Genome-wide association studies have yet to identify the majority of genetic variants involved in asthma. We hypothesized that expression quantitative trait locus (eQTL) mapping can identify novel asthma genes by enabling prioritization of putative functional variants for association testing.
We evaluated 6,706 cis-acting expression-associated variants (eSNP) identified through a genome-wide eQTL survey of CD4+ lymphocytes for association with asthma.
eSNP were tested for association with asthma in 359 asthma cases and 846 controls from the Childhood Asthma Management Program, with verification using family-based testing. Significant associations were tested for replication in 579 parent-child trios with asthma from Costa Rica. Further functional validation was performed by Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE)-qPCR and Chromatin-Immunoprecipitation (ChIP)-PCR in lung derived epithelial cell lines (Beas-2B and A549) and Jurkat cells, a leukemia cell line derived from T lymphocytes.
Cis-acting eSNP demonstrated associations with asthma in both cohorts. We confirmed the previously-reported association of ORMDL3/GSDMB variants with asthma (combined p=2.9 × 108). Reproducible associations were also observed for eSNP in three additional genes: FADS2 (p=0.002), NAGA (p=0.0002), and F13A1 (p=0.0001). We subsequently demonstrated that FADS2 mRNA is increased in CD4+ lymphocytes in asthmatics, and that the associated eSNPs reside within DNA segments with histone modifications that denote open chromatin status and confer enhancer activity.
Our results demonstrate the utility of eQTL mapping in the identification of novel asthma genes, and provide evidence for the importance of FADS2, NAGA, and F13A1 in the pathogenesis of asthma.
Asthma; CD4+; lymphocytes; regulatory variants; Expression Quantitative Trait Locus (eQTL); Haplotype; Integrative Genomics
Reversibility of airway obstruction in response to β2-agonists is highly variable among asthmatics, which is partially attributed to genetic factors. In a genome-wide association study of acute bronchodilator response (BDR) to inhaled albuterol, 534,290 single nucleotide polymorphisms (SNPs) were tested in 403 white trios from the Childhood Asthma Management Program using five statistical models to determine the most robust genetic associations. The primary replication phase included 1397 polymorphisms in three asthma trials (pooled n=764). The second replication phase tested 13 SNPs in three additional asthma populations (n=241, n=215, and n=592). An intergenic SNP on chromosome 10, rs11252394, proximal to several excellent biological candidates, significantly replicated (p=1.98×10−7) in the primary replication trials. An intronic SNP (rs6988229) in the collagen (COL22A1) locus also provided strong replication signals (p=8.51×10−6). This study applied a robust approach for testing the genetic basis of BDR and identified novel loci associated with this drug response in asthmatics.
pharmacogenetics; asthma; bronchodilator response; genome-wide association study; albuterol
Both asthma and obesity are complex disorders that are influenced by environmental and genetic factors. Shared genetic factors between asthma and obesity have been proposed to partly explain epidemiological findings of co-morbidity between these conditions.
To identify genetic variants that are associated with body mass index (BMI) in asthmatic children and adults, and to evaluate if there are differences between the genetics of BMI in asthmatics and healthy individuals.
In total, 19 studies contributed with genome-wide analysis study (GWAS) data from more than 23,000 individuals with predominantly European descent, of whom 8,165 are asthmatics.
We report associations between several DENND1B variants (p=2.2×10−7 for rs4915551) on chromosome 1q31 and BMI from a meta-analysis of GWAS data using 2,691 asthmatic children (screening data). The top DENND1B SNPs were next evaluated in seven independent replication data sets comprising 2,014 asthmatics, and rs4915551 was nominally replicated (p<0.05) in two of the seven studies and of borderline significance in one (p=0.059). However, strong evidence of effect heterogeneity was observed and overall, the association between rs4915551 and BMI was not significant in the total replication data set, p=0.71. Using a random effects model, BMI was overall estimated to increase by 0.30 kg/m2 (p=0.01 for combined screening and replication data sets, N=4,705) per additional G allele of this DENND1B SNP. FTO was confirmed as an important gene for adult and childhood BMI regardless of asthma status.
Conclusions and Clinical Relevance
DENND1B was recently identified as an asthma susceptibility gene in a GWAS on children, and here we find evidence that DENND1B variants may also be associated with BMI in asthmatic children. However, the association was overall not replicated in the independent data sets and the heterogeneous effect of DENND1B points to complex associations with the studied diseases that deserve further study.
Association; Asthma; BMI; Genetics; Genome-wide; Obesity
The relevance of allergic sensitization, judged by titers of serum IgE antibodies, to the risk of an asthma exacerbation caused by rhinovirus is unclear.
To examine the prevalence of rhinovirus infections in relation to the atopic status of children treated for wheezing in Costa Rica, a country with an increased asthma burden.
The children enrolled (n=287) were 7 through 12 years old. They included 96 with acute wheezing, 65 with stable asthma, and 126 non-asthmatic controls. PCR methods, including gene sequencing to identify rhinovirus strains, were used to identify viral pathogens in nasal washes. Results were examined in relation to wheezing, total IgE, allergen-specific IgE antibody, and levels of expired nitric oxide (FENO).
Sixty-four percent of wheezing children compared to 13% of children with stable asthma and 17% of the non-asthmatic controls tested positive for rhinovirus (p<0.001 for both comparisons). Among wheezing subjects, 75% of the rhinoviruses detected were Group C strains. High titers of IgE antibodies to dust mite allergen (especially Dermatophagoides sp) were common and correlated significantly with levels of total IgE and FENO. The greatest risk for wheezing was observed among children with titers of IgE antibodies to dust mite ≥17.5 IU/ml who tested positive for rhinovirus (odds ratio for wheezing: 31.5; 95% CI 8.3–108, p<0.001).
High titers of IgE antibody to dust mite allergen were common and significantly increased the risk for acute wheezing provoked by rhinovirus among asthmatic children.
acute asthma; dust mite specific IgE; emergency room visits; viral respiratory tract infections; rhinovirus strain C; total serum IgE; inhaled allergens; exhaled nitric oxide (FENO)
Vitamin D deficiency is becoming more apparent in many populations. Genetic factors may play a role in the maintenance of vitamin D levels. The objective of this study was to perform a genome-wide analysis (GWAS) of vitamin D levels, including replication of prior GWAS results. We measured 25-hydroxyvitamin D (25(OH)D) levels in serum collected at the time of enrollment and at year 4 in 572 Caucasian children with asthma, who were part of a multi-center clinical trial, the Childhood Asthma Management Program. Replication was performed in a second cohort of 592 asthmatics from Costa Rica and a third cohort of 516 Puerto Rican asthmatics. In addition, we attempted replication of three SNPs that were previously identified in a large GWAS of Caucasian individuals. The setting included data from a clinical trial of childhood asthmatics and two cohorts of asthmatics recruited for genetic studies of asthma. The main outcome measure was circulating 25(OH)D levels. The 25(OH)D levels at the two time-points were only modestly correlated with each other (intraclass correlation coefficient = 0.33) in the CAMP population. We identified SNPs that were nominally associated with 25(OH)D levels at two time-points in CAMP, and replicated four SNPs in the Costa Rican cohort: rs11002969, rs163221, rs1678849, and rs4864976. However, these SNPs were not significantly associated with 25(OH)D levels in a third population of Puerto Rican asthmatics. We were able to replicate the SNP with the strongest effect, previously reported in a large GWAS: rs2282679 (GC), and we were able to replicate another SNP, rs10741657 (CYP2R1), to a lesser degree. We were able to replicate two of three prior significant findings in a GWAS of 25(OH)D levels. Other SNPs may be additionally associated with 25(OH)D levels in certain populations.
It has recently been shown that vitamin D deficiency can increase asthma development and severity and that variations in vitamin D receptor genes are associated with asthma susceptibility.
We sought to find genetic factors that might interact with vitamin D levels to affect the risk of asthma exacerbation. Methods: We conducted a genome-wide study of gene–vitamin D interaction on asthma exacerbations using population-based and family-based approaches on 403 subjects and trios from the Childhood Asthma Management Program. Twenty-three polymorphisms with significant interactions were studied in a replication analysis in 584 children from a Costa Rican cohort. Results: We identified 3 common variants in the class I MHC–restricted T cell–associated molecule gene (CRTAM) that were associated with an increased rate of asthma exacerbations based on the presence of a low circulating vitamin D level. These results were replicated in a second independent population (unadjusted combined interaction, P =.00028–.00097; combined odds ratio, 3.28–5.38). One variant, rs2272094, is a nonsynonymous coding polymorphism of CRTAM. Functional studies on cell lines confirmed the interaction of vitamin D and rs2272094 on CRTAM expression. CRTAM is highly expressed in activated human CD8+ and natural killer T cells, both of which have been implicated in asthmatic patients.
The findings highlight an important gene-environment interaction that elucidates the role of vitamin D and CD8+ and natural killer T cells in asthma exacerbation in a genome-wide gene-environment interaction study that has been replicated in an independent population. The results suggest the potential importance of maintaining adequate vitamin D levels in subsets of high-risk asthmatic patients.
Gene-environment interaction; genome-wide association study; vitamin D; asthma exacerbation
Traditional genome-wide association studies (GWAS) of large cohort of subjects with chronic obstructive pulmonary disease (COPD) have successfully identified novel candidate genes, but several other plausible loci do not meet strict criteria for genome-wide significance after correction for multiple testing.
We hypothesize that by applying unbiased weights derived from unique populations we can identify additional COPD susceptibility loci.
We performed a homozygosity haplotype analysis on a group of subjects with and without COPD to identify regions of conserved homozygosity (RCHH). Weights were constructed based on the frequency of these RCHH in case vs. controls, and used to adjust the P values from a large collaborative GWAS of COPD.
We identified 2,318 regions of conserved homozygosity, of which 576 were significantly (P < .05) overrepresented in cases. After applying the weights constructed from these regions to a collaborative GWAS of COPD, we identified two single nucleotide polymorphisms in a novel gene (FGF7) that gained genome-wide significance by the false discovery rate method. In a follow-up analysis, both SNPs (rs12591300 and rs4480740) were significantly associated with COPD in an independent population (combined P values of 7.9E-07 and 2.8E-06 respectively). In another independent population, increased lung tissue FGF7 expression was associated with worse measures of lung function.
Weights constructed from a homozygosity haplotype analysis of an isolated population successfully identify novel genetic associations from a GWAS on a separate population. This method can be used to identify promising candidate genes that fail to meet strict correction for multiple testing.
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.
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.
Risk factors for allergic rhinitis (AR) in asthmatics are likely distinct from those for AR or asthma alone. We sought to identify clinical and environmental risk factors for AR in children with asthma.
We performed a cross-sectional study of 616 Costa Rican children aged 6–14 years with asthma. Candidate risk factors were drawn from questionnaire data, spirometry, methacholine challenge testing, skin testing, and serology. Two outcome measures, skin test reaction (STR)-positive AR and physician-diagnosed AR, were examined by logistic regression.
STR-positive AR had high prevalence (80%) in Costa Rican children with asthma, and its independent risk factors were nasal symptoms after exposure to dust or mold, parental history of AR, older age at asthma onset, oral steroid use in the past year, eosinophilia, and positive IgEs to dust mite and cockroach. Physician-diagnosed AR had lower prevalence (27%), and its independent risk factors were nasal symptoms after pollen exposure, STR to tree pollens, a parental history of AR, inhaled steroid and short-acting β2 agonist use in the past year, household mold/mildew, and fewer older siblings. A physician’s diagnosis was only 29.5% sensitive for STR-positive AR.
Risk factors for AR in children with asthma depend on the definition of AR. Indoor allergens drive risk for STR-positive AR. Outdoor allergens and home environmental conditions are risk factors for physician-diagnosed AR. We propose that children with asthma in Costa Rica and other Latin American nations undergo limited skin testing or specific IgE measurements to reduce the current under-diagnosis of AR.
allergic rhinitis; asthma; physician diagnosis; risk factor; skin test
Allergic rhinitis (AR) affects up to 80% of children with asthma and increases asthma severity. Thymic stromal lymphopoietin (TSLP) is a key mediator of allergic inflammation. The role of the TSLP gene (TSLP) in the pathogenesis of AR has not been studied.
To test for associations between variants in TSLP, TSLP-related genes, and AR in children with asthma.
We genotyped 15 single nucleotide polymorphisms (SNPs) in TSLP, OX40L, IL7R, and RXRα in three independent cohorts: 592 asthmatic Costa Rican children and their parents, 422 nuclear families of North American children with asthma, and 239 Swedish children with asthma. We tested for associations between these SNPs and AR. As we previously reported sex-specific effects for TSLP, we performed overall and sex-stratified analyses. We additionally performed secondary analyses for gene-by-gene interactions.
Across the three cohorts, the T allele of TSLP SNP rs1837253 was undertransmitted in boys with AR and asthma as compared to boys with asthma alone. The SNP was associated with reduced odds for AR (odds ratios ranging from 0.56 to 0.63, with corresponding Fisher's combined P value of 1.2 × 10-4). Our findings were significant after accounting for multiple comparisons. SNPs in OX40L, IL7R, and RXRα were not consistently associated with AR in children with asthma. There were nominally significant interactions between gene pairs.
TSLP SNP rs1837253 is associated with reduced odds for AR in boys with asthma. Our findings support a role for TSLP in the pathogenesis of AR in children with asthma.
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
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: Maternal vitamin D intake during pregnancy has been inversely associated with asthma symptoms in early childhood. However, no study has examined the relationship between measured vitamin D levels and markers of asthma severity in childhood.
Objectives: To determine the relationship between measured vitamin D levels and both markers of asthma severity and allergy in childhood.
Methods: We examined the relation between 25-hydroxyvitamin D levels (the major circulating form of vitamin D) and markers of allergy and asthma severity in a cross-sectional study of 616 Costa Rican children between the ages of 6 and 14 years. Linear, logistic, and negative binomial regressions were used for the univariate and multivariate analyses.
Measurements and Main Results: Of the 616 children with asthma, 175 (28%) had insufficient levels of vitamin D (<30 ng/ml). In multivariate linear regression models, vitamin D levels were significantly and inversely associated with total IgE and eosinophil count. In multivariate logistic regression models, a log10 unit increase in vitamin D levels was associated with reduced odds of any hospitalization in the previous year (odds ratio [OR], 0.05; 95% confidence interval [CI], 0.004–0.71; P = 0.03), any use of antiinflammatory medications in the previous year (OR, 0.18; 95% CI, 0.05–0.67; P = 0.01), and increased airway responsiveness (a ≤8.58-μmol provocative dose of methacholine producing a 20% fall in baseline FEV1 [OR, 0.15; 95% CI, 0.024–0.97; P = 0.05]).
Conclusions: Our results suggest that vitamin D insufficiency is relatively frequent in an equatorial population of children with asthma. In these children, lower vitamin D levels are associated with increased markers of allergy and asthma severity.
Rationale: Polymorphisms in the gene for transforming growth factor-β1 (TGFB1) have been associated with asthma, but not with airway responsiveness or disease exacerbations in subjects with asthma.
Objectives: To test for association between single nucleotide polymorphisms (SNPs) in TGFB1 and markers of asthma severity in childhood.
Methods: We tested for the association between nine SNPs in TGFB1 and indicators of asthma severity (lung function, airway responsiveness, and disease exacerbations) in two cohorts: 416 Costa Rican parent-child trios and 465 families of non-Hispanic white children in the Childhood Asthma Management Program (CAMP). We also tested for the interaction between these polymorphisms and exposure to dust mite allergen on asthma severity.
Measurements and Main Results: The A allele of promoter SNP rs2241712 was associated with increased airway responsiveness in Costa Rica (P = 0.0006) and CAMP (P = 0.005), and the C allele of an SNP in the promoter region (rs1800469) was associated with increased airway responsiveness in both cohorts (P ≤ 0.01). Dust mite exposure modified the effect of the C allele of exonic SNP rs1800471 on airway responsiveness (P = 0.03 for interactions in both cohorts). The T allele of a coding SNP (rs1982073) was associated with a reduced risk of asthma exacerbations in Costa Rica (P = 0.009) and CAMP (P = 0.005). Dust mite exposure also significantly modified the effect of the A allele of the promoter SNP rs2241712 on asthma exacerbations in both cohorts.
Conclusions: SNPs in TGFB1 are associated with airway responsiveness and disease exacerbations in children with asthma. Moreover, dust mite exposure may modify the effect of TGFB1 SNPs on airway responsiveness and asthma exacerbations.
airway responsiveness; asthma; dust mite allergen; single nucleotide polymorphisms; transforming growth factor-β1
Although asthma is highly prevalent among certain Hispanic subgroups, genetic determinants of asthma and asthma‐related traits have not been conclusively identified in Hispanic populations. A study was undertaken to identify genomic regions containing susceptibility loci for pulmonary function and bronchodilator responsiveness (BDR) in Costa Ricans.
Eight extended pedigrees were ascertained through schoolchildren with asthma in the Central Valley of Costa Rica. Short tandem repeat (STR) markers were genotyped throughout the genome at an average spacing of 8.2 cM. Multipoint variance component linkage analyses of forced expiratory volume in 1 second (FEV1) and FEV1/ forced vital capacity (FVC; both pre‐bronchodilator and post‐bronchodilator) and BDR were performed in these eight families (pre‐bronchodilator spirometry, n = 640; post‐bronchodilator spirometry and BDR, n = 624). Nine additional STR markers were genotyped on chromosome 7. Secondary analyses were repeated after stratification by cigarette smoking.
Among all subjects, the highest logarithm of the odds of linkage (LOD) score for FEV1 (post‐bronchodilator) was found on chromosome 7q34–35 (LOD = 2.45, including the additional markers). The highest LOD scores for FEV1/FVC (pre‐bronchodilator) and BDR were found on chromosomes 2q (LOD = 1.53) and 9p (LOD = 1.53), respectively. Among former and current smokers there was near‐significant evidence of linkage to FEV1/FVC (post‐bronchodilator) on chromosome 5p (LOD = 3.27) and suggestive evidence of linkage to FEV1 on chromosomes 3q (pre‐bronchodilator, LOD = 2.74) and 4q (post‐bronchodilator, LOD = 2.66).
In eight families of children with asthma in Costa Rica, there is suggestive evidence of linkage to FEV1 on chromosome 7q34–35. In these families, FEV1/FVC may be influenced by an interaction between cigarette smoking and a locus (loci) on chromosome 5p.
Rationale: The basis for gender influences on allergen-specific IgEs is unclear.
Objectives: To perform regular and sex-stratified genomewide linkage analyses of IgE to each of three allergens (Ascaris lumbricoides, Blatella germanica [German cockroach]), and Dermatophagoides pteronyssinus [dust mite]) and to conduct an association study of a candidate gene in a linked genomic region.
Methods: Genomewide linkage analyses of allergen-specific IgEs were conducted in 653 members of eight large families of Costa Rican children with asthma. An analysis of the association between single-nucleotide polymorphisms in thymic stromal lymphopoietin (TSLP) and IgE measurements was conducted in 417 parent–child trios in Costa Rica. Significant results were replicated in 470 families of white children in the Childhood Asthma Management Program (CAMP).
Measurements and Main Results: Among all subjects, there was suggestive evidence of linkage (LOD ⩾ 2.72) to IgE to Ascaris (on chromosome 7q) and IgE to dust mite (on chromosomes 7p and 12q). In a sex-stratified analysis, there was significant evidence of linkage to IgE to cockroach on chromosome 5q23 (peak LOD, 4.14 at 127 cM) in female subjects. TSLP is located within the 1.5 LOD-unit support interval for this linkage peak and has female-specific effects on lung disease in mice. In a sex-stratified analysis, the T allele of single-nucleotide polymorphism rs2289276 in TSLP was associated with reductions in IgE to cockroach (in Costa Rican girls) and total IgE (in girls in Costa Rica and in CAMP; P value for sex-by-genotype interaction, <0.01 in both studies).
Conclusions: Consistent with findings in murine models, a variant in TSLP may have female-specific effects on allergic phenotypes.
immunoglobulin E; linkage; thymic stromal lymphopoietin; single-nucleotide polymorphisms
Rationale: Replication of gene-disease associations has become a requirement in complex trait genetics.
Objectives: In studies of childhood asthma from two different ethnic groups, we attempted to replicate associations with five potential asthma susceptibility genes previously identified by positional cloning.
Methods: We analyzed two family-based samples ascertained through an asthmatic proband: 497 European-American children from the Childhood Asthma Management Program and 439 Hispanic children from the Central Valley of Costa Rica. We genotyped 98 linkage disequilibrium–tagging single-nucleotide polymorphisms (SNPs) in five genes: ADAM33, DPP10, GPR154 (HUGO name: NPSR1), HLA-G, and the PHF11 locus (includes genes SETDB2 and RCBTB1). SNPs were tested for association with asthma and two intermediate phenotypes: airway hyperresponsiveness and total serum immunoglobulin E levels.
Measurements and Main Results: Despite differing ancestries, linkage disequilibrium patterns were similar in both cohorts. Of the five evaluated genes, SNP-level replication was found only for GPR154 (NPSR1). In this gene, three SNPs were associated with asthma in both cohorts, although the opposite alleles were associated in either study. Weak evidence for locus-level replication with asthma was found in the PHF11 locus, although there was no overlap in the associated SNP across the two cohorts. No consistent associations were observed for the three other genes.
Conclusions: These results provide some further support for the role of genetic variation in GPR154 (NPSR1) and PHF11 in asthma susceptibility and also highlight the challenges of replicating genetic associations in complex traits such as asthma, even for genes identified by linkage analysis.
bronchial hyperreactivity; immunoglobulin E; linkage disequilibrium; NPSR1; single-nucleotide polymorphism
IL12A has been implicated in T-cell development and may thus influence the development of atopy and allergic diseases.
We tested for association between four linkage disequilibrium (LD)-tagging SNPs (rs2243123, rs2243151, rs668998, and rs17826053) in IL12A and asthma and allergy-related (serum total and allergen-specific IgE, and skin test reactivity [STR] to two common allergens) phenotypes in two samples: 417 Costa Rican children with asthma and their parents, and 470 families of 503 white children in the Childhood Asthma Management Program (CAMP). The analysis was conducted using the family-based association test (FBAT) statistic implemented in the PBAT program.
Among Costa Rican children with asthma, homozygosity for the minor allele of each of two SNPs in IL12A (rs2243123 and rs2243151) was associated with increased risks of STR to American cockroach (P ≤ 0.03 for both SNPs), STR to German cockroach (P ≤ 0.01 for both SNPs), and having a positive IgE to German cockroach (P < 0.05 for both SNPs). Among children in CAMP, homozygosity for the minor allele of SNP rs2243151 in IL12A was inversely associated with STR to German cockroach (P = 0.03) and homozygosity for the minor allele of SNP rs17826053 in IL12A was associated with increased risks of STR to American cockroach (P = 0.01) and STR to German cockroach (P = 0.007). There was no significant association between any SNP in IL12A and asthma, STR to dust mite, or total IgE in Costa Rica or CAMP.
Our findings suggest that variants in IL12A influence cockroach allergy among children with asthma.
A SNP upstream of the INSIG2 gene, rs7566605, was recently found to be associated with obesity as measured by body mass index (BMI) by Herbert and colleagues. The association between increased BMI and homozygosity for the minor allele was first observed in data from a genome-wide association scan of 86,604 SNPs in 923 related individuals from the Framingham Heart Study offspring cohort. The association was reproduced in four additional cohorts, but was not seen in a fifth cohort. To further assess the general reproducibility of this association, we genotyped rs7566605 in nine large cohorts from eight populations across multiple ethnicities (total n = 16,969). We tested this variant for association with BMI in each sample under a recessive model using family-based, population-based, and case-control designs. We observed a significant (p < 0.05) association in five cohorts but saw no association in three other cohorts. There was variability in the strength of association evidence across examination cycles in longitudinal data from unrelated individuals in the Framingham Heart Study Offspring cohort. A combined analysis revealed significant independent validation of this association in both unrelated (p = 0.046) and family-based (p = 0.004) samples. The estimated risk conferred by this allele is small, and could easily be masked by small sample size, population stratification, or other confounders. These validation studies suggest that the original association is less likely to be spurious, but the failure to observe an association in every data set suggests that the effect of SNP rs7566605 on BMI may be heterogeneous across population samples.
Obesity is an epidemic in the United States of America and developing world, portending an epidemic of related diseases such as diabetes and heart disease. While diet and lifestyle contribute to obesity, half of the population variation in body mass index, a common measure of obesity, is determined by inherited factors. Many studies have reported that common sequence variants in genes are associated with an increased risk for obesity, yet most of these are not reproducible in other study cohorts, suggesting that some are false. Recently, Herbert et al. reported a slightly increased risk of obesity for people carrying two copies of the minor allele at a common variant near INSIG2. We present our attempts to further evaluate this potential association with obesity in additional populations. We find evidence of increased risk of obesity for people carrying two copies of the minor allele in five out of nine cohorts tested, using both family- and population-based testing. We indicate possible reasons for the varied results, with the hope of encouraging a combined analysis across study cohorts to more precisely define the effect of this INSIG2 gene variant.