Sex-Based Genetic Association Study Identifies CELSR1 as a Possible Chronic Obstructive Pulmonary Disease Risk Locus among Women.

Chronic obstructive pulmonary disease (COPD) is a complex disease with strong environmental and genetic influences and sexually dimorphic features. Although genetic risk factors for COPD have been identified, much of the heritability remains unexplained. Sex-based genetic association studies may uncover additional COPD genetic risk factors. We studied current and former smokers from COPD case-control cohorts (COPDGene non-Hispanic whites and African Americans, Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-Points, and Genetics of Chronic Obstructive Lung Disease). COPD was defined as post-bronchodilator forced expiratory volume in 1 second/forced vital capacity less than 0.70 and forced expiratory volume in 1 second percent predicted less than 80. Testing was performed across all cohorts and combined in a meta-analysis adjusted for age, pack-years, and genetic ancestry. We first performed genome-wide single-nucleotide polymorphism (SNP)-by-sex interaction testing on the outcome of COPD affection status. We performed sex-stratified association testing for SNPs with interaction P less than 10-6. We examined over 8 million SNPs in four populations, including 6,260 subjects with COPD (40.6% female) and 5,269 smoking control subjects (47.3% female). The SNP rs9615358 in the cadherin gene CELSR1 approached genome-wide significance for an interaction with sex (P = 1.24 × 10-7). In the sex-stratified meta-analysis, this SNP was associated with COPD among females (odds ratio, 1.37 [95% confidence interval, 1.25-1.49]; P = 3.32 × 10-7) but not males (odds ratio, 0.90 [95% confidence interval, 0.79-1.01]; P = 0.06). CELSR1 is involved in fetal lung development. In a human fetal lung tissue dataset, we observed greater CELSR1 expression in female compared with male samples. This SNP-by-sex genome-wide association analysis identified the fetal lung development gene, CELSR1, as a potential sex-specific risk factor for COPD. Identifying sex-specific genetic risk factors may reveal new insights into sexually dimorphic features of COPD.

Genome-wide association analysis in asthma subjects identifies SPATS2L as a novel bronchodilator response gene.

Bronchodilator response (BDR) is an important asthma phenotype that measures reversibility of airway obstruction by comparing lung function (i.e. FEV(1)) before and after the administration of a short-acting ß(2)-agonist, the most common rescue medications used for the treatment of asthma. BDR also serves as a test of ß(2)-agonist efficacy. BDR is a complex trait that is partly under genetic control. A genome-wide association study (GWAS) of BDR, quantified as percent change in baseline FEV(1) after administration of a ß(2)-agonist, was performed with 1,644 non-Hispanic white asthmatic subjects from six drug clinical trials: CAMP, LOCCS, LODO, a medication trial conducted by Sepracor, CARE, and ACRN. Data for 469,884 single-nucleotide polymorphisms (SNPs) were used to measure the association of SNPs with BDR using a linear regression model, while adjusting for age, sex, and height. Replication of primary P-values was attempted in 501 white subjects from SARP and 550 white subjects from DAG. Experimental evidence supporting the top gene was obtained via siRNA knockdown and Western blotting analyses. The lowest overall combined P-value was 9.7E-07 for SNP rs295137, near the SPATS2L gene. Among subjects in the primary analysis, those with rs295137 TT genotype had a median BDR of 16.0 (IQR = [6.2, 32.4]), while those with CC or TC genotypes had a median BDR of 10.9 (IQR = [5.0, 22.2]). SPATS2L mRNA knockdown resulted in increased ß(2)-adrenergic receptor levels. Our results suggest that SPATS2L may be an important regulator of ß(2)-adrenergic receptor down-regulation and that there is promise in gaining a better understanding of the biological mechanisms of differential response to ß(2)-agonists through GWAS.

ITGB5 and AGFG1 variants are associated with severity of airway responsiveness.

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.

The impact of self-identified race on epidemiologic studies of gene expression.

Although population differences in gene expression have been established, the impact on differential gene expression studies in large populations is not well understood. We describe the effect of self-reported race on a gene expression study of lung function in asthma. We generated gene expression profiles for 254 young adults (205 non-Hispanic whites and 49 African Americans) with asthma on whom concurrent total RNA derived from peripheral blood CD4(+) lymphocytes and lung function measurements were obtained. We identified four principal components that explained 62% of the variance in gene expression. The dominant principal component, which explained 29% of the total variance in gene expression, was strongly associated with self-identified race (P<10(-16)). The impact of these racial differences was observed when we performed differential gene expression analysis of lung function. Using multivariate linear models, we tested whether gene expression was associated with a quantitative measure of lung function: pre-bronchodilator forced expiratory volume in one second (FEV(1)). Though unadjusted linear models of FEV(1) identified several genes strongly correlated with lung function, these correlations were due to racial differences in the distribution of both FEV(1) and gene expression, and were no longer statistically significant following adjustment for self-identified race. These results suggest that self-identified race is a critical confounding covariate in epidemiologic studies of gene expression and that, similar to genetic studies, careful consideration of self-identified race in gene expression profiling studies is needed to avoid spurious association.

Mapping of numerous disease-associated expression polymorphisms in primary peripheral blood CD4+ lymphocytes.

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.

Integration of mouse and human genome-wide association data identifies KCNIP4 as an asthma gene.

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.

Association of SERPINE2 with asthma.

BACKGROUND: The "Dutch hypothesis" suggests that asthma and COPD have common genetic determinants. The serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2 (SERPINE2) gene previously has been associated with COPD. We sought to determine whether SERPINE2 is associated with asthma and asthma-related phenotypes. METHODS: We measured the association of 39 SERPINE2 single-nucleotide polymorphisms (SNPs) with asthma-related phenotypes in 655 parent-child trios from the Childhood Asthma Management Program (CAMP), and we measured the association of 19 SERPINE2 SNPs with asthma in a case-control design of 359 CAMP probands and 846 population control subjects. We attempted to replicate primary asthma-related phenotype findings in one independent population and primary asthma affection status findings in two independent populations. We compared association results with CAMP proband expression quantitative trait loci. RESULTS: Nine of 39 SNPs had P < .05 for at least one phenotype in CAMP, and two of these replicated in an independent population of 426 people with childhood asthma. Six of 19 SNPs had P < .05 for association with asthma in CAMP/Illumina. None of these replicated in two independent populations. The expression quantitative trait loci revealed that five SNPs associated with asthma in CAMP/Illumina and one SNP associated with FEV(1) in CAMP are strongly correlated with SERPINE2 expression levels. Comparison of results to previous COPD studies identified five SNPs associated with both asthma- and COPD-related phenotypes. CONCLUSIONS: Our results weakly support SERPINE2 as a Dutch hypothesis candidate gene through nominally significant associations with asthma and related traits. Further study of SERPINE2 is necessary to verify its involvement in asthma and COPD.