explained or modified by vitamin D status. METHODS: Cross-sectional study of 582 children aged 6 to 14 years with (n = 304) and without (n = 278) asthma in San Juan, Puerto Rico. Folate deficiency was defined as plasma folate less than or equal to 20 ng/ml. Our outcomes were the number of positive skin tests to allergens (range, 0-15) in all children and (in children with asthma) one or more severe exacerbations in the previous year. Logistic and negative binomial regression models were used for the multivariate analysis. All multivariate models were adjusted for age, sex, household income, residential proximity to a major road, and (for atopy) case/control status; those for severe exacerbations were also adjusted for use of inhaled corticosteroids and vitamin D insufficiency (a plasma 25[OH]D < 30 ng/ml). MEASUREMENTS AND MAIN RESULTS: In a multivariate analysis, folate deficiency was significantly associated with an increased degree of atopy and 2.2 times increased odds of at least one severe asthma exacerbation (95% confidence interval for odds ratio, 1.1-4.6). Compared with children who had normal levels of both folate and vitamin D, those with both folate deficiency and vitamin D insufficiency had nearly eightfold increased odds of one or more severe asthma exacerbation (95% confidence interval for adjusted odds ratio, 2.7-21.6). CONCLUSIONS: Folate deficiency is associated with increased degree of atopy and severe asthma exacerbations in school-aged Puerto Ricans. Vitamin D insufficiency may further increase detrimental effects of folate deficiency on severe asthma exacerbations.

Diet, interleukin-17, and childhood asthma in Puerto Ricans.

BACKGROUND: Dietary patterns might influence the pathogenesis of asthma in Puerto Ricans, the ethnic group most affected by this disease in the United States. OBJECTIVE: To examine the association among diet, T-helper cell type 17 cytokines, and asthma in Puerto Rican children. METHODS: As part of a case-control study of 678 Puerto Rican children 6 to 14 years old in San Juan, participants completed a 75-item questionnaire on the child's food consumption in the prior week. Foods were aggregated into 7 groups: fruits, vegetables, grains, protein foods, dairy, fats, and sweets. Logistic regression was used to evaluate consumption frequency of each group, plasma T-helper cell type 17 cytokine levels, and asthma. Based on this analysis, a food score (range -2 [unhealthy diet: high consumption of dairy products and sweets, low consumption of vegetables and grains] to +2 [healthy diet: high consumption of grains and vegetables, low consumption of dairy and sweets]) was created to identify dietary patterns. RESULTS: High consumption of grains was associated with lower odds of asthma (adjusted odds ratio [aOR] 0.52, 95% confidence interval [CI] 0.33-0.82), whereas frequent consumption of dairy products (aOR 1.93, 95% CI 1.32-2.84) or sweets (aOR 1.82, 95% CI 1.08-2.72) was associated with higher odds of asthma. A healthier diet (each 1-point increment in food score) was associated with lower levels of interleukin-17F (ß = -1.48 pg/mL, 95% CI -1.78 to -1.20) and with 36% decreased odds of asthma (aOR 0.64, 95% CI 0.53-0.77). CONCLUSION: A healthy diet, with frequent consumption of vegetables and grains and low consumption of dairy products and sweets, was associated with lower levels of interleulin-17F and decreased odds of childhood asthma in Puerto Ricans.

Exposure to gun violence and asthma among children in Puerto Rico.

BACKGROUND AND OBJECTIVES: Although community violence may influence asthma morbidity by increasing stress, no study has assessed exposure to gun violence and childhood asthma. We examined whether exposure to gun violence is associated with asthma in children, particularly in those reporting fear of leaving their home. METHODS: Case-control study of 466 children aged 9-14 years with (n = 234) and without (n = 232) asthma in San Juan, Puerto Rico. Lifetime exposure to gun violence was defined as hearing a gunshot more than once. We also assessed whether the child was afraid to leave his/her home because of violence. Asthma was defined as physician-diagnosed asthma and wheeze in the prior year. We used logistic regression for the statistical analysis. All multivariate models were adjusted for age, gender, household income, parental asthma, environmental tobacco smoke, prematurity and residential distance from a major road. RESULTS: Cases were more likely to have heard a gunshot more than once than control subjects (n = 156 or 67.2% vs. n = 122 or 52.1%, P < 0.01). In a multivariate analysis, hearing a gunshot more than once was associated with asthma (odds ratio [OR] = 1.8, 95% confidence interval [CI] = 1.1-1.7, P = 0.01). Compared with children who had heard a gunshot not more than once and were not afraid to leave their home because of violence, those who had heard a gunshot more than once and were afraid to leave their home due to violence had 3.2 times greater odds of asthma (95% CI for OR = 2.2-4.4, P < 0.01). CONCLUSIONS: Exposure to gun violence is associated with asthma in Puerto Rican children, particularly in those afraid to leave their home. Stress from such violence may contribute to the high burden of asthma in Puerto Ricans.

Stress and Bronchodilator Response in Children with Asthma.

RATIONALE: Stress is associated with asthma morbidity in Puerto Ricans (PRs), who have reduced bronchodilator response (BDR). OBJECTIVES: To examine whether stress and/or a gene regulating anxiety (ADCYAP1R1) is associated with BDR in PR and non-PR children with asthma. METHODS: This was a cross-sectional study of stress and BDR (percent change in FEV1 after BD) in 234 PRs ages 9-14 years with asthma. We assessed child stress using the Checklist of Children's Distress Symptoms, and maternal stress using the Perceived Stress Scale. Replication analyses were conducted in two cohorts. Polymorphisms in ADCYAP1R1 were genotyped in our study and six replication studies. Multivariable models of stress and BDR were adjusted for age, sex, income, environmental tobacco smoke, and use of inhaled corticosteroids. MEASUREMENTS AND MAIN RESULTS: High child stress was associated with reduced BDR in three cohorts. PR children who were highly stressed (upper quartile, Checklist of Children's Distress Symptoms) and whose mothers had high stress (upper quartile, Perceived Stress Scale) had a BDR that was 10.2% (95% confidence interval, 6.1-14.2%) lower than children who had neither high stress nor a highly stressed mother. A polymorphism in ADCYAP1R1 (rs34548976) was associated with reduced BDR. This single-nucleotide polymorphism is associated with reduced expression of the gene for the ß2-adrenergic receptor (ADRB2) in CD4(+) lymphocytes of subjects with asthma, and it affects brain connectivity of the amygdala and the insula (a biomarker of anxiety). CONCLUSIONS: High child stress and an ADCYAP1R1 single-nucleotide polymorphism are associated with reduced BDR in children with asthma. This is likely caused by down-regulation of ADRB2 in highly stressed children.

Genome-wide expression profiles identify potential targets for gene-environment interactions in asthma severity.

BACKGROUND: Gene-environment interaction studies using genome-wide association study data are often underpowered after adjustment for multiple comparisons. Differential gene expression in response to the exposure of interest can capture the most biologically relevant genes at the genome-wide level. OBJECTIVE: We used differential genome-wide expression profiles from the Epidemiology of Home Allergens and Asthma birth cohort in response to Der f 1 allergen (sensitized vs nonsensitized) to inform a gene-environment study of dust mite exposure and asthma severity. METHODS: Polymorphisms in differentially expressed genes were identified in genome-wide association study data from the Childhood Asthma Management Program, a clinical trial in childhood asthmatic patients. Home dust mite allergen levels (<10 or ≥10 µg/g dust) were assessed at baseline, and (≥1) severe asthma exacerbation (emergency department visit or hospitalization for asthma in the first trial year) served as the disease severity outcome. The Genetics of Asthma in Costa Rica Study and a Puerto Rico/Connecticut asthma cohort were used for replication. RESULTS: IL9, IL5, and proteoglycan 2 expression (PRG2) was upregulated in Der f 1-stimulated PBMCs from dust mite-sensitized patients (adjusted P < .04). IL9 polymorphisms (rs11741137, rs2069885, and rs1859430) showed evidence for interaction with dust mite in the Childhood Asthma Management Program (P = .02 to .03), with replication in the Genetics of Asthma in Costa Rica Study (P = .04). Subjects with the dominant genotype for these IL9 polymorphisms were more likely to report a severe asthma exacerbation if exposed to increased dust mite levels. CONCLUSIONS: Genome-wide differential gene expression in response to dust mite allergen identified IL9, a biologically plausible gene target that might interact with environmental dust mite to increase severe asthma exacerbations in children.

Expression quantitative trait loci (eQTL) mapping in Puerto Rican children.

BACKGROUND: Expression quantitative trait loci (eQTL) have been identified using tissue or cell samples from diverse human populations, thus enhancing our understanding of regulation of gene expression. However, few studies have attempted to identify eQTL in racially admixed populations such as Hispanics. METHODS: We performed a systematic eQTL study to identify regulatory variants of gene expression in whole blood from 121 Puerto Rican children with (n = 63) and without (n = 58) asthma. Genome-wide genotyping was conducted using the Illumina Omni2.5M Bead Chip, and gene expression was assessed using the Illumina HT-12 microarray. After completing quality control, we performed a pair-wise genome analysis of ~15 K transcripts and ~1.3 M SNPs for both local and distal effects. This analysis was conducted under a regression framework adjusting for age, gender and principal components derived from both genotypic and mRNA data. We used a false discovery rate (FDR) approach to identify significant eQTL signals, which were next compared to top eQTL signals from existing eQTL databases. We then performed a pathway analysis for our top genes. RESULTS: We identified 36,720 local pairs in 3,391 unique genes and 1,851 distal pairs in 446 unique genes at FDR <0.05, corresponding to unadjusted P values lower than 1.5x10-4 and 4.5x10-9, respectively. A significant proportion of genes identified in our study overlapped with those identified in previous studies. We also found an enrichment of disease-related genes in our eQTL list. CONCLUSIONS: We present results from the first eQTL study in Puerto Rican children, who are members of a unique Hispanic cohort disproportionately affected with asthma, prematurity, obesity and other common diseases. Our study confirmed eQTL signals identified in other ethnic groups, while also detecting additional eQTLs unique to our study population. The identified eQTLs will help prioritize findings from future genome-wide association studies in Puerto Ricans.

A genome-wide association study of chronic obstructive pulmonary disease in Hispanics.

RATIONALE: Genome-wide association studies (GWAS) of chronic obstructive pulmonary disease (COPD) have identified disease-susceptibility loci, mostly in subjects of European descent. OBJECTIVES: We hypothesized that by studying Hispanic populations we would be able to identify unique loci that contribute to COPD pathogenesis in Hispanics but remain undetected in GWAS of non-Hispanic populations. METHODS: We conducted a metaanalysis of two GWAS of COPD in independent cohorts of Hispanics in Costa Rica and the United States (Multi-Ethnic Study of Atherosclerosis [MESA]). We performed a replication study of the top single-nucleotide polymorphisms in an independent Hispanic cohort in New Mexico (the Lovelace Smokers Cohort). We also attempted to replicate prior findings from genome-wide studies in non-Hispanic populations in Hispanic cohorts. MEASUREMENTS AND MAIN RESULTS: We found no genome-wide significant association with COPD in our metaanalysis of Costa Rica and MESA. After combining the top results from this metaanalysis with those from our replication study in the Lovelace Smokers Cohort, we identified two single-nucleotide polymorphisms approaching genome-wide significance for an association with COPD. The first (rs858249, combined P value = 6.1 × 10(-8)) is near the genes KLHL7 and NUPL2 on chromosome 7. The second (rs286499, combined P value = 8.4 × 10(-8)) is located in an intron of DLG2. The two most significant single-nucleotide polymorphisms in FAM13A from a previous genome-wide study in non-Hispanics were associated with COPD in Hispanics. CONCLUSIONS: We have identified two novel loci (in or near the genes KLHL7/NUPL2 and DLG2) that may play a role in COPD pathogenesis in Hispanic populations.

Breastfeeding duration and asthma in Puerto Rican children.

RATIONALE: Little is known about breastfeeding and asthma in Puerto Ricans, the ethnic group most affected by this disease in the US. We examined the relation between the currently recommended duration of breastfeeding and asthma in school-aged Puerto Rican children. METHODS: Case-control study of 1,127 Puerto Rican children aged 6-14 years living in Hartford, Connecticut (n = 449) and San Juan, Puerto Rico (n = 678). Parental recall of breastfeeding was categorized based on duration and according to current guidelines (i.e., none, 0-6 months, and >6 months). Asthma was defined as parental report of physician-diagnosed asthma and wheeze in the previous year. We used logistic regression for the multivariate analysis, which was conducted separately for each study site and for the combined cohort. All multivariate models were adjusted for age, gender, household income, atopy, maternal asthma, body mass index, early-life exposure to environmental tobacco smoke, and (for the combined cohort) study site. RESULTS: After adjustment for covariates, children who were breastfed for up to 6 months had 30% lower odds of asthma (95% CI = 0.5-1.0, P = 0.04) than those who were not breastfed. In this analysis, breastfeeding for longer than 6 months was not significantly associated with asthma (OR = 1.5, 95% CI = 1.0-2.4, P = 0.06). CONCLUSIONS: Our results suggest that breastfeeding for up to 6 months (as assessed by parental recall) is associated with decreased odds of asthma in Puerto Rican children, and that there is no additional beneficial effect of breastfeeding for over 6 months. These results support current recommendations on the duration of breastfeeding in an ethnic group at risk for asthma.

Mouse allergen exposure and decreased risk of allergic rhinitis in school-aged children.

BACKGROUND: Little is known about exposure to mouse allergen (Mus m 1) and allergic rhinitis (AR). OBJECTIVE: To evaluate the association between mouse allergen exposure and AR in children. METHODS: We examined the relation between mouse allergen level in house dust and AR in 511 children aged 6 to 14 years in San Juan, Puerto Rico. Study participants were chosen from randomly selected households using a multistage probability sample design. The study protocol included questionnaires, allergy skin testing, and collection of blood and dust samples. AR was defined as current rhinitis symptoms and skin test reactivity to at least one allergen. RESULTS: In the multivariate analyses, mouse allergen level was associated with a 25% decreased odds of AR in participating children (95% confidence interval, 0.62-0.92). Although endotoxin and mouse allergen levels were significantly correlated (r = 0.184, P < .001), the observed inverse association between Mus m 1 and AR was not explained by levels of endotoxin or other markers of microbial or fungal exposure (peptidoglycan and glucan). CONCLUSION: Mouse allergen exposure is associated with decreased odds of AR in Puerto Rican school-aged children.

Vitamin D and asthma-life after VIDA?

The vitamin D hypothesis postulates that lower vitamin D levels are causally associated with increased asthma risk and asthma severity. Multiple epidemiological studies have shown an inverse relationship between circulating vitamin D levels (in the form of 25-hydroxy vitamin D) and asthma severity and control and lung function. However, in the recently published vitamin D and asthma (VIDA) study, vitamin D supplementation failed to show an improvement in asthma control in adults. This article reviews the current epidemiological and trial evidence for vitamin D and asthma and explores some of the possible alternative explanations for previous findings (including "reverse causation" and the importance of studying children and adults). We also address some of the unique challenges of conducting vitamin D trials and potential ways to address them. Finally, I will argue for further clinical trials of vitamin D in asthma, especially in children, using knowledge gained from the VIDA trial.

Stress and asthma: novel insights on genetic, epigenetic, and immunologic mechanisms.

In the United States the economically disadvantaged and some ethnic minorities are often exposed to chronic psychosocial stressors and disproportionately affected by asthma. Current evidence suggests a causal association between chronic psychosocial stress and asthma or asthma morbidity. Recent findings suggest potential mechanisms underlying this association, including changes in the methylation and expression of genes that regulate behavioral, autonomic, neuroendocrine, and immunologic responses to stress. There is also evidence suggesting the existence of susceptibility genes that predispose chronically stressed youth to both post-traumatic stress disorder and asthma. In this review we critically examine published evidence and suggest future directions for research in this field.

Native American ancestry, lung function, and COPD in Costa Ricans.

BACKGROUND: Whether Native American ancestry (NAA) is associated with COPD or lung function in a racially admixed Hispanic population is unknown. METHODS: We recruited 578 Costa Ricans with and without COPD into a hybrid case-control/family-based cohort, including 316 members of families of index case subjects. All participants completed questionnaires and spirometry and gave a blood sample for DNA extraction. Genome-wide genotyping was conducted with the Illumina Human610-Quad and HumanOmniExpress BeadChip kits (Illumina Inc), and individual ancestral proportions were estimated from these genotypic data and reference panels. For unrelated individuals, linear or logistic regression was used for the analysis of NAA and COPD (GOLD [Global Initiative for Chronic Obstructive Lung Disease] stage II or greater) or lung function. For extended families, linear mixed models and generalized estimating equations were used for the analysis. All models were adjusted for age, sex, educational level, and smoking behavior; models for FEV1 were also adjusted for height. RESULTS: The average proportion of European, Native American, and African ancestry among participants was 62%, 35%, and 3%, respectively. After adjustment for current smoking and other covariates, NAA was inversely associated with COPD (OR per 10% increment, 0.55; 95% CI, 0.41-0.75) but positively associated with FEV1, FVC, and FEV1/FVC. After additional adjustment for pack-years of smoking, the association between NAA and COPD or lung function measures was slightly attenuated. We found that about 31% of the estimated effect of NAA on COPD is mediated by pack-years of smoking. CONCLUSIONS: NAA is inversely associated with COPD but positively associated with FEV1 or FVC in Costa Ricans. Ancestral effects on smoking behavior partly explain the findings for COPD but not for FEV1 or FVC.

Prematurity, atopy, and childhood asthma in Puerto Ricans.

BACKGROUND: Puerto Rican children share a disproportionate burden of prematurity and asthma in the United States. Little is known about prematurity and childhood asthma in Puerto Rican subjects. OBJECTIVE: We sought to examine whether prematurity is associated with asthma in Puerto Rican children. METHODS: We performed a case-control study of 678 children aged 6 to 14 years with (n = 351) and without (n = 327) asthma living in San Juan, Puerto Rico. Prematurity was defined by parental report for our primary analysis. In a secondary analysis, we only included children whose parents reported prematurity that required admission to the neonatal intensive care unit. Asthma was defined as physician-diagnosed asthma and wheeze in the prior year. We used logistic regression for analysis. All multivariate models were adjusted for age, sex, household income, atopy (≥1 positive IgE level to common allergens), maternal history of asthma, and early-life exposure to environmental tobacco smoke. RESULTS: In a multivariate analysis there was a significant interaction between prematurity and atopy on asthma (P = .006). In an analysis stratified by atopy, prematurity was associated with a nearly 5-fold increased odds of asthma in atopic children (adjusted odds ratio, 4.7; 95% CI, 1.5-14.3; P = .007). In contrast, there was no significant association between prematurity and asthma in nonatopic children. Similar results were obtained in our analysis of prematurity requiring admission to the neonatal intensive care unit and asthma. CONCLUSIONS: Our results suggest that atopy modifies the estimated effect of prematurity on asthma in Puerto Rican children. Prematurity might explain, in part, the high prevalence of atopic asthma in this ethnic group.