Multiancestry association study identifies new asthma risk loci that colocalize with immune-cell enhancer marks.

We examined common variation in asthma risk by conducting a meta-analysis of worldwide asthma genome-wide association studies (23,948 asthma cases, 118,538 controls) of individuals from ethnically diverse populations. We identified five new asthma loci, found two new associations at two known asthma loci, established asthma associations at two loci previously implicated in the comorbidity of asthma plus hay fever, and confirmed nine known loci. Investigation of pleiotropy showed large overlaps in genetic variants with autoimmune and inflammatory diseases. The enrichment in enhancer marks at asthma risk loci, especially in immune cells, suggested a major role of these loci in the regulation of immunologically related mechanisms.

Ethnic-specific associations of rare and low-frequency DNA sequence variants with asthma.

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.

Integrated genome-wide association, coexpression network, and expression single nucleotide polymorphism analysis identifies novel pathway in allergic rhinitis.

BACKGROUND: Allergic rhinitis is a common disease whose genetic basis is incompletely explained. We report an integrated genomic analysis of allergic rhinitis. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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 interaction studies reveal sex-specific asthma risk alleles.

Asthma is a complex disease with sex-specific differences in prevalence. Candidate gene studies have suggested that genotype-by-sex interaction effects on asthma risk exist, but this has not yet been explored at a genome-wide level. We aimed to identify sex-specific asthma risk alleles by performing a genome-wide scan for genotype-by-sex interactions in the ethnically diverse participants in the EVE Asthma Genetics Consortium. We performed male- and female-specific genome-wide association studies in 2653 male asthma cases, 2566 female asthma cases and 3830 non-asthma controls from European American, African American, African Caribbean and Latino populations. Association tests were conducted in each study sample, and the results were combined in ancestry-specific and cross-ancestry meta-analyses. Six sex-specific asthma risk loci had P-values < 1 × 10(-6), of which two were male specific and four were female specific; all were ancestry specific. The most significant sex-specific association in European Americans was at the interferon regulatory factor 1 (IRF1) locus on 5q31.1. We also identify a Latino female-specific association in RAP1GAP2. Both of these loci included single-nucleotide polymorphisms that are known expression quantitative trait loci and have been associated with asthma in independent studies. The IRF1 locus is a strong candidate region for male-specific asthma susceptibility due to the association and validation we demonstrate here, the known role of IRF1 in asthma-relevant immune pathways and prior reports of sex-specific differences in interferon responses.

Maternal microchimerism protects against the development of asthma.

BACKGROUND: Maternal asthma and child's sex are among the most significant and reproducible risk factors for the development of asthma. Although the mechanisms for these effects are unknown, they likely involve nonclassical genetic mechanisms. One such mechanism could involve the transfer and persistence of maternal cells to her offspring, a common occurrence known as maternal microchimerism (MMc). MMc has been associated with many autoimmune diseases but has not been investigated for a role in asthma or allergic disease. OBJECTIVE: We hypothesized that some of the observed risks for asthma may be due to different rates of transmission or persistence of maternal cells to children of mothers with asthma compared with children of mothers without asthma, or to sons compared with daughters. We further hypothesized that rates of MMc differ between children with and without asthma. METHODS: We tested these hypotheses in 317 subjects from 3 independent cohorts by using a real-time quantitative PCR assay to detect a noninherited HLA allele in the child. RESULTS: MMc was detected in 20.5% of the subjects (range 16.8%-27.1% in the 3 cohorts). We observed lower rates of asthma among MMc-positive subjects than among MMc-negative subjects (odds ratio, 0.38; 95% CI, 0.19-0.79; P = .029). Neither maternal asthma nor sex of the child was a significant predictor of MMc in the child (P = .81 and .15, respectively). CONCLUSIONS: Our results suggest for the first time that MMc may protect against the development of asthma.

Further replication studies of the EVE Consortium meta-analysis identifies 2 asthma risk loci in European Americans.

BACKGROUND: Genome-wide association studies of asthma have implicated many genetic risk factors, with well-replicated associations at approximately 10 loci that account for only a small proportion of the genetic risk. OBJECTIVES: We aimed to identify additional asthma risk loci by performing an extensive replication study of the results from the EVE Consortium meta-analysis. METHODS: We selected 3186 single nucleotide polymorphisms for replication based on the P values from the EVE Consortium meta-analysis. These single nucleotide polymorphisms were genotyped in ethnically diverse replication samples from 9 different studies, totaling 7202 cases, 6426 controls, and 507 case-parent trios. Association analyses were conducted within each participating study, and the resulting test statistics were combined in a meta-analysis. RESULTS: Two novel associations were replicated in European Americans: rs1061477 in the KLK3 gene on chromosome 19 (combined odds ratio = 1.18; 95% CI, 1.10-1.25) and rs9570077 (combined odds ratio =1.20; 95% CI, 1.12-1.29) on chromosome 13q21. We could not replicate any additional associations in the African Americans or Latinos. CONCLUSIONS: This extended replication study identified 2 additional asthma risk loci in populations of European descent. The absence of additional loci for African Americans and Latinos highlights the difficulty in replicating associations in admixed populations.

Genetic variation in vascular endothelial growth factor-a and lung function.

RATIONALE: Given the role of vascular endothelial growth factor (VEGF) in lung development, we hypothesized that polymorphisms in VEGF-A may be associated with lung function. OBJECTIVES: The current study was designed to assess the role of genetic variants in VEGF-A as determinants of airway function from infancy through early adulthood. METHODS: Association between five single-nucleotide polymorphisms (SNPs) in VEGF-A and lung function were assessed longitudinally in two unselected birth cohorts and cross-sectionally among infants. Replication with two SNPs was conducted in adults and children with asthma. We investigated the functionality of the SNP most consistently associated with lung function (rs3025028) using Western blotting to measure the ratio of plasma VEGF-A(165b)/panVEGF-A(165) among homozygotes. MEASUREMENTS AND MAIN RESULTS: In two populations in infancy, C-allele homozygotes of rs3025028 had significantly higher VmaxFRC, forced expiratory flow(50), and forced expiratory flow(25-75) compared with other genotype groups. Among preschool children (age 3 yr), C allele of rs3025028 was associated with significantly higher specific airway conductance, with similar findings observed for lung function in school-age children. For FEV(1)/FVC ratio similar findings were observed among adolescents and young adults (birth cohort), and then replicated in adults and schoolchildren with asthma (cross-sectional studies). For rs3025038, plasma VEGF-A(165b)/panVEGF-A(165) was significantly higher among CC versus GG homozygotes (P ≤ 0.02) at birth, in school-age children, and in adults. CONCLUSIONS: We report significant associations between VEGF-A SNP rs3025028 and parameters of airway function measured throughout childhood, with the effect persisting into adulthood. We propose that the mechanism may be mediated through the ratios of active and inhibitory isoforms of VEGF-A(165), which may be determined by alternative splicing.

Resequencing candidate genes implicates rare variants in asthma susceptibility.

Common variation in over 100 genes has been implicated in the risk of developing asthma, but the contribution of rare variants to asthma susceptibility remains largely unexplored. We selected nine genes that showed the strongest signatures of weak purifying selection from among 53 candidate asthma-associated genes, and we sequenced the coding exons and flanking noncoding regions in 450 asthmatic cases and 515 nonasthmatic controls. We observed an overall excess of p values <0.05 (p = 0.02), and rare variants in four genes (AGT, DPP10, IKBKAP, and IL12RB1) contributed to asthma susceptibility among African Americans. Rare variants in IL12RB1 were also associated with asthma susceptibility among European Americans, despite the fact that the majority of rare variants in IL12RB1 were specific to either one of the populations. The combined evidence of association with rare noncoding variants in IL12RB1 remained significant (p = 3.7 × 10(-4)) after correcting for multiple testing. Overall, the contribution of rare variants to asthma susceptibility was predominantly due to noncoding variants in sequences flanking the exons, although nonsynonymous rare variants in DPP10 and in IL12RB1 were associated with asthma in African Americans and European Americans, respectively. This study provides evidence that rare variants contribute to asthma susceptibility. Additional studies are required for testing whether prioritizing genes for resequencing on the basis of signatures of purifying selection is an efficient means of identifying novel rare variants that contribute to complex disease.

Meta-analysis of genome-wide association studies of asthma in ethnically diverse North American populations.

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 of asthma in 5,416 individuals with asthma (cases) including individuals of European American, African American or African Caribbean, and Latino ancestry, with replication in an additional 12,649 individuals from the same ethnic groups. We identified five susceptibility loci. Four were at previously reported loci on 17q21, near IL1RL1, TSLP and IL33, but we report for the first time, to our knowledge, that these loci are associated with asthma risk in three ethnic groups. In addition, we identified a new asthma susceptibility locus at PYHIN1, with the association being 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.

A SOCS-1 promoter variant is associated with total serum IgE levels.

SOCS-1 is a critical regulator of multiple signaling pathways, including those activated by cytokines that regulate Ig H chain class switching to IgE. Analysis of mice with mutations in the SOCS-1 gene demonstrated that IgE levels increase with loss of SOCS-1 alleles. This suggested that overall SOCS-1 acts as an inhibitor of IgE expression in vivo. A genetic association study was performed in 474 children enrolled in the Tucson Children's Respiratory Study to determine if genetic variation in the SOCS-1 locus correlates with altered levels of IgE. Carriers of the C-allele for a novel, 3' genomic single nucleotide polymorphism (SNP) in the SOCS-1 gene (SOCS1+1125G > C; rs33932899) were found to have significantly lower levels of serum IgE compared with those of homozygotes for the G-allele. Analysis demonstrated that the SOCS1+1125G > C SNP was in complete linkage disequilibrium with an SNP at position SOCS1-820G > T (rs33977706) of the SOCS-1 promoter. Carriers of the T-allele at the SOCS1-820G > T were also found to be associated with the decreased IgE. The promoter SNP increased transcriptional activity of the SOCS-1 promoter in reporter assays and human B cells. Consistent with this observation, the presence of this polymorphism within the promoter abolished binding of yin yang-1, which is identified as a negative regulator of SOCS-1 transcriptional activity. These data suggest that genetic variation in the SOCS-1 promoter may affect IgE production.

Identification of PCDH1 as a novel susceptibility gene for bronchial hyperresponsiveness.

RATIONALE: Asthma is a chronic inflammatory airway disease that affects more than 300 million individuals worldwide. Asthma is caused by interaction of genetic and environmental factors. Bronchial hyperresponsiveness (BHR) is a hallmark of asthma and results from increased sensitivity of the airways to physical or chemical stimulants. BHR and asthma are linked to chromosome 5q31-q33. OBJECTIVES: To identify a gene for BHR on chromosome 5q31-q33. METHODS: In 200 Dutch families with asthma, linkage analysis and fine mapping were performed, and the Protocadherin 1 gene (PCDH1) was identified. PCDH1 was resequenced in 96 subjects from ethnically diverse populations to identify novel sequence variants. Subsequent replication studies were undertaken in seven populations from The Netherlands, the United Kingdom, and the United States, including two general population samples, two family samples, and three case-control samples. PCDH1 mRNA and protein expression was investigated using polymerase chain reaction, Western blotting, and immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: In seven out of eight populations (n = 6,168) from The Netherlands, United Kingdom, and United States, PCHD1 gene variants were significantly associated with BHR (P values, 0.005-0.05) This association was present in both families with asthma and general populations. PCDH1 mRNA and protein were expressed in airway epithelial cells and in macrophages. CONCLUSIONS: PCDH1 is a novel gene for BHR in adults and children. The identification of PCDH1 as a BHR susceptibility gene may suggest that a structural defect in the integrity of the airway epithelium, the first line of defense against inhaled substances, contributes to the development of BHR.

Th2 cell-selective enhancement of human IL13 transcription by IL13-1112C>T, a polymorphism associated with allergic inflammation.

IL-13 is a central mediator of allergic inflammation. The single nucleotide polymorphism IL13-1112C>T (rs1800925) is associated with allergic phenotypes in ethnically distinct populations, but the underlying mechanism(s) remain unknown. Using in vivo, in vitro, and in silico analysis, we show that the IL13-1112T allele enhanced IL13 promoter activity in primary human and murine CD4(+) Th2 lymphocytes. Increased expression of IL13-1112T in Th2 cells was associated with the creation of a Yin-Yang 1 binding site that overlapped a STAT motif involved in negative regulation of IL13 expression and attenuated STAT6-mediated transcriptional repression. Because IL-13 secretion was increased in IL13-1112TT homozygotes, we propose that increased expression of IL13-1112T in vivo may underlie its association with susceptibility to allergic inflammation. Interestingly, IL13-1112T had opposite transcriptional effects in nonpolarized CD4(+) T cells, paralleled by distinct patterns of DNA-protein interactions at the IL13 promoter. Our findings suggest the nuclear milieu dictates the functional outcome of genetic variation.

Association of atopy and eczema with polymorphisms in T-cell immunoglobulin domain and mucin domain-IL-2-inducible T-cell kinase gene cluster in chromosome 5 q 33.

BACKGROUND: The T-cell immunoglobulin domain and mucin domain (TIM) gene family and the gene for IL-2-inducible T-cell kinase (ITK), located in chromosome 5 q 33 and potentially involved in the T-cell proliferation and differentiation, are good candidate genes for allergic diseases. OBJECTIVE: We assessed the role of polymorphisms in the TIM family genes and ITK in atopy, eczema, and asthma. METHODS: Twenty-one polymorphisms in the TIM-ITK gene cluster were genotyped in 564 children enrolled in the Tucson Children's Respiratory Study. Skin prick tests to common allergens were performed at age 6.1 years (n=508), age 10.8 years (n=539), and age 16.6 years (n=424). Asthma and eczema were assessed by questionnaire at these 3 points. Averaged relative risks were estimated. RESULTS: One 15-bp insertion/deletion in exon 4 of TIM 1 was significantly related to atopy and eczema (relative risk associated with carrying at least 1 rare allele=1.24 [1.07--1.45], P=.005; and 1.43 [1.01--2.01], P=.004, respectively). The 3 tested single nucleotide polymorphisms (SNPs) in TIM 3 were significantly related to atopy and eczema. One of them, at position +4259 calculated from the translation start site, predicts a putative change in the amino acid sequence of the protein, and was the most strongly related to atopy (relative risk=1.28 [1.12--1.47]; P=.0003). SNPs in the 5' genomic region in ITK, which show moderate linkage disequilibrium with those in TIM 3, had an independent effect on atopy. None of the polymorphisms studied was related to asthma. CONCLUSION: Our findings support a potential role for SNPs in TIM 1, TIM 3, and ITK, independent of each other, in allergic diseases.

Relation of beta2-adrenoceptor polymorphisms at codons 16 and 27 to persistence of asthma symptoms after the onset of puberty.

BACKGROUND: It has long been recognized that many children with asthma outgrow the disease after the onset of puberty, but little is known about genetic factors influencing this outcome. OBJECTIVES: The aim of the present study was to determine whether the polymorphisms at codons 16 and 27 of the beta2-adrenoceptor are significant predictors of the persistence of asthma during adolescence. DESIGN AND PARTICIPANTS: We used data from the prospective Tucson Children's Respiratory Study. Children were genotyped for the polymorphisms at codons 16 and 27. The presence of wheezing/asthma was assessed by questionnaire from age 6 years up to the reported onset of puberty (prepubertal period) and after the onset of puberty up to age 16 years (adolescence). RESULTS: Among children who wheezed in the prepubertal period (n = 168), subjects homozygous for Gly at codon 16 were at significantly increased risk for persistent wheezing after puberty, as compared with carriers of the other genotypes (relative risk [RR], 1.43; 95% confidence interval [CI], 1.06 to 1.92; p = 0.019). This relation was present among boys (RR, 2.17; 95% CI, 1.41 to 3.36) but not girls (RR, 0.85; 95% CI, 0.55 to 1.30), and increased linearly according to the frequency of wheezing episodes after the onset of puberty. These findings persisted after adjusting for ethnicity and other potential confounders and after selecting only white children. The polymorphism at codon 27 showed no relation with risk for persistent wheezing. CONCLUSIONS: This study provides evidence for a strong gender-specific effect of the Gly16 polymorphism on the persistence of asthma after the onset of puberty.