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1.  Ethnic-specific associations of rare and low-frequency DNA sequence variants with asthma 
Nature Communications  2015;6:5965.
Common variants at many loci have been robustly associated with asthma but explain little of the overall genetic risk. Here we investigate the role of rare (<1%) and low-frequency (1–5%) variants using the Illumina HumanExome BeadChip array in 4,794 asthma cases, 4,707 non-asthmatic controls and 590 case–parent trios representing European Americans, African Americans/African Caribbeans and Latinos. Our study reveals one low-frequency missense mutation in the GRASP gene that is associated with asthma in the Latino sample (P=4.31 × 10−6; OR=1.25; MAF=1.21%) and two genes harbouring functional variants that are associated with asthma in a gene-based analysis: GSDMB at the 17q12–21 asthma locus in the Latino and combined samples (P=7.81 × 10−8 and 4.09 × 10−8, respectively) and MTHFR in the African ancestry sample (P=1.72 × 10−6). Our results suggest that associations with rare and low-frequency variants are ethnic specific and not likely to explain a significant proportion of the ‘missing heritability’ of asthma.
Common variants account for only a small amount of the heritable risk for developing asthma. Using a meta-analysis approach, Igartua et al. identify one low-frequency missense mutation and two genes with functional variants that are associated with asthma, but only in specific ethnic groups.
doi:10.1038/ncomms6965
PMCID: PMC4309441  PMID: 25591454
2.  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
3.  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
4.  Further Replication Studies of the EVE Consortium Meta-Analysis Identifies Two Asthma Risk Loci in European Americans 
Background
Genome-wide association studies of asthma have implicated many genetic risk factors, with well-replicated associations at approximately 10 loci that account for only a small proportion of the genetic risk.
Objectives
We aimed to identify additional asthma risk loci by performing an extensive replication study of the results from the EVE Consortium meta-analysis.
Methods
We selected 3186 SNPs for replication based on the p-values from the EVE Consortium meta-analysis. These SNPs were genotyped in ethnically diverse replication samples from nine different studies, totaling to 7202 cases, 6426 controls, and 507 case-parent trios. Association analyses were conducted within each participating study and the resulting test statistics were combined in a meta-analysis.
Results
Two novel associations were replicated in European Americans: rs1061477 in the KLK3 gene on chromosome 19 (combined OR = 1.18; 95% CI 1.10 – 1.25) and rs9570077 (combined OR =1.20 95% CI 1.12–1.29) on chromosome 13q21. We could not replicate any additional associations in the African American or Latino individuals.
Conclusions
This extended replication study identified two additional asthma risk loci in populations of European descent. The absence of additional loci for African Americans and Latino individuals highlights the difficulty in replicating associations in admixed populations.
doi:10.1016/j.jaci.2012.07.054
PMCID: PMC3666859  PMID: 23040885
Asthma; genetic risk factors; meta-analysis; KLK3
5.  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
6.  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)
7.  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
8.  A Genome-Wide Association Study on African-Ancestry Populations For Asthma 
Background
Asthma is a complex disease characterized by striking ethnic disparities not explained entirely by environmental, social, cultural, or economic factors. Of the limited genetic studies performed on populations of African descent, notable differences in susceptibility allele frequencies have been observed.
Objectives
To test the hypothesis that some genes may contribute to the profound disparities in asthma.
Methods
We performed a genome-wide association study in two independent populations of African ancestry (935 African American asthma cases and controls from the Baltimore-Washington, D.C. area, and 929 African Caribbean asthmatics and their family members from Barbados) to identify single-nucleotide polymorphisms (SNPs) associated with asthma.
Results
Meta-analysis combining these two African-ancestry populations yielded three SNPs with a combined P-value <10-5 in genes of potential biological relevance to asthma and allergic disease: rs10515807, mapping to alpha-1B-adrenergic receptor (ADRA1B) gene on chromosome 5q33 (3.57×10-6); rs6052761, mapping to prion-related protein (PRNP) on chromosome 20pter-p12 (2.27×10-6); and rs1435879, mapping to dipeptidyl peptidase 10 (DPP10) on chromosome 2q12.3-q14.2. The generalizability of these findings was tested in family and case-control panels of UK and German origin, respectively, but none of the associations observed in the African groups were replicated in these European studies.
Conclusions
Evidence for association was also examined in four additional case-control studies of African Americans; however, none of the SNPs implicated in the discovery population were replicated. This study illustrates the complexity of identifying true associations for a complex and heterogeneous disease such as asthma in admixed populations, especially populations of African descent.
doi:10.1016/j.jaci.2009.08.031
PMCID: PMC3606015  PMID: 19910028
Asthma; GWAS; ADRA1B; PRNP; DPP10; African ancestry; ethnicity; polymorphism; genetic association
9.  Identification of a chronic obstructive pulmonary disease genetic determinant that regulates HHIP 
Human Molecular Genetics  2011;21(6):1325-1335.
Multiple intergenic single-nucleotide polymorphisms (SNPs) near hedgehog interacting protein (HHIP) on chromosome 4q31 have been strongly associated with pulmonary function levels and moderate-to-severe chronic obstructive pulmonary disease (COPD). However, whether the effects of variants in this region are related to HHIP or another gene has not been proven. We confirmed genetic association of SNPs in the 4q31 COPD genome-wide association study (GWAS) region in a Polish cohort containing severe COPD cases and healthy smoking controls (P = 0.001 to 0.002). We found that HHIP expression at both mRNA and protein levels is reduced in COPD lung tissues. We identified a genomic region located ∼85 kb upstream of HHIP which contains a subset of associated SNPs, interacts with the HHIP promoter through a chromatin loop and functions as an HHIP enhancer. The COPD risk haplotype of two SNPs within this enhancer region (rs6537296A and rs1542725C) was associated with statistically significant reductions in HHIP promoter activity. Moreover, rs1542725 demonstrates differential binding to the transcription factor Sp3; the COPD-associated allele exhibits increased Sp3 binding, which is consistent with Sp3's usual function as a transcriptional repressor. Thus, increased Sp3 binding at a functional SNP within the chromosome 4q31 COPD GWAS locus leads to reduced HHIP expression and increased susceptibility to COPD through distal transcriptional regulation. Together, our findings reveal one mechanism through which SNPs upstream of the HHIP gene modulate the expression of HHIP and functionally implicate reduced HHIP gene expression in the pathogenesis of COPD.
doi:10.1093/hmg/ddr569
PMCID: PMC3284120  PMID: 22140090
10.  Integration of Mouse and Human Genome-Wide Association Data Identifies KCNIP4 as an Asthma Gene 
PLoS ONE  2013;8(2):e56179.
Asthma is a common chronic respiratory disease characterized by airway hyperresponsiveness (AHR). The genetics of asthma have been widely studied in mouse and human, and homologous genomic regions have been associated with mouse AHR and human asthma-related phenotypes. Our goal was to identify asthma-related genes by integrating AHR associations in mouse with human genome-wide association study (GWAS) data. We used Efficient Mixed Model Association (EMMA) analysis to conduct a GWAS of baseline AHR measures from males and females of 31 mouse strains. Genes near or containing SNPs with EMMA p-values <0.001 were selected for further study in human GWAS. The results of the previously reported EVE consortium asthma GWAS meta-analysis consisting of 12,958 diverse North American subjects from 9 study centers were used to select a subset of homologous genes with evidence of association with asthma in humans. Following validation attempts in three human asthma GWAS (i.e., Sepracor/LOCCS/LODO/Illumina, GABRIEL, DAG) and two human AHR GWAS (i.e., SHARP, DAG), the Kv channel interacting protein 4 (KCNIP4) gene was identified as nominally associated with both asthma and AHR at a gene- and SNP-level. In EVE, the smallest KCNIP4 association was at rs6833065 (P-value 2.9e-04), while the strongest associations for Sepracor/LOCCS/LODO/Illumina, GABRIEL, DAG were 1.5e-03, 1.0e-03, 3.1e-03 at rs7664617, rs4697177, rs4696975, respectively. At a SNP level, the strongest association across all asthma GWAS was at rs4697177 (P-value 1.1e-04). The smallest P-values for association with AHR were 2.3e-03 at rs11947661 in SHARP and 2.1e-03 at rs402802 in DAG. Functional studies are required to validate the potential involvement of KCNIP4 in modulating asthma susceptibility and/or AHR. Our results suggest that a useful approach to identify genes associated with human asthma is to leverage mouse AHR association data.
doi:10.1371/journal.pone.0056179
PMCID: PMC3572953  PMID: 23457522
11.  Gene-by-environment effect of house dust mite on purinergic receptor P2Y12 (P2RY12) and lung function in children with asthma 
Clinical and Experimental Allergy  2012;42(2):229-237.
Background
Distinct receptors likely exist for leukotriene(LT)E4, a potent mediator of airway inflammation. Purinergic receptor P2Y12 is needed for LTE4-induced airways inflammation, and P2Y12 antagonism attenuates house dust mite-induced pulmonary eosinophilia in mice. Although experimental data support a role for P2Y12 in airway inflammation, its role in human asthma has never been studied.
Objective
To test for association between variants in the P2Y12 gene (P2RY12) and lung function in human subjects with asthma, and to examine for gene-by-environment interaction with house dust mite exposure.
Methods
19 single nucleotide polymorphisms (SNPs) in P2RY12 were genotyped in 422 children with asthma and their parents (n=1266). Using family-based methods, we tested for associations between these SNPs and five lung function measures. We performed haplotype association analyses and tested for gene-by-environment interactions using house dust mite exposure. We used the false discovery rate to account for multiple comparisons.
Results
Five SNPs in P2RY12 were associated with multiple lung function measures (P values 0.006–0.025). Haplotypes in P2RY12 were also associated with lung function (P values 0.0055–0.046). House dust mite exposure modulated associations between P2RY12 and lung function, with minor allele homozygotes exposed to house dust mite demonstrating worse lung function than those unexposed (significant interaction P values 0.0028–0.040).
Conclusions and clinical relevance
P2RY12 variants were associated with lung function in a large family-based asthma cohort. House dust mite exposure caused significant gene-by-environment effects. Our findings add the first human evidence to experimental data supporting a role for P2Y12 in lung function. P2Y12 could represent a novel target for asthma treatment.
doi:10.1111/j.1365-2222.2011.03874.x
PMCID: PMC3353543  PMID: 22010907
Purinergic receptor; leukotriene; asthma; house dust mite; lung function
12.  Meta-analysis of Genome-wide Association Studies of Asthma In Ethnically Diverse North American Populations 
Torgerson, Dara G. | Ampleford, Elizabeth J. | Chiu, Grace Y. | Gauderman, W. James | Gignoux, Christopher R. | Graves, Penelope E. | Himes, Blanca E. | Levin, Albert M. | Mathias, Rasika A. | Hancock, Dana B. | Baurley, James W. | Eng, Celeste | Stern, Debra A. | Celedón, Juan C. | Rafaels, Nicholas | Capurso, Daniel | Conti, David V. | Roth, Lindsey A. | Soto-Quiros, Manuel | Togias, Alkis | Li, Xingnan | Myers, Rachel A. | Romieu, Isabelle | Van Den Berg, David J. | Hu, Donglei | Hansel, Nadia N. | Hernandez, Ryan D. | Israel, Elliott | Salam, Muhammad T. | Galanter, Joshua | Avila, Pedro C. | Avila, Lydiana | Rodriquez-Santana, Jose R. | Chapela, Rocio | Rodriguez-Cintron, William | Diette, Gregory B. | Adkinson, N. Franklin | Abel, Rebekah A. | Ross, Kevin D. | Shi, Min | Faruque, Mezbah U. | Dunston, Georgia M. | Watson, Harold R. | Mantese, Vito J. | Ezurum, Serpil C. | Liang, Liming | Ruczinski, Ingo | Ford, Jean G. | Huntsman, Scott | Chung, Kian Fan | Vora, Hita | Li, Xia | Calhoun, William J. | Castro, Mario | Sienra-Monge, Juan J. | del Rio-Navarro, Blanca | Deichmann, Klaus A. | Heinzmann, Andrea | Wenzel, Sally E. | Busse, William W. | Gern, James E. | Lemanske, Robert F. | Beaty, Terri H. | Bleecker, Eugene R. | Raby, Benjamin A. | Meyers, Deborah A. | London, Stephanie J. | Gilliland, Frank D. | Burchard, Esteban G. | Martinez, Fernando D. | Weiss, Scott T. | Williams, L. Keoki | Barnes, Kathleen C. | Ober, Carole | Nicolae, Dan L.
Nature genetics  2011;43(9):887-892.
Asthma is a common disease with a complex risk architecture including both genetic and environmental factors. We performed a meta-analysis of North American genome-wide association studies (GWAS) of asthma in 5,416 asthma cases representing European Americans, African Americans/African Caribbeans, and Latinos, and replicated five regions among the most significant signals in 12,649 individuals from the same ethnic groups. Four were at previously reported loci on 17q21, and near the IL1RL1, TSLP, and IL33, genes, but we report for the first time that these loci are associated with asthma risk in three ethnic groups. In addition, we identified a novel association with asthma in the PYHIN1, gene that was specific to individuals of African descent (p=3.9×10−9). These results suggest that some asthma susceptibility loci are robust to differences in ancestry when sufficiently large samples sizes are investigated, and that ancestry-specific associations also contribute to the complex genetic architecture of asthma.
doi:10.1038/ng.888
PMCID: PMC3445408  PMID: 21804549
13.  Genetic Influences on Asthma Susceptibility in the Developing Lung 
Asthma is the leading serious pediatric chronic illness in the United States, affecting 7.1 million children. The prevalence of asthma in children under 4 years of age has increased dramatically in the last 2 decades. Existing evidence suggests that this increase in prevalence derives from early environmental exposures acting on a pre-existing asthma-susceptible genotype. We studied the origins of asthma susceptibility in developing lung in rat strains that model the distinct phenotypes of airway hyperresponsiveness (Fisher rats) and atopy (brown Norway [BN] rats). Postnatal BN rat lungs showed increased epithelial proliferation and tracheal goblet cell hyperplasia. Fisher pups showed increased lung resistance at age 2 weeks, with elevated neutrophils throughout the postnatal period. Diverse transcriptomic signatures characterized the distinct respiratory phenotypes of developing lung in both rat models. Linear regression across age and strain identified developmental variation in expression of 1,376 genes, and confirmed both strain and temporal regulation of lung gene expression. Biological processes that were heavily represented included growth and development (including the T Box 1 transcription factor [Tbx5], the epidermal growth factor receptor [Egfr], the transforming growth factor beta-1-induced transcript 1 [Tgfbr1i1]), extracellular matrix and cell adhesion (including collagen and integrin genes), and immune function (including lymphocyte antigen 6 (Ly6) subunits, IL-17b, Toll-interacting protein, and Ficolin B). Genes validated by quantitative RT-PCR and protein analysis included collagen III alpha 1 Col3a1, Ly6b, glucocorticoid receptor and Importin-13 (specific to the BN rat lung), and Serpina1 and Ficolin B (specific to the Fisher lung). Innate differences in patterns of gene expression in developing lung that contribute to individual variation in respiratory phenotype are likely to contribute to the pathogenesis of asthma.
doi:10.1165/rcmb.2009-0412OC
PMCID: PMC3159089  PMID: 20118217
asthma susceptibility; lung development; developmental gene expression
14.  In Utero Smoke Exposure and Impaired Response to Inhaled Corticosteroids in Children with Asthma 
Background
Few studies have examined the effects of in utero smoke exposure (IUS) on lung function in children with asthma, and there are no published data on the impact of IUS on treatment outcomes in asthmatic children.
Objectives
To explore whether IUS exposure is associated with increased airway responsiveness among children with asthma, and whether IUS modifies the response to treatment with inhaled corticosteroids (ICS).
Methods
To assess the impact of parent-reported IUS exposure on airway responsiveness in childhood asthma we performed a repeated-measures analysis of methacholine PC20 data from the Childhood Asthma Management Program (CAMP), a four-year, multicenter, randomized double masked placebo controlled trial of 1041 children ages 5–12 comparing the long term efficacy of ICS with mast cell stabilizing agents or placebo.
Results
Although improvement was seen in both groups, asthmatic children with IUS exposure had on average 26% less of an improvement in airway responsiveness over time compared to unexposed children (p=.01). Moreover, while children who were not exposed to IUS who received budesonide experienced substantial improvement in PC20 compared to untreated children (1.25 fold-increase, 95% CI 1.03, 1.50, p=.02) the beneficial effects of budesonide were attenuated among children with a history of IUS exposure (1.04 fold-increase, 95% CI 0.65, 1.68, p=.88).
Conclusions
IUS reduces age-related improvements in airway responsiveness among asthmatic children. Moreover, IUS appears to blunt the beneficial effects of ICS use on airways responsiveness. These results emphasize the importance of preventing this exposure through smoking cessation counseling efforts with pregnant women.
doi:10.1016/j.jaci.2010.06.016
PMCID: PMC2937829  PMID: 20673983
asthma; in utero smoke exposure; airway responsiveness; inhaled corticosteroids
15.  Global Analysis of the Impact of Environmental Perturbation on cis-Regulation of Gene Expression 
PLoS Genetics  2011;7(1):e1001279.
Genetic variants altering cis-regulation of normal gene expression (cis-eQTLs) have been extensively mapped in human cells and tissues, but the extent by which controlled, environmental perturbation influences cis-eQTLs is unclear. We carried out large-scale induction experiments using primary human bone cells derived from unrelated donors of Swedish origin treated with 18 different stimuli (7 treatments and 2 controls, each assessed at 2 time points). The treatments with the largest impact on the transcriptome, verified on two independent expression arrays, included BMP-2 (t = 2h), dexamethasone (DEX) (t = 24h), and PGE2 (t = 24h). Using these treatments and control, we performed expression profiling for 18,144 RefSeq transcripts on biological replicates of the complete study cohort of 113 individuals (ntotal = 782) and combined it with genome-wide SNP-genotyping data in order to map treatment-specific cis-eQTLs (defined as SNPs located within the gene ±250 kb). We found that 93% of cis-eQTLs at 1% FDR were observed in at least one additional treatment, and in fact, on average, only 1.4% of the cis-eQTLs were considered as treatment-specific at high confidence. The relative invariability of cis-regulation following perturbation was reiterated independently by genome-wide allelic expression tests where only a small proportion of variance could be attributed to treatment. Treatment-specific cis-regulatory effects were, however, 2- to 6-fold more abundant among differently expressed genes upon treatment. We further followed-up and validated the DEX–specific cis-regulation of the MYO6 and TNC loci and found top cis-regulatory variants located 180 kb and 250 kb upstream of the transcription start sites, respectively. Our results suggest that, as opposed to tissue-specificity of cis-eQTLs, the interactions between cellular environment and cis-variants are relatively rare (∼1.5%), but that detection of such specific interactions can be achieved by a combination of functional genomic approaches as described here.
Author Summary
Population variation in normal gene expression has been convincingly shown to be under strong genetic control where the main genetic variants are located within close proximity to the gene itself (so called cis-acting). However, the extent to which controlled, environmental stimuli influences cis-regulation of gene expression is unclear. Here, we combine different functional genomic approaches and examine the role of common genetic variants on induced gene expression in a population panel of primary human cells derived from ∼100 unrelated donors treated under multiple conditions. Using these approaches, we find that the interaction between cellular environment and cis-variants are relatively rare, with only a small proportion of the identified genetic variants being specific to treatment. However, although treatment-specific genetic regulation of gene expression seems to be infrequent, we prove its existence by thorough validation of treatment-specific effects of the glucocorticoid-specific regulation of TNC expression. Taken together, these findings indicate that the regulatory landscape within a cell is very stable but, by combining functional genomic tools gene-environmental interactions of clinical importance, can be detected and possibly used as biomarkers in future pharmacogenomic studies.
doi:10.1371/journal.pgen.1001279
PMCID: PMC3024267  PMID: 21283786
16.  Parsing the Effects of Individual SNPs in Candidate Genes with Family Data 
Human Heredity  2009;69(2):91-103.
We introduce a stepwise approach for family-based designs for selecting a set of markers in a gene that are independently associated with the disease. The approach is based on testing the effect of a set of markers conditional on another set of markers. Several likelihood-based approaches have been proposed for special cases, but no model-free based tests have been proposed. We propose two types of tests in a family-based framework that are applicable to arbitrary family structures and completely robust to population stratification. We propose methods for ascertained dichotomous traits and unascertained quantitative traits. We first propose a completely model-free extension of the FBAT main genetic effect test. Then, for power issues, we introduce two model-based tests, one for dichotomous traits and one for continuous traits. Lastly, we utilize these tests to analyze a continuous lung function phenotype as a proxy for asthma in the Childhood Asthma Management Program. The methods are implemented in the free R package fbati.
doi:10.1159/000264447
PMCID: PMC2956011  PMID: 19996607
Binary trait; Candidate gene analysis; Family-based association tests; FBAT-C; Linkage disequilibrium (LD); Model-based test; Model-free test; Nuclear families; Quantitative trait
17.  TSLP Polymorphisms are Associated with Asthma in a Sex-Specific Fashion 
Allergy  2010;65(12):1566-1575.
Background
Single nucleotide polymorphisms (SNPs) in thymic stromal lymphopoietin (TSLP) have been associated with IgE (in girls) and asthma (in general). We sought to determine whether TSLP SNPs are associated with asthma in a sex-specific fashion.
Methods
We conducted regular and sex-stratified analyses of association between SNPs in TSLP and asthma in families of asthmatic children in Costa Rica. Significant findings were replicated in white and African-American participants in the Childhood Asthma Management Program, in African Americans in the Genomic Research on Asthma in the African Diaspora study, in whites and Hispanics in the Children’s Health Study, and in whites in the Framingham Heart Study (FHS).
Main Results
Two SNPs in TSLP (rs1837253 and rs2289276) were significantly associated with a reduced risk of asthma in combined analyses of all cohorts (p values of 2×10−5 and 1×10−5, respectively). In a sex-stratified analysis, the T allele of rs1837253 was significantly associated with a reduced risk of asthma in males only (p= 3×10−6). Alternately, the T allele of rs2289276 was significantly associated with a reduced risk of asthma in females only (p= 2×10−4). Findings for rs2289276 were consistent in all cohorts except the FHS.
Conclusions
TSLP variants are associated with asthma in a sex-specific fashion.
doi:10.1111/j.1398-9995.2010.02415.x
PMCID: PMC2970693  PMID: 20560908
asthma; genetic association; sex-specific; thymic stromal lymphopoietin; TSLP
18.  MMP12, Lung Function, and COPD in High-Risk Populations 
The New England journal of medicine  2009;361(27):2599-2608.
BACKGROUND
Genetic variants influencing lung function in children and adults may ultimately lead to the development of chronic obstructive pulmonary disease (COPD), particularly in high-risk groups.
METHODS
We tested for an association between single-nucleotide polymorphisms (SNPs) in the gene encoding matrix metalloproteinase 12 (MMP12) and a measure of lung function (prebronchodilator forced expiratory volume in 1 second [FEV1]) in more than 8300 subjects in seven cohorts that included children and adults. Within the Normative Aging Study (NAS), a cohort of initially healthy adult men, we tested for an association between SNPs that were associated with FEV1 and the time to the onset of COPD. We then examined the relationship between MMP12 SNPs and COPD in two cohorts of adults with COPD or at risk for COPD.
RESULTS
The minor allele (G) of a functional variant in the promoter region of MMP12 (rs2276109 [−82A→G]) was positively associated with FEV1 in a combined analysis of children with asthma and adult former and current smokers in all cohorts (P=2×10−6). This allele was also associated with a reduced risk of the onset of COPD in the NAS cohort (hazard ratio, 0.65; 95% confidence interval [CI], 0.46 to 0.92; P = 0.02) and with a reduced risk of COPD in a cohort of smokers (odds ratio, 0.63; 95% CI, 0.45 to 0.88; P = 0.005) and among participants in a family-based study of early-onset COPD (P = 0.006).
CONCLUSIONS
The minor allele of a SNP in MMP12 (rs2276109) is associated with a positive effect on lung function in children with asthma and in adults who smoke. This allele is also associated with a reduced risk of COPD in adult smokers.
doi:10.1056/NEJMoa0904006
PMCID: PMC2904064  PMID: 20018959
19.  Assessing the Reproducibility of Asthma Candidate Gene Associations, Using Genome-wide Data 
Rationale: Association studies have implicated many genes in asthma pathogenesis, with replicated associations between single-nucleotide polymorphisms (SNPs) and asthma reported for more than 30 genes. Genome-wide genotyping enables simultaneous evaluation of most of this variation, and facilitates more comprehensive analysis of other common genetic variation around these candidate genes for association with asthma.
Objectives: To use available genome-wide genotypic data to assess the reproducibility of previously reported associations with asthma and to evaluate the contribution of additional common genetic variation surrounding these loci to asthma susceptibility.
Methods: Illumina Human Hap 550Kv3 BeadChip (Illumina, San Diego, CA) SNP arrays were genotyped in 422 nuclear families participating in the Childhood Asthma Management Program. Genes with at least one SNP demonstrating prior association with asthma in two or more populations were tested for evidence of association with asthma, using family-based association testing.
Measurements and Main Results: We identified 39 candidate genes from the literature, using prespecified criteria. Of the 160 SNPs previously genotyped in these 39 genes, 10 SNPs in 6 genes were significantly associated with asthma (including the first independent replication for asthma-associated integrin β3 [ITGB3]). Evaluation of 619 additional common variants included in the Illumina 550K array revealed additional evidence of asthma association for 15 genes, although none were significant after adjustment for multiple comparisons.
Conclusions: We replicated asthma associations for a minority of candidate genes. Pooling genome-wide association study results from multiple studies will increase the power to appreciate marginal effects of genes and further clarify which candidates are true “asthma genes.”
doi:10.1164/rccm.200812-1860OC
PMCID: PMC2695495  PMID: 19264973
asthma; replication; single-nucleotide polymorphism; integrin β3; association
20.  Using Canonical Correlation Analysis to Discover Genetic Regulatory Variants 
PLoS ONE  2010;5(5):e10395.
Background
Discovering genetic associations between genetic markers and gene expression levels can provide insight into gene regulation and, potentially, mechanisms of disease. Such analyses typically involve a linkage or association analysis in which expression data are used as phenotypes. This approach leads to a large number of multiple comparisons and may therefore lack power. We assess the potential of applying canonical correlation analysis to partitioned genomewide data as a method for discovering regulatory variants.
Methodology/Principal Findings
Simulations suggest that canonical correlation analysis has higher power than standard pairwise univariate regression to detect single nucleotide polymorphisms when the expression trait has low heritability. The increase in power is even greater under the recessive model. We demonstrate this approach using the Childhood Asthma Management Program data.
Conclusions/Significance
Our approach reduces multiple comparisons and may provide insight into the complex relationships between genotype and gene expression.
doi:10.1371/journal.pone.0010395
PMCID: PMC2869348  PMID: 20485529
21.  Variants in TGFB1, Dust Mite Exposure, and Disease Severity in Children with Asthma 
Rationale: Polymorphisms in the gene for transforming growth factor-β1 (TGFB1) have been associated with asthma, but not with airway responsiveness or disease exacerbations in subjects with asthma.
Objectives: To test for association between single nucleotide polymorphisms (SNPs) in TGFB1 and markers of asthma severity in childhood.
Methods: We tested for the association between nine SNPs in TGFB1 and indicators of asthma severity (lung function, airway responsiveness, and disease exacerbations) in two cohorts: 416 Costa Rican parent-child trios and 465 families of non-Hispanic white children in the Childhood Asthma Management Program (CAMP). We also tested for the interaction between these polymorphisms and exposure to dust mite allergen on asthma severity.
Measurements and Main Results: The A allele of promoter SNP rs2241712 was associated with increased airway responsiveness in Costa Rica (P = 0.0006) and CAMP (P = 0.005), and the C allele of an SNP in the promoter region (rs1800469) was associated with increased airway responsiveness in both cohorts (P ≤ 0.01). Dust mite exposure modified the effect of the C allele of exonic SNP rs1800471 on airway responsiveness (P = 0.03 for interactions in both cohorts). The T allele of a coding SNP (rs1982073) was associated with a reduced risk of asthma exacerbations in Costa Rica (P = 0.009) and CAMP (P = 0.005). Dust mite exposure also significantly modified the effect of the A allele of the promoter SNP rs2241712 on asthma exacerbations in both cohorts.
Conclusions: SNPs in TGFB1 are associated with airway responsiveness and disease exacerbations in children with asthma. Moreover, dust mite exposure may modify the effect of TGFB1 SNPs on airway responsiveness and asthma exacerbations.
doi:10.1164/rccm.200808-1268OC
PMCID: PMC2648908  PMID: 19096005
airway responsiveness; asthma; dust mite allergen; single nucleotide polymorphisms; transforming growth factor-β1
23.  On the frequency of copy number variants 
Bioinformatics  2008;24(20):2350-2355.
Motivation: Estimating the frequency distribution of copy number variants (CNVs) is an important aspect of the effort to characterize this new type of genetic variation. Currently, most studies report a strong skew toward low-frequency CNVs. In this article, our goal is to investigate the frequencies of CNVs. We employ a two-step procedure for the CNV frequency estimation process. We use family information a posteriori to select only the most reliable CNV regions, i.e. those showing high rates of Mendelian transmission.
Results: Our results suggest that the current skew toward low-frequency CNVs may not be representative of the true frequency distribution, but may be due, among other reasons, to the non-negligible false negative rates that characterize CNV detection methods. Moreover, false positives are also likely, as low-frequency CNVs are hard to detect with small sample sizes and technologies that are not ideally suited for their detection. Without appropriate validation methods, such as incorporation of biologically relevant information (for example, in our case, the transmission of heritable CNVs from parents to offspring), it is difficult to assess the validity of specific CNVs, and even harder to obtain reliable frequency estimates.
Availability: Software implementing the methods described in this article is available for download at the following address: http://www.isites.harvard.edu/icb/icb.do?keyword=k36162
Contact: iionita@hsph.harvard.edu
Supplementary informantion: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btn421
PMCID: PMC2562008  PMID: 18689430
24.  Genome-Wide Association Study Implicates Chromosome 9q21.31 as a Susceptibility Locus for Asthma in Mexican Children 
PLoS Genetics  2009;5(8):e1000623.
Many candidate genes have been studied for asthma, but replication has varied. Novel candidate genes have been identified for various complex diseases using genome-wide association studies (GWASs). We conducted a GWAS in 492 Mexican children with asthma, predominantly atopic by skin prick test, and their parents using the Illumina HumanHap 550 K BeadChip to identify novel genetic variation for childhood asthma. The 520,767 autosomal single nucleotide polymorphisms (SNPs) passing quality control were tested for association with childhood asthma using log-linear regression with a log-additive risk model. Eleven of the most significantly associated GWAS SNPs were tested for replication in an independent study of 177 Mexican case–parent trios with childhood-onset asthma and atopy using log-linear analysis. The chromosome 9q21.31 SNP rs2378383 (p = 7.10×10−6 in the GWAS), located upstream of transducin-like enhancer of split 4 (TLE4), gave a p-value of 0.03 and the same direction and magnitude of association in the replication study (combined p = 6.79×10−7). Ancestry analysis on chromosome 9q supported an inverse association between the rs2378383 minor allele (G) and childhood asthma. This work identifies chromosome 9q21.31 as a novel susceptibility locus for childhood asthma in Mexicans. Further, analysis of genome-wide expression data in 51 human tissues from the Novartis Research Foundation showed that median GWAS significance levels for SNPs in genes expressed in the lung differed most significantly from genes not expressed in the lung when compared to 50 other tissues, supporting the biological plausibility of our overall GWAS findings and the multigenic etiology of childhood asthma.
Author Summary
Asthma is a leading chronic childhood disease with a presumed strong genetic component, but no genes have been definitely shown to influence asthma development. Few genetic studies of asthma have included Hispanic populations. Here, we conducted a genome-wide association study of asthma in 492 Mexican children with asthma, predominantly atopic by skin prick test, and their parents to identify novel genetic variation for childhood asthma. We implicated several polymorphisms in or near TLE4 on chromosome 9q21.31 (a novel candidate region for childhood asthma) and replicated one polymorphism in an independent study of childhood-onset asthmatics with atopy and their parents of Mexican ethnicity. Hispanics have differing proportions of Native American, European, and African ancestries, and we found less Native American ancestry than expected at chromosome 9q21.31. This suggests that chromosome 9q21.31 may underlie ethnic differences in childhood asthma and that future replication would be most effective in populations with Native American ancestry. Analysis of publicly available genome-wide expression data revealed that association signals in genes expressed in the lung differed most significantly from genes not expressed in the lung when compared to 50 other tissues, supporting the biological plausibility of the overall GWAS findings and the multigenic etiology of asthma.
doi:10.1371/journal.pgen.1000623
PMCID: PMC2722731  PMID: 19714205
25.  Comprehensive Testing of Positionally Cloned Asthma Genes in Two Populations 
Rationale: Replication of gene-disease associations has become a requirement in complex trait genetics.
Objectives: In studies of childhood asthma from two different ethnic groups, we attempted to replicate associations with five potential asthma susceptibility genes previously identified by positional cloning.
Methods: We analyzed two family-based samples ascertained through an asthmatic proband: 497 European-American children from the Childhood Asthma Management Program and 439 Hispanic children from the Central Valley of Costa Rica. We genotyped 98 linkage disequilibrium–tagging single-nucleotide polymorphisms (SNPs) in five genes: ADAM33, DPP10, GPR154 (HUGO name: NPSR1), HLA-G, and the PHF11 locus (includes genes SETDB2 and RCBTB1). SNPs were tested for association with asthma and two intermediate phenotypes: airway hyperresponsiveness and total serum immunoglobulin E levels.
Measurements and Main Results: Despite differing ancestries, linkage disequilibrium patterns were similar in both cohorts. Of the five evaluated genes, SNP-level replication was found only for GPR154 (NPSR1). In this gene, three SNPs were associated with asthma in both cohorts, although the opposite alleles were associated in either study. Weak evidence for locus-level replication with asthma was found in the PHF11 locus, although there was no overlap in the associated SNP across the two cohorts. No consistent associations were observed for the three other genes.
Conclusions: These results provide some further support for the role of genetic variation in GPR154 (NPSR1) and PHF11 in asthma susceptibility and also highlight the challenges of replicating genetic associations in complex traits such as asthma, even for genes identified by linkage analysis.
doi:10.1164/rccm.200704-592OC
PMCID: PMC2048676  PMID: 17702965
bronchial hyperreactivity; immunoglobulin E; linkage disequilibrium; NPSR1; single-nucleotide polymorphism

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