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.
Allergic rhinitis is a common disease whose genetic basis is incompletely explained. We report an integrated genomic analysis of allergic rhinitis.
We performed genome wide association studies (GWAS) of allergic rhinitis in 5633 ethnically diverse North American subjects. Next, we profiled gene expression in disease-relevant tissue (peripheral blood CD4+ lymphocytes) collected from subjects who had been genotyped. We then integrated the GWAS and gene expression data using expression single nucleotide (eSNP), coexpression network, and pathway approaches to identify the biologic relevance of our GWAS.
GWAS revealed ethnicity-specific findings, with 4 genome-wide significant loci among Latinos and 1 genome-wide significant locus in the GWAS meta-analysis across ethnic groups. To identify biologic context for these results, we constructed a coexpression network to define modules of genes with similar patterns of CD4+ gene expression (coexpression modules) that could serve as constructs of broader gene expression. 6 of the 22 GWAS loci with P-value ≤ 1x10−6 tagged one particular coexpression module (4.0-fold enrichment, P-value 0.0029), and this module also had the greatest enrichment (3.4-fold enrichment, P-value 2.6 × 10−24) for allergic rhinitis-associated eSNPs (genetic variants associated with both gene expression and allergic rhinitis). The integrated GWAS, coexpression network, and eSNP results therefore supported this coexpression module as an allergic rhinitis module. Pathway analysis revealed that the module was enriched for mitochondrial pathways (8.6-fold enrichment, P-value 4.5 × 10−72).
Our results highlight mitochondrial pathways as a target for further investigation of allergic rhinitis mechanism and treatment. Our integrated approach can be applied to provide biologic context for GWAS of other diseases.
Genome-wide association study; Allergic rhinitis; Coexpression network; Expression single-nucleotide polymorphism; Coexpression module; Pathway; Mitochondria; Hay fever; Allergy
The investigation of complex disease heterogeneity has been challenging. Here, we introduce a network-based approach, using partial correlations, that analyzes the relationships among multiple disease-related phenotypes.
We applied this method to two large, well-characterized studies of chronic obstructive pulmonary disease (COPD). We also examined the associations between these COPD phenotypic networks and other factors, including case-control status, disease severity, and genetic variants. Using these phenotypic networks, we have detected novel relationships between phenotypes that would not have been observed using traditional epidemiological approaches.
Phenotypic network analysis of complex diseases could provide novel insights into disease susceptibility, disease severity, and genetic mechanisms.
Network medicine; Phenotypic networks; COPD; Genetic association analysis
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.
Our aim was to examine the clinical relevance and temporal stability of phenotypic clusters in children with asthma.
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.
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.
Phenotypic clustering can be used to identify longitudinally consistent and clinically relevant patient subgroups, with implications for targeted therapeutic strategies and clinical trials design.
Childhood asthma; asthma phenotypes; inhaled corticosteroids; cluster analysis; asthma classification; longitudinal study
Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis.
Hedgehog interacting protein (HHIP); Gene expression profiling; COPD (Chronic obstructive pulmonary disease); extracellular matrix (ECM); network modeling
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.
To identify the genetic predictors of serum total IgE levels.
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.
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.
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.
meta-analysis; genome wide association study; total immunoglobulin E; race-ethnicity; continental population groups
Genetic studies have identified numerous genes reproducibly associated with asthma, yet these studies have focused almost entirely on single nucleotide polymorphisms (SNPs), and virtually ignored another highly prevalent form of genetic variation: Copy Number Variants (CNVs).
To survey the prevalence of CNVs in genes previously associated with asthma, and to assess whether CNVs represent the functional asthma-susceptibility variants at these loci.
We genotyped 383 asthmatic trios participating in the Childhood Asthma Management Program (CAMP) using a competitive genomic hybridization (CGH) array designed to interrogate 20,092 CNVs. To ensure comprehensive assessment of all potential asthma candidate genes, we purposely used liberal asthma gene inclusion criteria, resulting in consideration of 270 candidate genes previously implicated in asthma. We performed statistical testing using FBAT-CNV.
Copy number variation in asthma candidate genes was prevalent, with 21% of tested genes residing near or within one of 69 CNVs. In 6 instances, the complete candidate gene sequence resides within the CNV boundaries. On average, asthmatic probands carried 6 asthma-candidate CNVs (range 1–29). However, the vast majority of identified CNVs were of rare frequency (< 5%), and were not statistically associated with asthma. Modest evidence for association with asthma was observed for 2 CNVs near NOS1 and SERPINA3. Linkage disequilibrium analysis suggests that CNV effects are unlikely to explain previously detected SNP associations with asthma.
Although a substantial proportion of asthma-susceptibility genes harbor polymorphic CNVs, the majority of these variants do not confer increased asthma risk. The lack of linkage disequilibrium (LD) between CNVs and asthma-associated SNPs suggests that these CNVs are unlikely to represent the functional variant responsible for most known asthma associations.
To identify metabolomic biomarkers predictive of Intensive Care Unit (ICU) mortality in adults.
Comprehensive metabolomic profiling of plasma at ICU admission to identify biomarkers associated with mortality has recently become feasible.
We performed metabolomic profiling of plasma from 90 ICU subjects enrolled in the BWH Registry of Critical Illness (RoCI). We tested individual metabolites and a Bayesian Network of metabolites for association with 28-day mortality, using logistic regression in R, and the CGBayesNets Package in MATLAB. Both individual metabolites and the network were tested for replication in an independent cohort of 149 adults enrolled in the Community Acquired Pneumonia and Sepsis Outcome Diagnostics (CAPSOD) study.
We tested variable metabolites for association with 28-day mortality. In RoCI, nearly one third of metabolites differed among ICU survivors versus those who died by day 28 (N = 57 metabolites, p<.05). Associations with 28-day mortality replicated for 31 of these metabolites (with p<.05) in the CAPSOD population. Replicating metabolites included lipids (N = 14), amino acids or amino acid breakdown products (N = 12), carbohydrates (N = 1), nucleotides (N = 3), and 1 peptide. Among 31 replicated metabolites, 25 were higher in subjects who progressed to die; all 6 metabolites that are lower in those who die are lipids. We used Bayesian modeling to form a metabolomic network of 7 metabolites associated with death (gamma-glutamylphenylalanine, gamma-glutamyltyrosine, 1-arachidonoylGPC(20:4), taurochenodeoxycholate, 3-(4-hydroxyphenyl) lactate, sucrose, kynurenine). This network achieved a 91% AUC predicting 28-day mortality in RoCI, and 74% of the AUC in CAPSOD (p<.001 in both populations).
Both individual metabolites and a metabolomic network were associated with 28-day mortality in two independent cohorts. Metabolomic profiling represents a valuable new approach for identifying novel biomarkers in critically ill patients.
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.
We aimed to identify additional asthma risk loci by performing an extensive replication
study of the results from the EVE Consortium meta-analysis.
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.
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.
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.
Asthma; genetic risk factors; meta-analysis; KLK3
Past studies have demonstrated that single nucleotide polymorphisms (SNPs) of the sodium-bicarbonate co-transporter gene (SLC4A5) are associated with hypertension. We tested the hypothesis that SNPs in SLC4A5 are associated with salt-sensitivity of blood pressure (BP) in 185 Caucasians consuming an isocaloric constant diet with a randomized order of 7 days low Na+ (10 mmol/d) and 7 days high Na+ (300 mmol/d) intake. Salt-sensitivity was defined as a ≥7mm Hg increase in mean arterial pressure (MAP) during a randomized transition between high and low Na+ diet.
A total of 35 polymorphisms in 17 candidate genes were assayed, 25 of which were tested for association. Association analyses with salt-sensitivity revealed three variants that associated with salt-sensitivity, two in SLC4A5 (P <0.001), and one in GRK4 (P = 0.020). Of these, two SNPs in SLC4A5 (rs7571842 and rs10177833) demonstrated highly significant results and large effects sizes, using logistic regression. These two SNPs had P values of 1.0×10−4 and 3.1×10−4 with odds ratios of 0.221 and 0.221 in unadjusted regression models, respectively, with the G allele at both sites conferring protection. These SNPs remained significant after adjusting for BMI and age, (P = 8.9×10−5 and 2.6×10−4 and odds ratios 0.210 and 0.286, respectively). Further, the association of these SNPS with salt-sensitivity was replicated in a second hypertensive population. Meta-analysis demonstrated significant associations of both SNPs with salt-sensitivity [rs7571842 (P=1.2×10−5); rs1017783 (P=1.1×10−4)]. In conclusion, SLC4A5 variants are strongly associated with salt-sensitivity of BP in two separate Caucasian populations.
Genetics-human; Genetics-association studies; Cardiovascular disease; Hypertension (Kidney); Blood Pressure; Sodium-bicarbonate co-transporter; Salt-sensitivity; Hypertension; Genetics
drug response; genetic variants; pharmacogenomics; vitamin D receptor
Airway hyperresponsiveness (AHR), a primary characteristic of asthma, involves increased airway smooth muscle contractility in response to certain exposures. We sought to determine whether common genetic variants were associated with AHR severity.
A genome-wide association study (GWAS) of AHR, quantified as the natural log of the dosage of methacholine causing a 20% drop in FEV1, was performed with 994 non-Hispanic white asthmatic subjects from three drug clinical trials: CAMP, CARE, and ACRN. Genotyping was performed on Affymetrix 6.0 arrays, and imputed data based on HapMap Phase 2, was used to measure the association of SNPs with AHR using a linear regression model. Replication of primary findings was attempted in 650 white subjects from DAG, and 3,354 white subjects from LHS. Evidence that the top SNPs were eQTL of their respective genes was sought using expression data available for 419 white CAMP subjects.
The top primary GWAS associations were in rs848788 (P-value 7.2E-07) and rs6731443 (P-value 2.5E-06), located within the ITGB5 and AGFG1 genes, respectively. The AGFG1 result replicated at a nominally significant level in one independent population (LHS P-value 0.012), and the SNP had a nominally significant unadjusted P-value (0.0067) for being an eQTL of AGFG1.
Based on current knowledge of ITGB5 and AGFG1, our results suggest that variants within these genes may be involved in modulating AHR. Future functional studies are required to confirm that our associations represent true biologically significant findings.
Asthma; Airway hyperresponsiveness; Genome-wide association study; ITGB5; AGFG1
Hypertension represents a complex heritable disease in which environmental factors may directly affect gene function via epigenetic mechanisms. The aim of this study was to test the hypothesis that dietary salt influences the activity of a histone modifying enzyme, lysine-specific demethylase 1 (LSD-1), which in turn is associated with salt-sensitivity of blood pressure (BP).
Animal and human studies were performed. Salt-sensitivity of LSD-1 expression was assessed in wild-type and LSD-1 heterozygote knockout (LSD-1+/−) mice. Clinical relevance was tested by multivariate associations between single nuclear polymorphisms (SNPs) in the LSD-1 gene and salt-sensitivity of BP, with control of dietary sodium, in a primary African-American hypertensive cohort and two replication hypertensive cohorts (Caucasian and Mexican-American).
LSD1 expression was modified by dietary salt in wild-type mice with lower levels associated with liberal salt intake. LSD-1+/− mice expressed lower LSD-1 protein levels than wild-type mice in kidney tissue. Similar to LSD-1+/− mice, African-American minor allele carriers of two LSD-1 SNPs displayed greater change in systolic BP in response to change from low to liberal salt diet (rs671357, p=0.01; rs587168, p=0.005). This association was replicated in the Hispanic (rs587168, p=0.04) but not the Caucasian cohort. Exploratory analyses demonstrated decreased serum aldosterone concentrations in African-American minor allele carriers similar to findings in the LSD-1+/− mice, decreased alpha-EnaC expression in LSD-1+/− mice, and impaired renovascular responsiveness to salt loading in minor allele carriers.
The results of this translational research study support a role for LSD1 in the pathogenesis of salt-sensitive hypertension.
Hypertension; Salt-sensitivity; LSD1; Genetics; Epigenetic
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 (FEV1). Though unadjusted linear models of FEV1 identified several genes strongly correlated with lung function, these correlations were due to racial differences in the distribution of both FEV1 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.
ancestry; gene expression; population stratification; self-identified race
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.
interleukin 10 (IL10); single nucleotide polymorphism (SNP); genetic association; family-based association test (FBAT); haplotype; promoter; 3′; untranslated region (3′UTR)
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.
To identify genetic variants associated with earlier onset of childhood asthma.
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.
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.
We have identified two SNPs associated with earlier onset of childhood asthma in four independent cohorts.
Asthma; pediatrics; age of onset; asthma genetics; C1orf100; genome-wide association study; pediatric asthma
In recent years there has been a growing interest in the role of copy number variations (CNV) in genetic diseases. Though there has been rapid development of technologies and statistical methods devoted to detection in CNVs from array data, the inherent challenges in data quality associated with most hybridization techniques remains a challenging problem in CNV association studies.
To help address these data quality issues in the context of family-based association studies, we introduce a statistical framework for the intensity-based array data that takes into account the family information for copy-number assignment. The method is an adaptation of traditional methods for modeling SNP genotype data that assume Gaussian mixture model, whereby CNV calling is performed for all family members simultaneously and leveraging within family-data to reduce CNV calls that are incompatible with Mendelian inheritance while still allowing de-novo CNVs. Applying this method to simulation studies and a genome-wide association study in asthma, we find that our approach significantly improves CNV calls accuracy, and reduces the Mendelian inconsistency rates and false positive genotype calls. The results were validated using qPCR experiments.
In conclusion, we have demonstrated that the use of family information can improve the quality of CNV calling and hopefully give more powerful association test of CNVs.
Insulin resistance (IR) is a complex disorder caused by an interplay of both genetic and environmental factors. Recent studies identified a significant interaction between body mass index (BMI) and the rs1800795 polymorphism of the Interleukin-6 (IL-6) gene that influences both IR and onset of type 2 diabetes mellitus (T2DM) with obese individuals homozygous for the C allele demonstrating the highest level of IR and greatest risk for T2DM. Replication of a gene-environment interaction is important to confirm the validity of the initial finding and extends the generalizability of the results to other populations. Thus, the objective of this study was to replicate this gene-environment interaction on IR in a hypertensive population and perform a meta-analysis with prior published results.
Material and Methods
The replication analysis was performed using Caucasian individuals with hypertension (HTN) from the HyperPATH cohort (N=311), genotyped for rs1800795. Phenotype studies were conducted after participants consumed two diets: high sodium (HS) (200mmol/day) and low sodium (LS) (10mmol/day) for 7 days each. Measurements for plasma glucose, insulin, and IL-6 were obtained after 8 hours of fasting. IR was characterized by the homeostatic model assessment (HOMA-IR).
In HyperPATH, BMI was a significant effect modifier of the relationship between rs1800795 and HOMA-IR; higher BMI was associated with higher HOMA-IR among homozygote CC individuals when compared to major allele G carriers (p=0.003). Further, the meta-analysis in 1028 individuals confirmed the result demonstrating the same significant interaction between rs1800795 and BMI on HOMA-IR (p=1.05×10−6).
This rare replication of a gene-environment interaction extends the generalizability of the results to HTN while highlighting this polymorphism as a marker of IR in obese individuals.
Interleukin-6 gene; Hypertension; Obesity; Insulin Resistance
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.
To test the hypothesis that some genes may contribute to the profound disparities in asthma.
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.
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.
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.
Asthma; GWAS; ADRA1B; PRNP; DPP10; African ancestry; ethnicity; polymorphism; genetic association
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
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.
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