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.
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.
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.
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.
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.
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.
Purinergic receptor; leukotriene; asthma; house dust mite; lung function
The renin gene has been previously reported to be associated with essential hypertension in a variety of ethnic groups. However, no studies have systematically evaluated the relationship between single nucleotide polymorphisms (SNPs) representing coverage of the entire renin gene and hypertension risk. To evaluate the association between renin gene variation and hypertension we investigated data on HyperPath cohort with 570 hypertensive and 222 normotensive Caucasian subjects. Six tagging SNPs and resultant haplotypes were tested for associations with hypertension risk, followed by mean arterial pressure (MAP), plasma renin activity (PRA) and the change in MAP in response to angiotensin II infusion (AngII ΔMAP). The A allele of SNP rs6693954 and the haplotype containing rs6696954A were significantly associated with higher risk for hypertension (OR=1.98, P=0.0001; OR=1.63 P=0.0005, respectively). The same haplotype block was also associated with altered PRA levels and blunted AngII ΔMAP (global P value=0.02, 0.047, respectively). Our results confirm that polymorphisms in the REN are associated with increased risk for hypertension in an independent cohort, and that the underlying mechanism may reside in the interaction of renin activity and vascular responsiveness to angiotensin II.
Renin; SNP; haplotype; blood pressure and Hypertension
The purpose of this study was to clarify the association of the angiotensinogen gene (AGT) with insulin sensitivity using SNP and haplotype analyses in a Caucasian cohort.
Material and Methods
A candidate gene association study was conducted in Caucasians with and without hypertension (N=449). Seventeen single nucleotide polymorphisms (SNPs) of the AGT gene and their haplotypes were analyzed for an association with HOMA-IR. Multivariate regression model accounting for age, gender, BMI, hypertension status, study site, and sibling relatedness was used to test the hypothesis.
Nine of the seventeen SNPs were significantly associated with lower HOMA-IR levels. Homozygous minor allele carriers of the most significant SNP rs2493134 (GG), a surrogate for the gain of function mutation rs699 [AGT p.M268T], had significantly lower HOMA-IR levels (p=0.0001) than heterozygous or homozygous major allele carriers (GC, AA). Direct genotyping of rs699 in a subset of the population showed similar results with minor allele carriers exhibiting significantly decreased HOMA-IR levels (p=0.003). Haplotype analysis demonstrated that haplotypes rs2493137A|rs5050A|rs3789678G|rs2493134A and rs2004776G|rs11122576A|rs699T|rs6687360G were also significantly associated with HOMA-IR (p=0.0009, p=0.02) and these results were driven by rs2493134 and rs699.
This study confirms an association between the AGT gene and insulin sensitivity in Caucasian humans. Haplotype analysis extends this finding and implicates SNPs rs2493134 and rs699 as the most influential. Thus, AGT gene variants, previously shown to be associated with AGT levels, are also associated with insulin sensitivity; suggesting a relationship between the AGT gene, AGT levels, and insulin sensitivity in humans.
insulin resistance; hypertension; angiotensinogen; genetics
Williams-Beuren Syndrome (WBS) is caused by a submicroscopic deletion on chromosome 7q11.23 that encompasses the entire elastin (ELN) gene. Elastin, a key component of elastic fibers within the lung, is progressively destroyed in emphysema. Defects in the elastin gene have been associated with increased susceptibility towards developing chronic obstructive pulmonary disease (COPD) and emphysema in both humans and mice. We postulate that hemizygosity at the elastin gene locus may increase susceptibility towards the development of COPD and emphysema in subjects with WBS. We describe an adult subject with WBS who was a lifelong non-smoker and was found to have moderate emphysema. We also examined the pulmonary function of a separate cohort of adolescents and young adults with WBS. Although no significant spirometric abnormalities were identified, a significant proportion of subjects reported respiratory symptoms. Thus while significant obstructive disease does not appear to be common in relatively young adults with WBS, subclinical emphysema and lung disease may exist which possibly could worsen with advancing age. Further investigation may elucidate the pathogenesis of non-smoking related emphysema.
Elastin; emphysema; pulmonary function tests; Williams Syndrome
We propose a new approach for the analysis of copy number variants (CNVs)for genome-wide association studies in family-based designs. Our new overall association test combines the between-family component and the within-family component of the data so that the new test statistic is fully efficient and, at the same time, achieves the complete robustness against population-admixture and stratification, as classical family-based association tests that are based only on the between-family component. Although all data are incorporated into the test statistic, an adjustment for genetic confounding is not needed, not even for the between-family component. The new test statistic is valid for testing either quantitative or dichotomous phenotypes. If external CNV data are available, the approach can also be used in completely ascertained samples. Similar to the approach by Ionita-Laza et al.(1), the proposed test statistic does not required a CNV-calling algorithm and is based directly on the CNV probe intensity data. We show, via simulation studies, that our methodology increases the power of the FBAT statistic to levels comparable to those of population-based designs. The advantages of the approach in practice are demonstrated by an application to a genome-wide association study for body mass index (BMI).
Genome-wide association studies of human gene expression promise to identify functional regulatory genetic variation that contributes to phenotypic diversity. However, it is unclear how useful this approach will be for the identification of disease-susceptibility variants. We generated gene expression profiles for 22 184 mRNA transcripts using RNA derived from peripheral blood CD4+ lymphocytes, and genome-wide genotype data for 516 512 autosomal markers in 200 subjects. We screened for cis-acting variants by testing variants mapping within 50 kb of expressed transcripts for association with transcript abundance using generalized linear models. Significant associations were identified for 1585 genes at a false discovery rate of 0.05 (corresponding to P-values ranging from 1 × 10−91 to 7 × 10−4). Importantly, we identified evidence of regulatory variation for 119 previously mapped disease genes, including 24 examples where the variant with the strongest evidence of disease-association demonstrates strong association with specific transcript abundance. The prevalence of cis-acting variants among disease-associated genes was 63% higher than the genome-wide rate in our data set (P = 6.41 × 10−6), and although many of the implicated loci were associated with immune-related diseases (including asthma, connective tissue disorders and inflammatory bowel disease), associations with genes implicated in non-immune-related diseases including lipid profiles, anthropomorphic measurements, cancer and neurologic disease were also observed. Genetic variants that confer inter-individual differences in gene expression represent an important subset of variants that contribute to disease susceptibility. Population-based integrative genetic approaches can help identify such variation and enhance our understanding of the genetic basis of complex traits.
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.
asthma susceptibility; lung development; developmental gene expression
Linkage- and association-based methods have been proposed for mapping disease-causing rare variants. Based on the family information provided in the Genetic Analysis Workshop 17 data set, we formulate a two-pronged approach that combines both methods. Using the identity-by-descent information provided for eight extended pedigrees (n = 697) and the simulated quantitative trait Q1, we explore various traditional nonparametric linkage analysis methods; the best result is obtained by assuming between-family heterogeneity and applying the Haseman-Elston regression to each pedigree separately. We discover strong signals from two genes in two different families and weaker signals for a third gene from two other families. As an exploratory approach, we apply an association test based on a modified family-based association test statistic to all rare variants (frequency < 1% or < 3%) designated as causal for Q1. Family-based association tests correctly identified causal single-nucleotide polymorphisms for four genes (KDR, VEGFA, VEGFC, and FLT1). Our results suggest that both linkage and association tests with families show promise for identifying rare variants.
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.
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).
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.
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).
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.
asthma; in utero smoke exposure; airway responsiveness; inhaled corticosteroids
Network modeling of whole transcriptome expression data enables characterization of complex epistatic (gene-gene) interactions that underlie cellular functions. Though numerous methods have been proposed and successfully implemented to develop these networks, there are no formal methods for comparing differences in network connectivity patterns as a function of phenotypic trait.
Here we describe a novel approach for quantifying the differences in gene-gene connectivity patterns across disease states based on Graphical Gaussian Models (GGMs). We compare the posterior probabilities of connectivity for each gene pair across two disease states, expressed as a posterior odds-ratio (postOR) for each pair, which can be used to identify network components most relevant to disease status. The method can also be generalized to model differential gene connectivity patterns within previously defined gene sets, gene networks and pathways. We demonstrate that the GGM method reliably detects differences in network connectivity patterns in datasets of varying sample size. Applying this method to two independent breast cancer expression data sets, we identified numerous reproducible differences in network connectivity across histological grades of breast cancer, including several published gene sets and pathways. Most notably, our model identified two gene hubs (MMP12 and CXCL13) that each exhibited differential connectivity to more than 30 transcripts in both datasets. Both genes have been previously implicated in breast cancer pathobiology, but themselves are not differentially expressed by histologic grade in either dataset, and would thus have not been identified using traditional differential gene expression testing approaches. In addition, 16 curated gene sets demonstrated significant differential connectivity in both data sets, including the matrix metalloproteinases, PPAR alpha sequence targets, and the PUFA synthesis pathway.
Our results suggest that GGM can be used to formally evaluate differences in global interactome connectivity across disease states, and can serve as a powerful tool for exploring the molecular events that contribute to disease at a systems level.
The relationships between total serum IgE levels and gene expression patterns in peripheral blood CD4+ T cells (in all subjects and within each sex specifically) are not known.
Peripheral blood CD4+ T cells from 223 participants from the Childhood Asthma Management Program (CAMP) with simultaneous measurement of IgE. Total RNA was isolated, and expression profiles were generated with Illumina HumanRef8 v2 BeadChip arrays. Modeling of the relationship between genome-wide gene transcript levels and IgE levels was performed in all subjects, and stratified by sex.
Among all subjects, significant evidence for association between gene transcript abundance and IgE was identified for a single gene, the interleukin 17 receptor B (IL17RB), explaining 12% of the variance (r2) in IgE measurement (p value = 7 × 10-7, 9 × 10-3 after adjustment for multiple testing). Sex stratified analyses revealed that the correlation between IL17RB and IgE was restricted to males only (r2 = 0.19, p value = 8 × 10-8; test for sex-interaction p < 0.05). Significant correlation between gene transcript abundance and IgE level was not found in females. Additionally we demonstrated substantial sex-specific differences in IgE when considering multi-gene models, and in canonical pathway analyses of IgE level.
Our results indicate that IL17RB may be the only gene expressed in CD4+ T cells whose transcript measurement is correlated with the variation in IgE level in asthmatics. These results provide further evidence sex may play a role in the genomic regulation of IgE.
Rationale: Animal models demonstrate that aberrant gene expression in utero can result in abnormal pulmonary phenotypes.
Objectives: We sought to identify genes that are differentially expressed during in utero airway development and test the hypothesis that variants in these genes influence lung function in patients with asthma.
Methods: Stage 1 (Gene Expression): Differential gene expression analysis across the pseudoglandular (n = 27) and canalicular (n = 9) stages of human lung development was performed using regularized t tests with multiple comparison adjustments. Stage 2 (Genetic Association): Genetic association analyses of lung function (FEV1, FVC, and FEV1/FVC) for variants in five differentially expressed genes were conducted in 403 parent-child trios from the Childhood Asthma Management Program (CAMP). Associations were replicated in 583 parent-child trios from the Genetics of Asthma in Costa Rica study.
Measurements and Main Results: Of the 1,776 differentially expressed genes between the pseudoglandular (gestational age: 7–16 wk) and the canalicular (gestational age: 17–26 wk) stages, we selected 5 genes in the Wnt pathway for association testing. Thirteen single nucleotide polymorphisms in three genes demonstrated association with lung function in CAMP (P < 0.05), and associations for two of these genes were replicated in the Costa Ricans: Wnt1-inducible signaling pathway protein 1 with FEV1 (combined P = 0.0005) and FVC (combined P = 0.0004), and Wnt inhibitory factor 1 with FVC (combined P = 0.003) and FEV1/FVC (combined P = 0.003).
Conclusions: Wnt signaling genes are associated with impaired lung function in two childhood asthma cohorts. Furthermore, gene expression profiling of human fetal lung development can be used to identify genes implicated in the pathogenesis of lung function impairment in individuals with asthma.
asthma; lung development; lung function; genetic variation; gene expression
Prior studies suggest a role for a variant (rs5743836) in the promoter of toll-like receptor 9 (TLR9) in asthma and other inflammatory diseases. We performed detailed genetic association studies of the functional variant rs5743836 with asthma susceptibility and asthma-related phenotypes in three independent cohorts.
rs5743836 was genotyped in two family-based cohorts of children with asthma and a case-control study of adult asthmatics. Association analyses were performed using chi square, family-based and population-based testing. A luciferase assay was performed to investigate whether rs5743836 genotype influences TLR9 promoter activity.
Contrary to prior reports, rs5743836 was not associated with asthma in any of the three cohorts. Marginally significant associations were found with FEV1 and FVC (p = 0.003 and p = 0.008, respectively) in one of the family-based cohorts, but these associations were not significant after correcting for multiple comparisons. Higher promoter activity of the CC genotype was demonstrated by luciferase assay, confirming the functional importance of this variant.
Although rs5743836 confers regulatory effects on TLR9 transcription, this variant does not appear to be an important asthma-susceptibility locus.
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.
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.
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.
Binary trait; Candidate gene analysis; Family-based association tests; FBAT-C; Linkage disequilibrium (LD); Model-based test; Model-free test; Nuclear families; Quantitative trait
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.
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).
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.
TSLP variants are associated with asthma in a sex-specific fashion.
asthma; genetic association; sex-specific; thymic stromal lymphopoietin; TSLP
The glutathione S-transferase M1 (GSTM1) null variant is a common copy number variant associated with adverse pulmonary outcomes, including asthma and airflow obstruction, with evidence of important gene-by-environment interactions with exposures to oxidative stress.
To explore the joint interactive effects of GSTM1 copy number and tobacco smoke exposure on the development of asthma and asthma-related phenotypes in a family-based cohort of childhood asthmatics.
We performed quantitative PCR-based genotyping for GSTM1 copy number in children of self-reported white ancestry with mild to moderate asthma in the Childhood Asthma Management Program. Questionnaire data regarding intrauterine (IUS) and postnatal, longitudinal environmental tobacco smoke exposure were available. We performed both family-based and population-based tests of association for the interaction between GSTM1 copy number and tobacco smoke exposure with asthma and asthma-related phenotypes.
Associations of GSTM1 null variants with asthma (p= .03), younger age of asthma symptom onset (p=.03), and greater airflow obstruction (reduced FEV1/FVC, p=.01) were observed among the 50 children (10% of the cohort) with exposure to IUS. In contrast, no associations were observed between GSTM1 null variants and asthma-related phenotypes among children without IUS exposure. Presence of at least one copy of GSTM1 conferred protection.
These findings support an important gene-by-environment interaction between two common factors: increased risk of asthma and asthma-related phenotypes conferred by GSTM1-null homozygosity in children is restricted to those with a history of IUS exposure.
Asthma; GSTM1; copy number variation (CNV); gene by environment; intrauterine smoke exposure; tobacco smoke
Asthma is a chronic respiratory disease whose genetic basis has been explored for over two decades, most recently via genome-wide association studies. We sought to find asthma-susceptibility variants by using probands from a single population in both family-based and case-control association designs.
We used probands from the Childhood Asthma Management Program (CAMP) in two primary genome-wide association study designs: (1) probands were combined with publicly available population controls in a case-control design, and (2) probands and their parents were used in a family-based design. We followed a two-stage replication process utilizing three independent populations to validate our primary findings.
We found that single nucleotide polymorphisms with similar case-control and family-based association results were more likely to replicate in the independent populations, than those with the smallest p-values in either the case-control or family-based design alone. The single nucleotide polymorphism that showed the strongest evidence for association to asthma was rs17572584, which replicated in 2/3 independent populations with an overall p-value among replication populations of 3.5E-05. This variant is near a gene that encodes an enzyme that has been implicated to act coordinately with modulators of Th2 cell differentiation and is expressed in human lung.
Our results suggest that using probands from family-based studies in case-control designs, and combining results of both family-based and case-control approaches, may be a way to augment our ability to find SNPs associated with asthma and other complex diseases.
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.
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.
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).
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.
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.”
asthma; replication; single-nucleotide polymorphism; integrin β3; association
Summary: Associations between DNA polymorphisms and mRNA abundance are a natural target of genetic investigations, and microarrays facilitate genome-wide and transcriptome-wide surveys of these associations. This work is motivated by emerging requirements for data architectures and algorithm interfaces to allow flexible exploration of public and private archives of genotyping and expression arrays. Using R/Bioconductor facilities, Phase II HapMap genotypes and Illumina 47K expression assay results archived on multiple populations may be interactively explored and analyzed using commodity hardware.
Availability and Implementation: Open Source. Bioconductor 2.3 packages GGtools, GGBase, GGdata, hmyriB36. Freely available on the web at http://www.bioconductor.org
The destruction of elastic fibers has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Emphysema has been described in autosomal dominant cutis laxa, which can be caused by mutations in the elastin gene. Previously, a rare functional mutation in the terminal exon of elastin was found in a case of severe, early-onset COPD. To test the hypothesis that other similar elastin mutations may predispose to COPD, we screened 90 probands from the Boston Early-Onset COPD Study and 90 smoking control subjects from the Normative Aging Study for mutations in elastin exons using high-resolution DNA melt analysis followed by resequencing. Rare nonsynonymous single-nucleotide polymorphisms (SNPs) seen only in cases were examined for segregation with airflow obstruction within pedigrees. Common nonsynonymous SNPs were tested for association with COPD in a family-based analysis of 949 subjects from the Boston Early-Onset COPD Study, and in a case–control analysis in 389 COPD cases from the National Emphysema Treatment Trial and 472 control subjects from the Normative Aging Study. Of 28 elastin variants found, 3 were nonsynonymous SNPs found only in cases. The previously described Gly773Asp mutation was found in another proband. The other two SNPs did not clearly segregate with COPD within families. Two common nonsynonymous SNPs did not demonstrate significant associations in either a family-based or case–control analysis. Exonic SNPs in the elastin gene do not appear to be common risk factors for severe COPD.
elastin; chronic obstructive pulmonary disease; emphysema; genetic polymorphism
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.
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.
Our approach reduces multiple comparisons and may provide insight into the complex relationships between genotype and gene expression.