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1.  Integrated genome-wide association, coexpression network, and expression single nucleotide polymorphism analysis identifies novel pathway in allergic rhinitis 
BMC Medical Genomics  2014;7:48.
Allergic rhinitis is a common disease whose genetic basis is incompletely explained. We report an integrated genomic analysis of allergic rhinitis.
We performed genome wide association studies (GWAS) of allergic rhinitis in 5633 ethnically diverse North American subjects. Next, we profiled gene expression in disease-relevant tissue (peripheral blood CD4+ lymphocytes) collected from subjects who had been genotyped. We then integrated the GWAS and gene expression data using expression single nucleotide (eSNP), coexpression network, and pathway approaches to identify the biologic relevance of our GWAS.
GWAS revealed ethnicity-specific findings, with 4 genome-wide significant loci among Latinos and 1 genome-wide significant locus in the GWAS meta-analysis across ethnic groups. To identify biologic context for these results, we constructed a coexpression network to define modules of genes with similar patterns of CD4+ gene expression (coexpression modules) that could serve as constructs of broader gene expression. 6 of the 22 GWAS loci with P-value ≤ 1x10−6 tagged one particular coexpression module (4.0-fold enrichment, P-value 0.0029), and this module also had the greatest enrichment (3.4-fold enrichment, P-value 2.6 × 10−24) for allergic rhinitis-associated eSNPs (genetic variants associated with both gene expression and allergic rhinitis). The integrated GWAS, coexpression network, and eSNP results therefore supported this coexpression module as an allergic rhinitis module. Pathway analysis revealed that the module was enriched for mitochondrial pathways (8.6-fold enrichment, P-value 4.5 × 10−72).
Our results highlight mitochondrial pathways as a target for further investigation of allergic rhinitis mechanism and treatment. Our integrated approach can be applied to provide biologic context for GWAS of other diseases.
PMCID: PMC4127082  PMID: 25085501
Genome-wide association study; Allergic rhinitis; Coexpression network; Expression single-nucleotide polymorphism; Coexpression module; Pathway; Mitochondria; Hay fever; Allergy
2.  Vitamin D related genes in lung development and asthma pathogenesis 
BMC Medical Genomics  2013;6:47.
Poor maternal vitamin D intake is a risk factor for subsequent childhood asthma, suggesting that in utero changes related to vitamin D responsive genes might play a crucial role in later disease susceptibility. We hypothesized that vitamin D pathway genes are developmentally active in the fetal lung and that these developmental genes would be associated with asthma susceptibility and regulation in asthma.
Vitamin D pathway genes were derived from PubMed and Gene Ontology surveys. Principal component analysis was used to identify characteristic lung development genes.
Vitamin D regulated genes were markedly over-represented in normal human (odds ratio OR 2.15, 95% confidence interval CI: 1.69-2.74) and mouse (OR 2.68, 95% CI: 2.12-3.39) developing lung transcriptomes. 38 vitamin D pathway genes were in both developing lung transcriptomes with >63% of genes more highly expressed in the later than earlier stages of development. In immortalized B-cells derived from 95 asthmatics and their unaffected siblings, 12 of the 38 (31.6%) vitamin D pathway lung development genes were significantly differentially expressed (OR 3.00, 95% CI: 1.43-6.21), whereas 11 (29%) genes were significantly differentially expressed in 43 control versus vitamin D treated immortalized B-cells from Childhood Asthma Management Program subjects (OR 2.62, 95% CI: 1.22-5.50). 4 genes, LAMP3, PIP5K1B, SCARB2 and TXNIP were identified in both groups; each displays significant biologic plausibility for a role in asthma.
Our findings demonstrate a significant association between early lung development and asthma–related phenotypes for vitamin D pathway genes, supporting a genomic mechanistic basis for the epidemiologic observations relating maternal vitamin D intake and childhood asthma susceptibility.
PMCID: PMC4228235  PMID: 24188128
Vitamin D; Cholecalciferol; Lung development; Asthma; Fetal programming
3.  Delivery by caesarean section and risk of obesity in preschool age children: a prospective cohort study 
Archives of disease in childhood  2012;97(7):610-616.
To examine whether delivery by caesarean section is a risk factor for childhood obesity.
Prospective pre-birth cohort study (Project Viva).
Eight outpatient multi-specialty practices based in the Boston, Massachusetts area.
We recruited women during early pregnancy between 1999 and 2002, and followed their children after birth. We included 1255 children with body composition measured at 3 years of age.
Main outcome measures
Body mass index (BMI) z-score, obesity (BMI for age and sex ≥ 95th percentile), and sum of triceps + subscapular skinfold thicknesses, at 3 years of age.
284 children (22.6 percent) were delivered by caesarean section. At age 3, 15.7% of children delivered by caesarean section were obese, compared with 7.5% of children born vaginally. In multivariable logistic and linear regression models adjusting for maternal pre-pregnancy BMI, birth weight, and other covariates, birth by caesarean section was associated with a higher odds of obesity at age 3 (OR 2.10, 95%CI 1.36 to 3.23), higher mean BMI z-score (0.20 units, 95% CI 0.07 to 0.33), and higher sum of triceps + subscapular skinfold thicknesses (0.94 mm, 95% CI 0.36 to 1.51).
Infants delivered by caesarean section may be at increased risk of childhood obesity. Further studies are needed to confirm our findings and to explore mechanisms underlying this association.
PMCID: PMC3784307  PMID: 22623615
4.  ITGB5 and AGFG1 variants are associated with severity of airway responsiveness 
BMC Medical Genetics  2013;14:86.
Airway hyperresponsiveness (AHR), a primary characteristic of asthma, involves increased airway smooth muscle contractility in response to certain exposures. We sought to determine whether common genetic variants were associated with AHR severity.
A genome-wide association study (GWAS) of AHR, quantified as the natural log of the dosage of methacholine causing a 20% drop in FEV1, was performed with 994 non-Hispanic white asthmatic subjects from three drug clinical trials: CAMP, CARE, and ACRN. Genotyping was performed on Affymetrix 6.0 arrays, and imputed data based on HapMap Phase 2, was used to measure the association of SNPs with AHR using a linear regression model. Replication of primary findings was attempted in 650 white subjects from DAG, and 3,354 white subjects from LHS. Evidence that the top SNPs were eQTL of their respective genes was sought using expression data available for 419 white CAMP subjects.
The top primary GWAS associations were in rs848788 (P-value 7.2E-07) and rs6731443 (P-value 2.5E-06), located within the ITGB5 and AGFG1 genes, respectively. The AGFG1 result replicated at a nominally significant level in one independent population (LHS P-value 0.012), and the SNP had a nominally significant unadjusted P-value (0.0067) for being an eQTL of AGFG1.
Based on current knowledge of ITGB5 and AGFG1, our results suggest that variants within these genes may be involved in modulating AHR. Future functional studies are required to confirm that our associations represent true biologically significant findings.
PMCID: PMC3765944  PMID: 23984888
Asthma; Airway hyperresponsiveness; Genome-wide association study; ITGB5; AGFG1
5.  The Pharmacogenetics and Pharmacogenomics of Asthma Therapy 
The Pharmacogenomics Journal  2011;11(6):383-392.
Despite the availability of several classes of asthma medications and their overall effectiveness, a significant portion of patients fail to respond to these therapeutic agents. Evidence suggests that genetic factors may partly mediate the heterogeneity in asthma treatment response. This review discusses important findings in asthma pharmacogenetics and pharmacogenomics studies conducted to date, examines limitations of these studies and finally, proposes future research directions in this field. The focus will be on the three major classes of asthma medications: β-adrenergic receptor agonists, inhaled corticosteroids and leukotriene modifiers. Although many studies are limited by small sample sizes and replication of the findings is needed, several candidate genes have been identified. High-throughput technologies is also allowing for large-scale genetic investigations. Thus, the future is promising for a personalized treatment of asthma, which will improve therapeutic outcomes, minimize side effects and lead to a more cost-effective care.
PMCID: PMC3298891  PMID: 21987090
asthma; pharmacogenetics; pharmacogenomics
6.  Folate network genetic variation, plasma homocysteine, and global genomic methylation content: a genetic association study 
BMC Medical Genetics  2011;12:150.
Sequence variants in genes functioning in folate-mediated one-carbon metabolism are hypothesized to lead to changes in levels of homocysteine and DNA methylation, which, in turn, are associated with risk of cardiovascular disease.
330 SNPs in 52 genes were studied in relation to plasma homocysteine and global genomic DNA methylation. SNPs were selected based on functional effects and gene coverage, and assays were completed on the Illumina Goldengate platform. Age-, smoking-, and nutrient-adjusted genotype--phenotype associations were estimated in regression models.
Using a nominal P ≤ 0.005 threshold for statistical significance, 20 SNPs were associated with plasma homocysteine, 8 with Alu methylation, and 1 with LINE-1 methylation. Using a more stringent false discovery rate threshold, SNPs in FTCD, SLC19A1, and SLC19A3 genes remained associated with plasma homocysteine. Gene by vitamin B-6 interactions were identified for both Alu and LINE-1 methylation, and epistatic interactions with the MTHFR rs1801133 SNP were identified for the plasma homocysteine phenotype. Pleiotropy involving the MTHFD1L and SARDH genes for both plasma homocysteine and Alu methylation phenotypes was identified.
No single gene was associated with all three phenotypes, and the set of the most statistically significant SNPs predictive of homocysteine or Alu or LINE-1 methylation was unique to each phenotype. Genetic variation in folate-mediated one-carbon metabolism, other than the well-known effects of the MTHFR c.665C>T (known as c.677 C>T, rs1801133, p.Ala222Val), is predictive of cardiovascular disease biomarkers.
PMCID: PMC3266217  PMID: 22103680
7.  Genome Wide Association Study to predict severe asthma exacerbations in children using random forests classifiers 
BMC Medical Genetics  2011;12:90.
Personalized health-care promises tailored health-care solutions to individual patients based on their genetic background and/or environmental exposure history. To date, disease prediction has been based on a few environmental factors and/or single nucleotide polymorphisms (SNPs), while complex diseases are usually affected by many genetic and environmental factors with each factor contributing a small portion to the outcome. We hypothesized that the use of random forests classifiers to select SNPs would result in an improved predictive model of asthma exacerbations. We tested this hypothesis in a population of childhood asthmatics.
In this study, using emergency room visits or hospitalizations as the definition of a severe asthma exacerbation, we first identified a list of top Genome Wide Association Study (GWAS) SNPs ranked by Random Forests (RF) importance score for the CAMP (Childhood Asthma Management Program) population of 127 exacerbation cases and 290 non-exacerbation controls. We predict severe asthma exacerbations using the top 10 to 320 SNPs together with age, sex, pre-bronchodilator FEV1 percentage predicted, and treatment group.
Testing in an independent set of the CAMP population shows that severe asthma exacerbations can be predicted with an Area Under the Curve (AUC) = 0.66 with 160-320 SNPs in comparison to an AUC score of 0.57 with 10 SNPs. Using the clinical traits alone yielded AUC score of 0.54, suggesting the phenotype is affected by genetic as well as environmental factors.
Our study shows that a random forests algorithm can effectively extract and use the information contained in a small number of samples. Random forests, and other machine learning tools, can be used with GWAS studies to integrate large numbers of predictors simultaneously.
PMCID: PMC3148549  PMID: 21718536
8.  Expression analysis of asthma candidate genes during human and murine lung development 
Respiratory Research  2011;12(1):86.
Little is known about the role of most asthma susceptibility genes during human lung development. Genetic determinants for normal lung development are not only important early in life, but also for later lung function.
To investigate the role of expression patterns of well-defined asthma susceptibility genes during human and murine lung development. We hypothesized that genes influencing normal airways development would be over-represented by genes associated with asthma.
Asthma genes were first identified via comprehensive search of the current literature. Next, we analyzed their expression patterns in the developing human lung during the pseudoglandular (gestational age, 7-16 weeks) and canalicular (17-26 weeks) stages of development, and in the complete developing lung time series of 3 mouse strains: A/J, SW, C57BL6.
In total, 96 genes with association to asthma in at least two human populations were identified in the literature. Overall, there was no significant over-representation of the asthma genes among genes differentially expressed during lung development, although trends were seen in the human (Odds ratio, OR 1.22, confidence interval, CI 0.90-1.62) and C57BL6 mouse (OR 1.41, CI 0.92-2.11) data. However, differential expression of some asthma genes was consistent in both developing human and murine lung, e.g. NOD1, EDN1, CCL5, RORA and HLA-G. Among the asthma genes identified in genome wide association studies, ROBO1, RORA, HLA-DQB1, IL2RB and PDE10A were differentially expressed during human lung development.
Our data provide insight about the role of asthma susceptibility genes during lung development and suggest common mechanisms underlying lung morphogenesis and pathogenesis of respiratory diseases.
PMCID: PMC3141421  PMID: 21699702
Asthma; Development; Expression; Genetics; Lung
9.  Modulation of Lgl1 by steroid, retinoic acid, and Vitamin D models complex transcriptional regulation during alveolarization 
Pediatric research  2010;67(4):375-381.
Alveolarization depends on circulating glucocorticoid (GC), retinoid (RA) and Vitamin D (VitD). Bronchopulmonary dysplasia (BPD), a leading cause of neonatal morbidity, is associated with arrested alveolarization. In hyperoxia-exposed rats displaying features of BPD, reduced levels of Lgl1 normalize during recovery. We show that GC (100nM) stimulates (7–115 fold) and VitD (100µM) suppresses (2 fold) Lgl1 expression. RA (all trans/9-cis, 10µM) effects are biphasic. From postnatal (PN) days 7–10, RA was stimulatory (2 fold) at 24h, after which effects were inhibitory (3–15 fold). Lgl1 promoter-luciferase reporter assays confirmed that these agents operated at the transcriptional level. Interestingly, the individual inhibitory effects of VitD and RA on GC induction of Lgl1 were abrogated when both agents were present, suggesting that steric hindrance may influence promoter accessibility. Analysis of the proximity (<50 base pairs) of binding sites for overlapping VitD and RA receptors to that of the GC receptor identified 81% of promoters in 66 genes (including Lgl1) important in human lung development compared to 48% in a random set of 1000 genes. Complex integration of the effects of GC, RA, and VitD on gene expression in the postnatal lung is likely to contribute to the timely advance of alveolarization without attendant inflammation.
PMCID: PMC3104505  PMID: 20057335
10.  Thymic stromal lymphopoietin (TSLP) is associated with allergic rhinitis in children with asthma 
Allergic rhinitis (AR) affects up to 80% of children with asthma and increases asthma severity. Thymic stromal lymphopoietin (TSLP) is a key mediator of allergic inflammation. The role of the TSLP gene (TSLP) in the pathogenesis of AR has not been studied.
To test for associations between variants in TSLP, TSLP-related genes, and AR in children with asthma.
We genotyped 15 single nucleotide polymorphisms (SNPs) in TSLP, OX40L, IL7R, and RXRα in three independent cohorts: 592 asthmatic Costa Rican children and their parents, 422 nuclear families of North American children with asthma, and 239 Swedish children with asthma. We tested for associations between these SNPs and AR. As we previously reported sex-specific effects for TSLP, we performed overall and sex-stratified analyses. We additionally performed secondary analyses for gene-by-gene interactions.
Across the three cohorts, the T allele of TSLP SNP rs1837253 was undertransmitted in boys with AR and asthma as compared to boys with asthma alone. The SNP was associated with reduced odds for AR (odds ratios ranging from 0.56 to 0.63, with corresponding Fisher's combined P value of 1.2 × 10-4). Our findings were significant after accounting for multiple comparisons. SNPs in OX40L, IL7R, and RXRα were not consistently associated with AR in children with asthma. There were nominally significant interactions between gene pairs.
TSLP SNP rs1837253 is associated with reduced odds for AR in boys with asthma. Our findings support a role for TSLP in the pathogenesis of AR in children with asthma.
PMCID: PMC3032752  PMID: 21244681
11.  Maternal diet vs lack of exposure to sunlight as the cause of the epidemic of asthma, allergies and other autoimmune diseases 
Thorax  2007;62(9):746-748.
Role of vitamin D deficiency in allergic and autoimmune diseases
PMCID: PMC2117306  PMID: 17726168
12.  Asthma-susceptibility variants identified using probands in case-control and family-based analyses 
BMC Medical Genetics  2010;11:122.
Asthma is a chronic respiratory disease whose genetic basis has been explored for over two decades, most recently via genome-wide association studies. We sought to find asthma-susceptibility variants by using probands from a single population in both family-based and case-control association designs.
We used probands from the Childhood Asthma Management Program (CAMP) in two primary genome-wide association study designs: (1) probands were combined with publicly available population controls in a case-control design, and (2) probands and their parents were used in a family-based design. We followed a two-stage replication process utilizing three independent populations to validate our primary findings.
We found that single nucleotide polymorphisms with similar case-control and family-based association results were more likely to replicate in the independent populations, than those with the smallest p-values in either the case-control or family-based design alone. The single nucleotide polymorphism that showed the strongest evidence for association to asthma was rs17572584, which replicated in 2/3 independent populations with an overall p-value among replication populations of 3.5E-05. This variant is near a gene that encodes an enzyme that has been implicated to act coordinately with modulators of Th2 cell differentiation and is expressed in human lung.
Our results suggest that using probands from family-based studies in case-control designs, and combining results of both family-based and case-control approaches, may be a way to augment our ability to find SNPs associated with asthma and other complex diseases.
PMCID: PMC2927535  PMID: 20698975
13.  Predicting response to short-acting bronchodilator medication using Bayesian networks 
Pharmacogenomics  2009;10(9):1393-1412.
Bronchodilator response tests measure the effect of β2-agonists, the most commonly used short-acting reliever drugs for asthma. We sought to relate candidate gene SNP data with bronchodilator response and measure the predictive accuracy of a model constructed with genetic variants.
Materials & methods
Bayesian networks, multivariate models that are able to account for simultaneous associations and interactions among variables, were used to create a predictive model of bronchodilator response using candidate gene SNP data from 308 Childhood Asthma Management Program Caucasian subjects.
The model found that 15 SNPs in 15 genes predict bronchodilator response with fair accuracy, as established by a fivefold cross-validation area under the receiver-operating characteristic curve of 0.75 (standard error: 0.03).
Bayesian networks are an attractive approach to analyze large-scale pharmacogenetic SNP data because of their ability to automatically learn complex models that can be used for the prediction and discovery of novel biological hypotheses.
PMCID: PMC2804237  PMID: 19761364
asthma; Bayesian networks; β2-agonists; bronchodilator response; prediction
14.  Predictors of poor response during asthma therapy differ with definition of outcome 
Pharmacogenomics  2009;10(8):1231-1242.
To evaluate phenotypic and genetic variables associated with a poor long-term response to inhaled corticosteroid therapy for asthma, based independently on lung function changes or asthma exacerbations.
Materials & methods
We tested 17 phenotypic variables and polymorphisms in FCER2 and CRHR1 in 311 children (aged 5–12 years) randomized to a 4-year course of inhaled corticosteroid during the Childhood Asthma Management Program (CAMP).
Predictors of recurrent asthma exacerbations are distinct from predictors of poor lung function response. A history of prior asthma exacerbations, younger age and a higher IgE level (p < 0.05) are associated with recurrent exacerbations. By contrast, lower bronchodilator response to albuterol and the minor alleles of RS242941 in CRHR1 and T2206C in FCER2 (p < 0.05) are associated with poor lung function response. Poor lung function response does not increase the risk of exacerbations and vice versa (p = 0.72).
Genetic and phenotypic predictors of a poor long-term response to inhaled corticosteroids differ markedly depending on definition of outcome (based on exacerbations vs lung function). These findings are important in comparing outcomes of clinical trials and in designing future pharmacogenetic studies.
PMCID: PMC2746392  PMID: 19663668
asthma; corticosteroid; exacerbation; lung function; pharmacogenetics
15.  Asthma and genes encoding components of the vitamin D pathway 
Respiratory Research  2009;10(1):98.
Genetic variants at the vitamin D receptor (VDR) locus are associated with asthma and atopy. We hypothesized that polymorphisms in other genes of the vitamin D pathway are associated with asthma or atopy.
Eleven candidate genes were chosen for this study, five of which code for proteins in the vitamin D metabolism pathway (CYP27A1, CYP27B1, CYP2R1, CYP24A1, GC) and six that are known to be transcriptionally regulated by vitamin D (IL10, IL1RL1, CD28, CD86, IL8, SKIIP). For each gene, we selected a maximally informative set of common SNPs (tagSNPs) using the European-derived (CEU) HapMap dataset. A total of 87 SNPs were genotyped in a French-Canadian family sample ascertained through asthmatic probands (388 nuclear families, 1064 individuals) and evaluated using the Family Based Association Test (FBAT) program. We then sought to replicate the positive findings in four independent samples: two from Western Canada, one from Australia and one from the USA (CAMP).
A number of SNPs in the IL10, CYP24A1, CYP2R1, IL1RL1 and CD86 genes were modestly associated with asthma and atopy (p < 0.05). Two-gene models testing for both main effects and the interaction were then performed using conditional logistic regression. Two-gene models implicating functional variants in the IL10 and VDR genes as well as in the IL10 and IL1RL1 genes were associated with asthma (p < 0.0002). In the replicate samples, SNPs in the IL10 and CYP24A1 genes were again modestly associated with asthma and atopy (p < 0.05). However, the SNPs or the orientation of the risk alleles were different between populations. A two-gene model involving IL10 and VDR was replicated in CAMP, but not in the other populations.
A number of genes involved in the vitamin D pathway demonstrate modest levels of association with asthma and atopy. Multilocus models testing genes in the same pathway are potentially more effective to evaluate the risk of asthma, but the effects are not uniform across populations.
PMCID: PMC2779188  PMID: 19852851
16.  Importin-13 genetic variation is associated with improved airway responsiveness in childhood asthma 
Respiratory Research  2009;10(1):67.
Glucocorticoid function is dependent on efficient translocation of the glucocorticoid receptor (GR) from the cytoplasm to the nucleus of cells. Importin-13 (IPO13) is a nuclear transport receptor that mediates nuclear entry of GR. In airway epithelial cells, inhibition of IPO13 expression prevents nuclear entry of GR and abrogates anti-inflammatory effects of glucocorticoids. Impaired nuclear entry of GR has been documented in steroid-non-responsive asthmatics. We hypothesize that common IPO13 genetic variation influences the anti-inflammatory effects of inhaled corticosteroids for the treatment of asthma, as measured by change in methacholine airway hyperresponsiveness (AHR-PC20).
10 polymorphisms were evaluated in 654 children with mild-to-moderate asthma participating in the Childhood Asthma Management Program (CAMP), a clinical trial of inhaled anti-inflammatory medications (budesonide and nedocromil). Population-based association tests with repeated measures of PC20 were performed using mixed models and confirmed using family-based tests of association.
Among participants randomized to placebo or nedocromil, IPO13 polymorphisms were associated with improved PC20 (i.e. less AHR), with subjects harboring minor alleles demonstrating an average 1.51–2.17 fold increase in mean PC20 at 8-months post-randomization that persisted over four years of observation (p = 0.01–0.005). This improvement was similar to that among children treated with long-term inhaled corticosteroids. There was no additional improvement in PC20 by IPO13 variants among children treated with inhaled corticosteroids.
IPO13 variation is associated with improved AHR in asthmatic children. The degree of this improvement is similar to that observed with long-term inhaled corticosteroid treatment, suggesting that IPO13 variation may improve nuclear bioavailability of endogenous glucocorticoids.
PMCID: PMC2724419  PMID: 19619331
17.  Recommendations for using standardised phenotypes in genetic association studies 
Human Genomics  2009;3(4):308-319.
Genetic association studies of complex traits often rely on standardised quantitative phenotypes, such as percentage of predicted forced expiratory volume and body mass index to measure an underlying trait of interest (eg lung function, obesity). These phenotypes are appealing because they provide an easy mechanism for comparing subjects, although such standardisations may not be the best way to control for confounders and other covariates. We recommend adjusting raw or standardised phenotypes within the study population via regression. We illustrate through simulation that optimal power in both population- and family-based association tests is attained by using the residuals from within-study adjustment as the complex trait phenotype. An application of family-based association analysis of forced expiratory volume in one second, and obesity in the Childhood Asthma Management Program data, illustrates that power is maintained or increased when adjusted phenotype residuals are used instead of typical standardised quantitative phenotypes.
PMCID: PMC3525193  PMID: 19706362
body mass index; confounding factors; covariate adjustment; forced expiratory volume; heritable quantitative traits
18.  Platform dependence of inference on gene-wise and gene-set involvement in human lung development 
BMC Bioinformatics  2009;10:189.
With the recent development of microarray technologies, the comparability of gene expression data obtained from different platforms poses an important problem. We evaluated two widely used platforms, Affymetrix U133 Plus 2.0 and the Illumina HumanRef-8 v2 Expression Bead Chips, for comparability in a biological system in which changes may be subtle, namely fetal lung tissue as a function of gestational age.
We performed the comparison via sequence-based probe matching between the two platforms. "Significance grouping" was defined as a measure of comparability. Using both expression correlation and significance grouping as measures of comparability, we demonstrated that despite overall cross-platform differences at the single gene level, increased correlation between the two platforms was found in genes with higher expression level, higher probe overlap, and lower p-value. We also demonstrated that biological function as determined via KEGG pathways or GO categories is more consistent across platforms than single gene analysis.
We conclude that while the comparability of the platforms at the single gene level may be increased by increasing sample size, they are highly comparable ontologically even for subtle differences in a relatively small sample size. Biologically relevant inference should therefore be reproducible across laboratories using different platforms.
PMCID: PMC2711081  PMID: 19545372
19.  A graphical model approach for inferring large-scale networks integrating gene expression and genetic polymorphism 
BMC Systems Biology  2009;3:55.
Graphical models (e.g., Bayesian networks) have been used frequently to describe complex interaction patterns and dependent structures among genes and other phenotypes. Estimation of such networks has been a challenging problem when the genes considered greatly outnumber the samples, and the situation is exacerbated when one wishes to consider the impact of polymorphisms (SNPs) in genes.
Here we describe a multistep approach to infer a gene-SNP network from gene expression and genotyped SNP data. Our approach is based on 1) construction of a graphical Gaussian model (GGM) based on small sample estimation of partial correlation and false-discovery rate multiple testing; 2) extraction of a subnetwork of genes directly linked to a target candidate gene of interest; 3) identification of cis-acting regulatory variants for the genes composing the subnetwork; and 4) evaluating the identified cis-acting variants for trans-acting regulatory effects of the target candidate gene. This approach identifies significant gene-gene and gene-SNP associations not solely on the basis of gene co-expression but rather through whole-network modeling. We demonstrate the method by building two complex gene-SNP networks around Interferon Receptor 12B2 (IL12RB2) and Interleukin 1B (IL1B), two biologic candidates in asthma pathogenesis, using 534,290 genotyped variants and gene expression data on 22,177 genes from total RNA derived from peripheral blood CD4+ lymphocytes from 154 asthmatics.
Our results suggest that graphical models based on integrative genomic data are computationally efficient, work well with small samples, and can describe complex interactions among genes and polymorphisms that could not be identified by pair-wise association testing.
PMCID: PMC2694152  PMID: 19473523
20.  Diversity of the gut microbiota and eczema in early life 
A modest number of prospective studies of the composition of the intestinal microbiota and eczema in early life have yielded conflicting results.
To examine the relationship between the bacterial diversity of the gut and the development of eczema in early life by methods other than stool culture.
Fecal samples were collected from 21 infants at 1 and 4 months of life. Nine infants were diagnosed with eczema by the age of 6 months (cases) and 12 infants were not (controls). After conducting denaturating gradient gel electrophoresis (DGGE) of stool samples, we compared the microbial diversity of cases and controls using the number of electrophoretic bands and the Shannon index of diversity (H') as indicators.
Control subjects had significantly greater fecal microbial diversity than children with eczema at ages 1 (mean H' for controls = 0.75 vs. 0.53 for cases, P = 0.01) and 4 months (mean H' for controls = 0.92 vs. 0.59 for cases, P = 0.02). The increase in diversity from 1 to 4 months of age was significant in controls (P = 0.04) but not in children who developed eczema by 6 months of age (P = 0.32).
Our findings suggest that reduced microbial diversity is associated with the development of eczema in early life.
PMCID: PMC2562383  PMID: 18808715
21.  Practice-Level Effects of Interventions to Improve Asthma Care in Primary Care Settings: The Pediatric Asthma Care Patient Outcomes Research Team 
Health Services Research  2005;40(6 Pt 1):1737-1757.
To assess the practice-level effects of (1) a physician peer leader intervention and (2) peer leaders in combination with the introduction of asthma education nurses to facilitate care improvement. And, to compare findings with previously reported patient-level outcomes of trial enrollees.
Study Setting
Data were included on children 5–17 years old with asthma in 40 primary care practices, affiliated with managed health care plans enrolled in the Pediatric Asthma Care Patient Outcomes Research Team (PORT) randomized trial.
Study Design
Primary care practices were randomly assigned to one of two care improvement arms or to usual care. Automated claims data were analyzed for 12-month periods using a repeated cross-sectional design. The primary outcome was evidence of at least one controller medication dispensed among patients with persistent asthma. Secondary outcomes included controller dispensing among all identified asthmatics, evidence of chronic controller use, and the dispensing of oral steroids. Health service utilization outcomes included numbers of ambulatory visits and hospital-based events.
Principal Findings
The proportion of children with persistent asthma prescribed controllers increased in all study arms. No effect of the interventions on the proportion receiving controllers was detected (peer leader intervention effect 0.01, 95 percent confidence interval [CI]: −0.07, 0.08; planned care intervention effect −0.03, 95 percent CI: −0.09, 0.02). A statistical trend was seen toward an increased number of oral corticosteroid bursts dispensed in intervention practices. Significant adjusted increases in ambulatory visits of 0.08–0.10 visits per child per year were seen in the first intervention year, but only a statistical trend in these outcomes persisted into the second year of follow-up. No differences in hospital-based events were detected.
This analysis showed a slight increase in ambulatory asthma visits as a result of asthma care improvement interventions, using automated data. The absence of detectable impact on medication use at the practice level differs from the positive intervention effect observed in patient self-reported data from trial enrollees. Analysis of automated data on nonenrollees adds information about practice-level impact of care improvement strategies. Benefits of practice-level interventions may accrue disproportionately to the subgroup of trial enrollees. The effect of such interventions may be less apparent at the level of practices or health plans.
PMCID: PMC1361234  PMID: 16336546
Asthma care; randomized controlled trial; chronic care model; physician behavior change
22.  Maternal antioxidant intake in pregnancy and wheezing illnesses in children at 2 y of age2 
Low intakes of dietary antioxidants may contribute to increases in asthma and allergy.
We investigated the association of maternal total intakes (foods + supplements) of 10 antioxidant nutrients during pregnancy with wheezing and eczema in 2-y-old children.
Subjects were 1290 mother-child pairs in an ongoing cohort study. Maternal dietary and supplement intakes were assessed by using a validated food-frequency questionnaire administered in the first and second trimesters. Antioxidant nutrient intakes were calculated, and the mean for each nutrient was considered to be the exposure during pregnancy. The outcomes of interest were any wheezing by the child during either the first or second year of life, recurrent wheezing in both years, and eczema in either the first or second year.
No association was observed between maternal total intake of any antioxidant nutrient and eczema. In multivariate logistic regression models, the highest quartile compared with the lowest quartile of maternal total intakes of vitamin E [odds ratio (OR): 0.70; 95% CI: 0.48, 1.03] and zinc (OR: 0.59; 95% CI: 0.41, 0.88) was inversely associated with any wheezing at 2 y of age (P for trend = 0.06 and 0.01 over quartiles of intake for vitamin E and zinc, respectively). Similar results were obtained for recurrent wheezing at 2 y of age with vitamin E (OR: 0.49; 95% CI: 0.27, 0.90) and zinc (OR: 0.49; 95% CI: 0.27, 0.87) (P for trend = 0.05 and 0.06 over quartiles of intake for vitamin E and zinc, respectively).
Our results suggest that higher maternal total intakes of antioxidants during pregnancy may decrease the risks for wheezing illnesses in early childhood.
PMCID: PMC1994925  PMID: 17023719
Asthma; diet; antioxidants; eczema; childhood wheezing
23.  The Association of a SNP Upstream of INSIG2 with Body Mass Index is Reproduced in Several but Not All Cohorts 
PLoS Genetics  2007;3(4):e61.
A SNP upstream of the INSIG2 gene, rs7566605, was recently found to be associated with obesity as measured by body mass index (BMI) by Herbert and colleagues. The association between increased BMI and homozygosity for the minor allele was first observed in data from a genome-wide association scan of 86,604 SNPs in 923 related individuals from the Framingham Heart Study offspring cohort. The association was reproduced in four additional cohorts, but was not seen in a fifth cohort. To further assess the general reproducibility of this association, we genotyped rs7566605 in nine large cohorts from eight populations across multiple ethnicities (total n = 16,969). We tested this variant for association with BMI in each sample under a recessive model using family-based, population-based, and case-control designs. We observed a significant (p < 0.05) association in five cohorts but saw no association in three other cohorts. There was variability in the strength of association evidence across examination cycles in longitudinal data from unrelated individuals in the Framingham Heart Study Offspring cohort. A combined analysis revealed significant independent validation of this association in both unrelated (p = 0.046) and family-based (p = 0.004) samples. The estimated risk conferred by this allele is small, and could easily be masked by small sample size, population stratification, or other confounders. These validation studies suggest that the original association is less likely to be spurious, but the failure to observe an association in every data set suggests that the effect of SNP rs7566605 on BMI may be heterogeneous across population samples.
Author Summary
Obesity is an epidemic in the United States of America and developing world, portending an epidemic of related diseases such as diabetes and heart disease. While diet and lifestyle contribute to obesity, half of the population variation in body mass index, a common measure of obesity, is determined by inherited factors. Many studies have reported that common sequence variants in genes are associated with an increased risk for obesity, yet most of these are not reproducible in other study cohorts, suggesting that some are false. Recently, Herbert et al. reported a slightly increased risk of obesity for people carrying two copies of the minor allele at a common variant near INSIG2. We present our attempts to further evaluate this potential association with obesity in additional populations. We find evidence of increased risk of obesity for people carrying two copies of the minor allele in five out of nine cohorts tested, using both family- and population-based testing. We indicate possible reasons for the varied results, with the hope of encouraging a combined analysis across study cohorts to more precisely define the effect of this INSIG2 gene variant.
PMCID: PMC1857727  PMID: 17465681
24.  Mode of delivery and cord blood cytokines: a birth cohort study 
The mechanisms for the association between birth by cesarean section and atopy and asthma are largely unknown.
To examine whether cesarean section results in neonatal secretion of cytokines that are associated with increased risk of atopy and/or asthma in childhood. To examine whether the association between mode of delivery and neonatal immune responses is explained by exposure to the maternal gut flora (a marker of the vaginal flora).
CBMCs were isolated from 37 neonates at delivery, and secretion of IL-13, IFN-γ, and IL-10 (at baseline and after stimulation with antigens [dust mite and cat dander allergens, phytohemagglutinin, and lipopolysaccharide]) was quantified by ELISA. Total and specific microbes were quantified in maternal stool. The relation between mode of delivery and cord blood cytokines was examined by linear regression. The relation between maternal stool microbes and cord blood cytokines was examined by Spearman's correlation coefficients.
Cesarean section was associated with increased levels of IL-13 and IFN-γ. In multivariate analyses, cesarean section was associated with an increment of 79.4 pg/ml in secretion of IL-13 by CBMCs after stimulation with dust mite allergen (P < 0.001). Among children born by vaginal delivery, gram-positive anaerobes and total anaerobes in maternal stool were positively correlated with levels of IL-10, and gram-negative aerobic bacteria in maternal stool were negatively correlated with levels of IL-13 and IFN-γ.
Cesarean section is associated with increased levels of IL-13 and IFN-γ, perhaps because of lack of labor and/or reduced exposure to specific microbes (e.g., gram-positive anaerobes) at birth.
PMCID: PMC1592116  PMID: 17002791
25.  Polymorphisms in signal transducer and activator of transcription 3 and lung function in asthma 
Respiratory Research  2005;6(1):52.
Identifying genetic determinants for lung function is important in providing insight into the pathophysiology of asthma. Signal transducer and activator of transcription 3 is a transcription factor latent in the cytoplasm; the gene (STAT3) is activated by a wide range of cytokines, and may play a role in lung development and asthma pathogenesis.
We genotyped six single nucleotide polymorphisms (SNPs) in the STAT3 gene in a cohort of 401 Caucasian adult asthmatics. The associations between each SNP and forced expiratory volume in 1 second (FEV1), as a percent of predicted, at the baseline exam were tested using multiple linear regression models. Longitudinal analyses involving repeated measures of FEV1 were conducted with mixed linear models. Haplotype analyses were conducted using imputed haplotypes. We completed a second association study by genotyping the same six polymorphisms in a cohort of 652 Caucasian children with asthma.
We found that three polymorphisms were significantly associated with baseline FEV1: homozygotes for the minor alleles of each polymorphism had lower FEV1 than homozygotes for the major alleles. Moreover, these associations persisted when we performed an analysis on repeated measures of FEV1 over 8 weeks. A haplotypic analysis based on the six polymorphisms indicated that two haplotypes were associated with baseline FEV1. Among the childhood asthmatics, one polymorphism was associated with both baseline FEV1 and the repeated measures of FEV1 over 4 years.
Our results indicate that genetic variants in STAT3, independent of asthma treatment, are determinants of FEV1 in both adults and children with asthma, and suggest that STAT3 may participate in inflammatory pathways that have an impact on level of lung function.
PMCID: PMC1180474  PMID: 15935090

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