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1.  Prenatal Tobacco Smoke Exposure Is Associated with Childhood DNA CpG Methylation 
PLoS ONE  2014;9(6):e99716.
Background
Smoking while pregnant is associated with a myriad of negative health outcomes in the child. Some of the detrimental effects may be due to epigenetic modifications, although few studies have investigated this hypothesis in detail.
Objectives
To characterize site-specific epigenetic modifications conferred by prenatal smoking exposure within asthmatic children.
Methods
Using Illumina HumanMethylation27 microarrays, we estimated the degree of methylation at 27,578 distinct DNA sequences located primarily in gene promoters using whole blood DNA samples from the Childhood Asthma Management Program (CAMP) subset of Asthma BRIDGE childhood asthmatics (n = 527) ages 5–12 with prenatal smoking exposure data available. Using beta-regression, we screened loci for differential methylation related to prenatal smoke exposure, adjusting for gender, age and clinical site, and accounting for multiple comparisons by FDR.
Results
Of 27,578 loci evaluated, 22,131 (80%) passed quality control assessment and were analyzed. Sixty-five children (12%) had a history of prenatal smoke exposure. At an FDR of 0.05, we identified 19 CpG loci significantly associated with prenatal smoke, of which two replicated in two independent populations. Exposure was associated with a 2% increase in mean CpG methylation in FRMD4A (p = 0.01) and Cllorf52 (p = 0.001) compared to no exposure. Four additional genes, XPNPEP1, PPEF2, SMPD3 and CRYGN, were nominally associated in at least one replication group.
Conclusions
These data suggest that prenatal exposure to tobacco smoke is associated with reproducible epigenetic changes that persist well into childhood. However, the biological significance of these altered loci remains unknown.
doi:10.1371/journal.pone.0099716
PMCID: PMC4070909  PMID: 24964093
2.  RNA-Seq Transcriptome Profiling Identifies CRISPLD2 as a Glucocorticoid Responsive Gene that Modulates Cytokine Function in Airway Smooth Muscle Cells 
PLoS ONE  2014;9(6):e99625.
Asthma is a chronic inflammatory respiratory disease that affects over 300 million people worldwide. Glucocorticoids are a mainstay therapy for asthma because they exert anti-inflammatory effects in multiple lung tissues, including the airway smooth muscle (ASM). However, the mechanism by which glucocorticoids suppress inflammation in ASM remains poorly understood. Using RNA-Seq, a high-throughput sequencing method, we characterized transcriptomic changes in four primary human ASM cell lines that were treated with dexamethasone—a potent synthetic glucocorticoid (1 µM for 18 hours). Based on a Benjamini-Hochberg corrected p-value <0.05, we identified 316 differentially expressed genes, including both well known (DUSP1, KLF15, PER1, TSC22D3) and less investigated (C7, CCDC69, CRISPLD2) glucocorticoid-responsive genes. CRISPLD2, which encodes a secreted protein previously implicated in lung development and endotoxin regulation, was found to have SNPs that were moderately associated with inhaled corticosteroid resistance and bronchodilator response among asthma patients in two previously conducted genome-wide association studies. Quantitative RT-PCR and Western blotting showed that dexamethasone treatment significantly increased CRISPLD2 mRNA and protein expression in ASM cells. CRISPLD2 expression was also induced by the inflammatory cytokine IL1β, and small interfering RNA-mediated knockdown of CRISPLD2 further increased IL1β-induced expression of IL6 and IL8. Our findings offer a comprehensive view of the effect of a glucocorticoid on the ASM transcriptome and identify CRISPLD2 as an asthma pharmacogenetics candidate gene that regulates anti-inflammatory effects of glucocorticoids in the ASM.
doi:10.1371/journal.pone.0099625
PMCID: PMC4057123  PMID: 24926665
3.  CGBayesNets: Conditional Gaussian Bayesian Network Learning and Inference with Mixed Discrete and Continuous Data 
PLoS Computational Biology  2014;10(6):e1003676.
Bayesian Networks (BN) have been a popular predictive modeling formalism in bioinformatics, but their application in modern genomics has been slowed by an inability to cleanly handle domains with mixed discrete and continuous variables. Existing free BN software packages either discretize continuous variables, which can lead to information loss, or do not include inference routines, which makes prediction with the BN impossible. We present CGBayesNets, a BN package focused around prediction of a clinical phenotype from mixed discrete and continuous variables, which fills these gaps. CGBayesNets implements Bayesian likelihood and inference algorithms for the conditional Gaussian Bayesian network (CGBNs) formalism, one appropriate for predicting an outcome of interest from, e.g., multimodal genomic data. We provide four different network learning algorithms, each making a different tradeoff between computational cost and network likelihood. CGBayesNets provides a full suite of functions for model exploration and verification, including cross validation, bootstrapping, and AUC manipulation. We highlight several results obtained previously with CGBayesNets, including predictive models of wood properties from tree genomics, leukemia subtype classification from mixed genomic data, and robust prediction of intensive care unit mortality outcomes from metabolomic profiles. We also provide detailed example analysis on public metabolomic and gene expression datasets. CGBayesNets is implemented in MATLAB and available as MATLAB source code, under an Open Source license and anonymous download at http://www.cgbayesnets.com.
doi:10.1371/journal.pcbi.1003676
PMCID: PMC4055564  PMID: 24922310
4.  Classification of childhood asthma phenotypes and long-term clinical responses to inhaled anti-inflammatory medications 
Background
Although recent studies have identified the presence of phenotypic clusters in asthmatic patients, the clinical significance and temporal stability of these clusters have not been explored.
Objective
Our aim was to examine the clinical relevance and temporal stability of phenotypic clusters in children with asthma.
Methods
We applied spectral clustering to clinical data from 1041 children with asthma participating in the Childhood Asthma Management Program. Posttreatment randomization follow-up data collected over 48 months were used to determine the effect of these clusters on pulmonary function and treatment response to inhaled anti-inflammatory medication.
Results
We found 5 reproducible patient clusters that could be differentiated on the basis of 3 groups of features: atopic burden, degree of airway obstruction, and history of exacerbation. Cluster grouping predicted long-term asthma control, as measured by the need for oral prednisone (P < .0001) or additional controller medications (P = .001), as well as longitudinal differences in pulmonary function (P < .0001). We also found that the 2 clusters with the highest rates of exacerbation had different responses to inhaled corticosteroids when compared with the other clusters. One cluster demonstrated a positive response to both budesonide (P = .02) and nedocromil (P = .01) compared with placebo, whereas the other cluster demonstrated minimal responses to both budesonide (P = .12) and nedocromil (P = .56) compared with placebo.
Conclusion
Phenotypic clustering can be used to identify longitudinally consistent and clinically relevant patient subgroups, with implications for targeted therapeutic strategies and clinical trials design.
PMCID: PMC4047642  PMID: 24892144
Childhood asthma; asthma phenotypes; inhaled corticosteroids; cluster analysis; asthma classification; longitudinal study
5.  Germline Variants and Advanced Colorectal Adenomas: Adenoma Prevention with Celecoxib Trial Genomewide Association Study 
Purpose
Identification of single nucleotide polymorphisms (SNPs) associated with development of advanced colorectal adenomas.
Experimental Design
Discovery Phase: 1,406 Caucasian patients (139 advanced adenoma cases and 1,267 controls) from the Adenoma Prevention with Celecoxib (APC) trial were included in a genome-wide association study (GWAS) to identify variants associated with post-polypectomy disease recurrence. Genome-wide significance was defined as false discovery rate < 0.05, unadjusted p=7.4×10−7. Validation Phase: Results were further evaluated using 4,175 familial colorectal adenoma or CRC cases and 5,036 controls from patients of European ancestry (COloRectal Gene Identification consortium, Scotland, Australia and VQ58).
Results
Our study identified eight SNPs associated with advanced adenoma risk in the APC trial (rs2837156, rs7278863, rs2837237, rs2837241, rs2837254, rs741864 at 21q22.2, and rs1381392 and rs17651822 at 3p24.1, at p<10–7 level with odds ratio – OR>2). Five variants in strong pairwise linkage disequilbrium (rs7278863, rs2837237, rs741864, rs741864 and rs2837241, r2=0.8–1) are in or near the coding region for the tight junction adhesion protein, IGSF5. An additional variant associated with advanced adenomas, rs1535989 (minor allele frequency 0.11; OR 2.09; 95% confidence interval 1.50–2.91), also predicted CRC development in a validation analysis (p=0.019) using a series of adenoma cases or CRC (CORGI study) and 3 sets of CRC cases and controls (Scotland, VQ58 and Australia, N=9,211).
Conclusions
Our results suggest that common polymorphisms contribute to the risk of developing advanced adenomas and might also contribute to the risk of developing CRC. The variant at rs1535989 may identify patients whose risk for neoplasia warrants increased colonoscopic surveillance.
doi:10.1158/1078-0432.CCR-13-0550
PMCID: PMC4037290  PMID: 24084763
Colorectal adenomas; colorectal cancer screening; genetic predisposition
6.  A Meta-analysis of Genome-wide Association Studies for Serum Total IgE in Diverse Study Populations 
Background
Immunoglobulin E (IgE) is both a marker and mediator of allergic inflammation. Despite reported differences in serum total IgE levels by race-ethnicity, African American and Latino individuals have not been well represented in genetic studies of total IgE.
Objective
To identify the genetic predictors of serum total IgE levels.
Methods
We used genome wide association (GWA) data from 4,292 individuals (2,469 African Americans, 1,564 European Americans, and 259 Latinos) in the EVE Asthma Genetics Consortium. Tests for association were performed within each cohort by race-ethnic group (i.e., African American, Latino, and European American) and asthma status. The resulting p-values were meta-analyzed accounting for sample size and direction of effect. Top single nucleotide polymorphism (SNP) associations from the meta-analysis were reassessed in six additional cohorts comprising 5,767 individuals.
Results
We identified 10 unique regions where the combined association statistic was associated with total serum IgE levels (P-value <5.0×10−6) and the minor allele frequency was ≥5% in two or more population groups. Variant rs9469220, corresponding to HLA-DQB1, was the most significantly associated SNP with serum total IgE levels when assessed in both the replication cohorts and the discovery and replication sets combined (P-value = 0.007 and 2.45×10−7, respectively). In addition, findings from earlier GWA studies were also validated in the current meta-analysis.
Conclusion
This meta-analysis independently identified a variant near HLA-DQB1 as a predictor of total serum IgE in multiple race-ethnic groups. This study also extends and confirms the findings of earlier GWA analyses in African American and Latino individuals.
doi:10.1016/j.jaci.2012.10.002
PMCID: PMC3596497  PMID: 23146381
meta-analysis; genome wide association study; total immunoglobulin E; race-ethnicity; continental population groups
7.  The Temporal Version of the Pediatric Sepsis Biomarker Risk Model 
PLoS ONE  2014;9(3):e92121.
Background
PERSEVERE is a risk model for estimating mortality probability in pediatric septic shock, using five biomarkers measured within 24 hours of clinical presentation.
Objective
Here, we derive and test a temporal version of PERSEVERE (tPERSEVERE) that considers biomarker values at the first and third day following presentation to estimate the probability of a “complicated course”, defined as persistence of ≥2 organ failures at seven days after meeting criteria for septic shock, or death within 28 days.
Methods
Biomarkers were measured in the derivation cohort (n = 225) using serum samples obtained during days 1 and 3 of septic shock. Classification and Regression Tree (CART) analysis was used to derive a model to estimate the risk of a complicated course. The derived model was validated in the test cohort (n = 74), and subsequently updated using the combined derivation and test cohorts.
Results
A complicated course occurred in 23% of the derivation cohort subjects. The derived model had a sensitivity for a complicated course of 90% (95% CI 78–96), specificity was 70% (62–77), positive predictive value was 47% (37–58), and negative predictive value was 96% (91–99). The area under the receiver operating characteristic curve was 0.85 (0.79–0.90). Similar test characteristics were observed in the test cohort. The updated model had a sensitivity of 91% (81–96), a specificity of 70% (64–76), a positive predictive value of 47% (39–56), and a negative predictive value of 96% (92–99).
Conclusions
tPERSEVERE reasonably estimates the probability of a complicated course in children with septic shock. tPERSEVERE could potentially serve as an adjunct to physiological assessments for monitoring how risk for poor outcomes changes during early interventions in pediatric septic shock.
doi:10.1371/journal.pone.0092121
PMCID: PMC3953585  PMID: 24626215
8.  Testing the Prognostic Accuracy of the Updated Pediatric Sepsis Biomarker Risk Model 
PLoS ONE  2014;9(1):e86242.
Background
We previously derived and validated a risk model to estimate mortality probability in children with septic shock (PERSEVERE; PEdiatRic SEpsis biomarkEr Risk modEl). PERSEVERE uses five biomarkers and age to estimate mortality probability. After the initial derivation and validation of PERSEVERE, we combined the derivation and validation cohorts (n = 355) and updated PERSEVERE. An important step in the development of updated risk models is to test their accuracy using an independent test cohort.
Objective
To test the prognostic accuracy of the updated version PERSEVERE in an independent test cohort.
Methods
Study subjects were recruited from multiple pediatric intensive care units in the United States. Biomarkers were measured in 182 pediatric subjects with septic shock using serum samples obtained during the first 24 hours of presentation. The accuracy of PERSEVERE 28-day mortality risk estimate was tested using diagnostic test statistics, and the net reclassification improvement (NRI) was used to test whether PERSEVERE adds information to a physiology-based scoring system.
Results
Mortality in the test cohort was 13.2%. Using a risk cut-off of 2.5%, the sensitivity of PERSEVERE for mortality was 83% (95% CI 62–95), specificity was 75% (68–82), positive predictive value was 34% (22–47), and negative predictive value was 97% (91–99). The area under the receiver operating characteristic curve was 0.81 (0.70–0.92). The false positive subjects had a greater degree of organ failure burden and longer intensive care unit length of stay, compared to the true negative subjects. When adding PERSEVERE to a physiology-based scoring system, the net reclassification improvement was 0.91 (0.47–1.35; p<0.001).
Conclusions
The updated version of PERSEVERE estimates mortality probability reliably in a heterogeneous test cohort of children with septic shock and provides information over and above a physiology-based scoring system.
doi:10.1371/journal.pone.0086242
PMCID: PMC3906040  PMID: 24489704
9.  Very important pharmacogene summary for VDR 
Pharmacogenetics and genomics  2012;22(10):758-763.
doi:10.1097/FPC.0b013e328354455c
PMCID: PMC3678550  PMID: 22588316
drug response; genetic variants; pharmacogenomics; vitamin D receptor
11.  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.
Objective
To examine whether delivery by caesarean section is a risk factor for childhood obesity.
Design
Prospective pre-birth cohort study (Project Viva).
Setting
Eight outpatient multi-specialty practices based in the Boston, Massachusetts area.
Participants
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.
Results
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).
Conclusions
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.
doi:10.1136/archdischild-2011-301141
PMCID: PMC3784307  PMID: 22623615
12.  Effect of Vitamin D and Inhaled Corticosteroid Treatment on Lung Function in Children 
Rationale: Low vitamin D levels are associated with asthma and decreased airway responsiveness. Treatment with inhaled corticosteroids improves airway responsiveness and asthma control.
Objectives: To assess the effect of vitamin D levels on prebronchodilator FEV1, bronchodilator response, and responsiveness to methacholine (PC20, provocative concentration of methacholine producing a 20% decline in FEV1) in patients with asthma treated with inhaled corticosteroids.
Methods: We measured 25-hydroxyvitamin D levels in the serum of children with persistent asthma at the time of enrollment in the Childhood Asthma Management Program. We divided subjects into the vitamin D sufficiency (>30 ng/ml), insufficiency (20–30 ng/ml), and deficiency (<20 ng/ml) groups. Covariates included age, treatment, sex, body mass index, race, history of emergency department visits, hospitalizations, and season that vitamin D specimen was drawn. Our main outcome measures were change in prebronchodilator FEV1, bronchodilator response, and PC20 from enrollment to 8–12 months.
Measurements and Main Results: Of the 1,024 subjects, 663 (65%) were vitamin D sufficient, 260 (25%) were insufficient, and 101 (10%) were deficient. Vitamin D–deficient subjects were more likely to be older, African American, and have a higher body mass index compared with the vitamin D–sufficient and insufficient subjects. In the inhaled corticosteroid treatment group, prebronchodilator FEV1 increased from randomization to 12 months by 140 ml in the vitamin D–deficient group and prebronchodilator FEV1 increased by 330 ml in the vitamin D insufficiency group and by 290 ml in the vitamin D sufficiency group (P = 0.0072), in adjusted models.
Conclusions: In children with asthma treated with inhaled corticosteroids, vitamin D deficiency is associated with poorer lung function than in children with vitamin D insufficiency or sufficiency.
doi:10.1164/rccm.201202-0351OC
PMCID: PMC3480528  PMID: 22798322
asthma; vitamin D; lung function; forced expiratory volume; children
13.  Genome-wide association analysis of circulating vitamin D levels in children with asthma 
Human genetics  2012;131(9):1495-1505.
Vitamin D deficiency is becoming more apparent in many populations. Genetic factors may play a role in the maintenance of vitamin D levels. The objective of this study was to perform a genome-wide analysis (GWAS) of vitamin D levels, including replication of prior GWAS results. We measured 25-hydroxyvitamin D (25(OH)D) levels in serum collected at the time of enrollment and at year 4 in 572 Caucasian children with asthma, who were part of a multi-center clinical trial, the Childhood Asthma Management Program. Replication was performed in a second cohort of 592 asthmatics from Costa Rica and a third cohort of 516 Puerto Rican asthmatics. In addition, we attempted replication of three SNPs that were previously identified in a large GWAS of Caucasian individuals. The setting included data from a clinical trial of childhood asthmatics and two cohorts of asthmatics recruited for genetic studies of asthma. The main outcome measure was circulating 25(OH)D levels. The 25(OH)D levels at the two time-points were only modestly correlated with each other (intraclass correlation coefficient = 0.33) in the CAMP population. We identified SNPs that were nominally associated with 25(OH)D levels at two time-points in CAMP, and replicated four SNPs in the Costa Rican cohort: rs11002969, rs163221, rs1678849, and rs4864976. However, these SNPs were not significantly associated with 25(OH)D levels in a third population of Puerto Rican asthmatics. We were able to replicate the SNP with the strongest effect, previously reported in a large GWAS: rs2282679 (GC), and we were able to replicate another SNP, rs10741657 (CYP2R1), to a lesser degree. We were able to replicate two of three prior significant findings in a GWAS of 25(OH)D levels. Other SNPs may be additionally associated with 25(OH)D levels in certain populations.
doi:10.1007/s00439-012-1185-z
PMCID: PMC3648789  PMID: 22673963
14.  ITGB5 and AGFG1 variants are associated with severity of airway responsiveness 
BMC Medical Genetics  2013;14:86.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2350-14-86
PMCID: PMC3765944  PMID: 23984888
Asthma; Airway hyperresponsiveness; Genome-wide association study; ITGB5; AGFG1
15.  Key findings and clinical implications from The Epidemiology and Natural History of Asthma: Outcomes and Treatment Regimens (TENOR) study 
Patients with severe or difficult-to-treat asthma are an understudied population but account for considerable asthma morbidity, mortality, and costs. The Epidemiology and Natural History of Asthma: Outcomes and Treatment Regimens (TENOR) study was a large, 3-year, multicenter, observational cohort study of 4756 patients (n = 3489 adults ≥18 years of age, n = 497 adolescents 13-17 years of age, and n = 770 children 6-12 years of age) with severe or difficult-to-treat asthma. TENOR's primary objective was to characterize the natural history of disease in this cohort. Data assessed semiannually and annually included demographics, medical history, comorbidities, asthma control, asthma-related health care use, medication use, lung function, IgE levels, self-reported asthma triggers, and asthma-related quality of life. We highlight the key findings and clinical implications from more than 25 peer-reviewed TENOR publications. Regardless of age, patients with severe or difficult-to-treat asthma demonstrated high rates of health care use and substantial asthma burden despite receiving multiple long-term controller medications. Recent exacerbation history was the strongest predictor of future asthma exacerbations. Uncontrolled asthma, as defined by the 2007 National Heart, Lung, and Blood Institute guidelines’ impairment domain, was highly prevalent and predictive of future asthma exacerbations; this assessment can be used to identify high-risk patients. IgE and allergen sensitization played a role in the majority of severe or difficult-to-treat asthmatic patients.
doi:10.1016/j.jaci.2012.04.014
PMCID: PMC3622643  PMID: 22694932
TENOR; severe or difficult-to-treat asthma; asthma control; asthma exacerbations; burden; medication; quality of life; allergy; IgE
16.  IL1B Polymorphisms Modulate Cystic Fibrosis Lung Disease 
Pediatric pulmonology  2009;44(6):580-593.
Summary
Rationale: Variability in pulmonary disease severity is found in patients with cystic fibrosis (CF) who have identical mutations in the CF transmembrane conductance regulator (CFTR) gene. We hypothesized that one factor accounting for heterogeneity in pulmonary disease severity is variation in the family of genes affecting the biology of interleukin-1 (IL-1), which impacts acquisition and maintenance of Pseudomonas aeruginosa infection in animal models of chronic infection. Methods: We genotyped 58 single nucleotide polymorphisms (SNPs) in the IL-1 gene cluster in 808 CF subjects from the University of North Carolina and Case Western Reserve University (UNC/CWRU) joint cohort. All were homozygous for ΔF508, and categories of “severe” (cases) or “mild” (control subjects) lung disease were defined by the lowest or highest quartile of forced expired volume (FEV1) for age in the CF population. After adjustment for age and gender, genotypic data were tested for association with lung disease severity. Odds ratios (ORs) comparing severe versus mild CF were also calculated for each genotype (with the homozygote major allele as the reference group) for all 58 SNPs. From these analyses, nine SNPs with a moderate effect size, OR ≤ 0.5or > 1.5, were selected for further testing. To replicate the case-control study results, we genotyped the same nine SNPs in a second population of CF parent-offspring trios (recruited from Children’s Hospital Boston), in which the offspring had similar pulmonary phenotypes. For the trio analysis, both family-based and population-based associations were performed. Results: SNPs rs1143634 and rs1143639 in the IL1B gene demonstrated a consistent association with lung disease severity categories (P < 0.10) and longitudinal analysis of lung disease severity (P < 0.10) in CF in both the case-control and family-based studies. In females, there was a consistent association (false discovery rate adjusted joint P-value < 0.06 for both SNPs) in both the analysis of lung disease severity in the UNC/CWRU cohort and the family-based analysis of affection status. Conclusion: Our findings suggest that IL1β is a clinically relevant modulator of CF lung disease.
doi:10.1002/ppul.21026
PMCID: PMC3716579  PMID: 19431193
gene modifiers; cystic fibrosis; CFTR; IL-1 gene family
17.  IL10 Gene Polymorphisms Are Associated With Asthma Phenotypes in Children 
Genetic epidemiology  2004;26(2):155-165.
IL10 is an anti-inflammatory cytokine that has been found to have lower production in macrophages and mononuclear cells from asthmatics. Since reduced IL10 levels may influence the severity of asthma phenotypes, we examined IL10 single-nucleotide polymorphisms (SNPs) for association with asthma severity and allergy phenotypes as quantitative traits. Utilizing DNA samples from 518 Caucasian asthmatic children from the Childhood Asthma Management Program (CAMP) and their parents, we genotyped six IL10 SNPs: 3 in the promoter, 2 in introns, and one in the 3′ UTR. Using family-based association tests, each SNP was tested for association with asthma and allergy phenotypes individually. Population-based association analysis was performed with each SNP locus, the promoter haplotypes and the 6-loci haplotypes. The 3′ UTR SNP was significantly associated with FEV1 as a percent of predicted (FEV1PP) (P=0.0002) in both the family and population analyses. The promoter haplotype GCC was positively associated with IgE levels and FEV1PP (P=0.007 and 0.012, respectively). The promoter haplotype ATA was negatively associated with lnPC20 and FEV1PP (P=0.008 and 0.043, respectively). Polymorphisms in IL10 are associated with asthma phenotypes in this cohort. Further studies of variation in the IL10 gene may help elucidate the mechanism of asthma development in children.
doi:10.1002/gepi.10298
PMCID: PMC3705717  PMID: 14748015
interleukin 10 (IL10); single nucleotide polymorphism (SNP); genetic association; family-based association test (FBAT); haplotype; promoter; 3′; untranslated region (3′UTR)
18.  Corticosteroid use and bone mineral accretion in children with asthma: effect modification by vitamin D 
Background
The adverse effects of corticosteroids on bone mineral accretion (BMA) have been well documented. Vitamin D insufficiency, a prevalent condition in the pediatric population, has also been associated with decreased bone mineral density (BMD).
Objective
To determine whether children with asthma who have lower vitamin D levels are more susceptible to the negative effects of corticosteroids on BMD over time.
Methods
Children aged 5–12 years with mild-to-moderate asthma who participated in the Childhood Asthma Management Program were followed for a mean of 4.3 years. Total doses of inhaled and oral corticosteroids (OCS) were recorded, serum 25-hydroxyvitamin D3 levels were measured at the beginning of the trial and serial DEXA scans of the lumbar spine were performed. Annual BMA rates were defined as: [(BMD at 4 years follow-up − BMD at baseline)/4 years].
Results
BMA was calculated for 780 subjects. In boys, baseline vitamin D levels significantly modified the relationship between OCS and BMA (vitamin D x OCS interaction, p=0.023). Stratification by vitamin D levels showed a decrease in BMA with increased use of OCS in vitamin D insufficient boys only (p<0.001). Compared to vitamin D sufficient boys, vitamin D insufficient boys exposed to more than 2 courses of oral corticosteroids per year had twice the decrease in BMA rate (relative to boys who were OCS-unexposed).
Conclusions
Vitamin D levels significantly modified the effect of oral corticosteroids on bone mineral accretion in boys. Further research is needed to examine whether vitamin D supplementation in children with poorly controlled asthma may confer benefits to bone health.
doi:10.1016/j.jaci.2012.04.005
PMCID: PMC3387323  PMID: 22608570
Asthma; vitamin D; bone mineral density; corticosteroids
19.  Genomewide association study of the age of onset of childhood asthma 
BACKGROUND
Childhood asthma is a complex disease with known heritability and phenotypic diversity. Although an earlier onset has been associated with more severe disease, there has been no genome-wide association study of the age of onset of asthma in children.
OBJECTIVE
To identify genetic variants associated with earlier onset of childhood asthma.
METHODS
We conducted the first genome-wide association study (GWAS) of the age of onset of childhood asthma among participants in the Childhood Asthma Management Program (CAMP), and used three independent cohorts from North America, Costa Rica, and Sweden for replication.
RESULTS
Two SNPs were associated with earlier onset of asthma in the combined analysis of CAMP and the replication cohorts: : rs9815663 (Fisher’s P value=2.31 × 10−8) and rs7927044 (P=6.54 × 10−9). Of these two SNPs, rs9815663 was also significantly associated with earlier asthma onset in an analysis including only the replication cohorts. Ten SNPs in linkage disequilibrium with rs9815663 were also associated with earlier asthma onset (2.24 × 10−7 < P < 8.22 ×10−6). Having ≥1 risk allele of the two SNPs of interest (rs9815663 and rs7927044) was associated with lower lung function and higher asthma medication use during 4 years of follow-up in CAMP.
CONCLUSIONS
We have identified two SNPs associated with earlier onset of childhood asthma in four independent cohorts.
doi:10.1016/j.jaci.2012.03.020
PMCID: PMC3387331  PMID: 22560479
Asthma; pediatrics; age of onset; asthma genetics; C1orf100; genome-wide association study; pediatric asthma
20.  Evaluation of Asymmetric Dimethylarginine, Arginine, and Carnitine Metabolism in Pediatric Sepsis 
Pediatric Critical Care Medicine  2012;13(4):e210-e218.
Objective
Increased plasma concentrations of the endogenous nitric oxide (NO) synthase inhibitor, asymmetric dimethylarginine (ADMA), decreased arginine bioavailability, and mitochondrial dysfunction have been reported in adult sepsis. We studied whether ADMA, arginine, and carnitine metabolism (a measure of mitochondrial dysfunction) are altered in pediatric sepsis and whether these are clinically useful biomarkers.
Design
Prospective, observational study
Setting
Pediatric intensive care unit at an academic medical center
Patients
Ninety patients ≤ 18 years-old—30 with severe sepsis or septic shock compared with thirty age-matched febrile and thirty age-matched healthy controls.
Interventions
None.
Measurements and Main Results
Plasma ADMA and whole blood arginine, citrulline, ornithine, and acylcarnitine:free carnitine (AC:FC) ratio were measured daily for septic patients and once for controls using tandem mass spectrometry. Plasma ADMA concentration (median, IQR µmol/L) on day 1 was lower in severe sepsis and septic shock (0.38, 0.30–0.56) compared with febrile (0.45, 0.40–0.59) and healthy (0.60, 0.54–0.67) controls (p<0.001), though decreased ADMA was predominantly found in neutropenic patients. Day 1 arginine was lower in septic (10, IQR 7–20 µmol/L) compared with healthy patients (32, IQR 23–40; p<0.001), and the arginine:ornithine ratio was decreased in sepsis, indicating increased arginase activity (an alternative pathway for arginine metabolism). The arginine:ADMA and AC:FC ratios did not differ between septic and control patients. ADMA was inversely correlated with organ dysfunction by PELOD score (r=−0.50, p=0.009), interleukin-6 (r=−0.55, p=0.01), and interleukin-8 (r=−0.52, p=0.03) on admission. Arginine, arginine:ADMA, and AC:FC were not associated with organ dysfunction or outcomes.
Conclusions
ADMA was decreased in pediatric sepsis and was inversely associated with inflammation and organ dysfunction. This suggests that inhibition of NO synthase by ADMA accumulation is unlikely to impact sepsis pathophysiology in septic children despite decreased arginine bioavailability. We did not find an association of ADMA with altered carnitine metabolism, nor were ADMA, arginine, and AC:FC useful as clinical biomarkers.
doi:10.1097/PCC.0b013e318238b5cd
PMCID: PMC3392424  PMID: 22460770
Nitric oxide; nitric oxide synthase; arginine; carnitine; sepsis; intensive care units, pediatric
21.  Predictors of Mucoid Pseudomonas Colonization in Cystic Fibrosis Patients 
Pediatric pulmonology  2008;43(5):463-471.
Summary
Rationale: Chronic mucoid Pseudomonas aeruginosa within the airway in cystic fibrosis (CF) patients can determine prognosis. Understanding the risk factors of mucoid P. aeruginosa acquisition may change how we deliver care. This study aims to evaluate whether presence of risk factors reported to predict disease severity including gender, CFTR genotype, bacterial organisms in airway cultures, and serum levels of vitamins A and E, albumin, C-reactive protein, alpha 1-antitrypsin, and immunoglobulins increased the risk of mucoid P. aeruginosa acquisition. Methods: Primary endpoint was age at first transition from negative to positive culture for mucoid P. aeruginosa. Cox proportional hazards regression with time-dependent covariates examined development of mucoid P. aeruginosa infection and its association with longitudinally measured serum biomarkers, pulmonary function, and culture results for other organisms. Results: Median ages at CF diagnosis and at first culture were 0.55 and 5.7 years, respectively. Median number of cultures/patient was 17. Of the 323 subjects, 150 developed mucoid P. aeruginosa during a median 8.1 years’ follow-up. In multivariate analysis, gender (relative hazard [RH] 0.55 for male vs. female, P=0.001), number of DF508 alleles (RH 1.66 for1 or 2 vs. 0, P=0.04), FEV1 % (RH 1.16 for 10% decrease, P=0.008), and most recent Staphylococcus aureus status (RH 0.24 for positive vs. negative, P< 0.0001) remained statistically significant. Conclusion: Female gender, number of DF508 alleles, decreased lung function, and lack of S. aureus on recent sputum culture are important risk factors for early detection of mucoid P. aeruginosa.
doi:10.1002/ppul.20794
PMCID: PMC3693457  PMID: 18361452
cystic fibrosis; Pseudomonas colonization
22.  Genome-wide Association Identifies the T Gene as a Novel Asthma Pharmacogenetic Locus 
Rationale: To date, most studies aimed at discovering genetic factors influencing treatment response in asthma have focused on biologic candidate genes. Genome-wide association studies (GWAS) can rapidly identify novel pharmacogenetic loci.
Objectives: To investigate if GWAS can identify novel pharmacogenetic loci in asthma.
Methods: Using phenotypic and GWAS genotype data available through the NHLBI-funded Single-nucleotide polymorphism Health association-Asthma Resource Project, we analyzed differences in FEV1 in response to inhaled corticosteroids in 418 white subjects with asthma. Of the 444,088 single nucleotide polymorphisms (SNPs) analyzed, the lowest 50 SNPs by P value were genotyped in an independent clinical trial population of 407 subjects with asthma.
Measurements and Main Results: The lowest P value for the GWAS analysis was 2.09 × 10−6. Of the 47 SNPs successfully genotyped in the replication population, three were associated under the same genetic model in the same direction, including two of the top four SNPs ranked by P value. Combined P values for these SNPs were 1.06 × 10−5 for rs3127412 and 6.13 × 10−6 for rs6456042. Although these two were not located within a gene, they were tightly correlated with three variants mapping to potentially functional regions within the T gene. After genotyping, each T gene variant was also associated with lung function response to inhaled corticosteroids in each of the trials associated with rs3127412 and rs6456042 in the initial GWAS analysis. On average, there was a twofold to threefold difference in FEV1 response for those subjects homozygous for the wild-type versus mutant alleles for each T gene SNP.
Conclusions: Genome-wide association has identified the T gene as a novel pharmacogenetic locus for inhaled corticosteroid response in asthma.
doi:10.1164/rccm.201111-2061OC
PMCID: PMC3381232  PMID: 22538805
polymorphism; genome; pharmacogenomics; glucocorticoid
23.  Maternal intestinal flora and wheeze in early childhood 
Background
Increasing evidence links altered intestinal flora in infancy to eczema and asthma. No studies have investigated the influence of maternal intestinal flora on wheezing and eczema in early childhood.
Objective
To investigate the link between maternal intestinal flora during pregnancy and development of wheeze and eczema in infancy.
Methods
Sixty pregnant women from the Boston area gave stool samples during the third trimester of their pregnancy and answered questions during pregnancy about their own health, and about their children’s health when the child was 2 and 6 months of age. Quantitative culture was performed on stool samples and measured in log10colony-forming units(CFU)/gram stool. Primary outcomes included infant wheeze and eczema in the first 6 months of life. Atopic wheeze, defined as wheeze and eczema, was analyzed as a secondary outcome.
Results
In multivariate models adjusted for breastfeeding, daycare attendance and maternal atopy, higher counts of maternal total aerobes (TA) and enterococci (E) were associated with increased risk of infant wheeze (TA: OR 2.32 for 1 log increase in CFU/g stool [95% CI 1.22, 4.42]; E: OR 1.57 [95% CI 1.06, 2.31]). No organisms were associated with either eczema or atopic wheeze.
Conclusions & Clinical Relevance
In our cohort, higher maternal total aerobes and enterococci were related to increased risk of infant wheeze. Maternal intestinal flora may be an important environmental exposure in early immune system development.
doi:10.1111/j.1365-2222.2011.03950.x
PMCID: PMC3428746  PMID: 22909161
infant wheeze; eczema; asthma; microbiota; intestinal flora; maternal flora
24.  Genomewide Association between GLCCI1 and Response to Glucocorticoid Therapy in Asthma 
The New England journal of medicine  2011;365(13):1173-1183.
BACKGROUND
The response to treatment for asthma is characterized by wide interindividual variability, with a significant number of patients who have no response. We hypothesized that a genomewide association study would reveal novel pharmacogenetic determinants of the response to inhaled glucocorticoids.
METHODS
We analyzed a small number of statistically powerful variants selected on the basis of a family-based screening algorithm from among 534,290 single-nucleotide polymorphisms (SNPs) to determine changes in lung function in response to inhaled glucocorticoids. A significant, replicated association was found, and we characterized its functional effects.
RESULTS
We identified a significant pharmacogenetic association at SNP rs37972, replicated in four independent populations totaling 935 persons (P = 0.0007), which maps to the glucocorticoid-induced transcript 1 gene (GLCCI1) and is in complete linkage disequilibrium (i.e., perfectly correlated) with rs37973. Both rs37972 and rs37973 are associated with decrements in GLCCI1 expression. In isolated cell systems, the rs37973 variant is associated with significantly decreased luciferase reporter activity. Pooled data from treatment trials indicate reduced lung function in response to inhaled glucocorticoids in subjects with the variant allele (P = 0.0007 for pooled data). Overall, the mean (± SE) increase in forced expiratory volume in 1 second in the treated subjects who were homozygous for the mutant rs37973 allele was only about one third of that seen in similarly treated subjects who were homozygous for the wild-type allele (3.2 ± 1.6% vs. 9.4 ± 1.1%), and their risk of a poor response was significantly higher (odds ratio, 2.36; 95% confidence interval, 1.27 to 4.41), with genotype accounting for about 6.6% of overall inhaled glucocorticoid response variability.
CONCLUSIONS
A functional GLCCI1 variant is associated with substantial decrements in the response to inhaled glucocorticoids in patients with asthma. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT00000575.)
doi:10.1056/NEJMoa0911353
PMCID: PMC3667396  PMID: 21991891
25.  Development of a Pharmacogenetic Predictive Test in asthma: proof of concept 
Pharmacogenetics and genomics  2010;20(2):86-93.
Objective
To assess the feasibility of developing a Combined Clinical and Pharmacogenetic Predictive Test, comprised of multiple single nucleotide polymorphisms (SNPs) that is associated with poor bronchodilator response (BDR).
Methods
We genotyped SNPs that tagged the whole genome of the parents and children in the Childhood Asthma Management Program (CAMP) and implemented an algorithm using a family-based association test that ranked SNPs by statistical power. The top eight SNPs that were associated with BDR comprised the Pharmacogenetic Predictive Test. The Clinical Predictive Test was comprised of baseline forced expiratory volume in 1 s (FEV1). We evaluated these predictive tests and a Combined Clinical and Pharmacogenetic Predictive Test in three distinct populations: the children of the CAMP trial and two additional clinical trial populations of asthma. Our outcome measure was poor BDR, defined as BDR of less than 20th percentile in each population. BDR was calculated as the percent difference between the prebronchodilator and postbronchodilator (two puffs of albuterol at 180 μg/puff) FEV1 value. To assess the predictive ability of the test, the corresponding area under the receiver operating characteristic curves (AUROCs) were calculated for each population.
Results
The AUROC values for the Clinical Predictive Test alone were not significantly different from 0.50, the AUROC of a random classifier. Our Combined Clinical and Pharmacogenetic Predictive Test comprised of genetic polymorphisms in addition to FEV1 predicted poor BDR with an AUROC of 0.65 in the CAMP children (n= 422) and 0.60 (n= 475) and 0.63 (n= 235) in the two independent populations. Both the Combined Clinical and Pharmacogenetic Predictive Test and the Pharmacogenetic Predictive Test were significantly more accurate than the Clinical Predictive Test (AUROC between 0.44 and 0.55) in each of the populations.
Conclusion
Our finding that genetic polymorphisms with a clinical trait are associated with BDR suggests that there is promise in using multiple genetic polymorphisms simultaneously to predict which asthmatics are likely to respond poorly to bronchodilators.
doi:10.1097/FPC.0b013e32833428d0
PMCID: PMC3654515  PMID: 20032818
asthma; bronchodilator response; personalized medicine; pharmacogenetic test; predictive medicine

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