Vanin-1 expression and methylation discriminate pediatric asthma corticosteroid treatment response.

BACKGROUND: There is considerable heterogeneity in asthma treatment response. OBJECTIVE: We sought to identify biomarkers of corticosteroid treatment response in children with asthma and evaluate the utility and mechanistic basis of these biomarkers. METHODS: Children (5-18 years) presenting to the emergency department with an acute asthma exacerbation were recruited and followed during hospitalization. Nasal epithelial cells were collected on presentation to the emergency department (T0) and 18 to 24 hours later (T1), and T1/T0 gene expression ratios were analyzed to identify genes associated with good and poor corticosteroid treatment response phenotypes. The utility of these genes in discriminating between systemic corticosteroid treatment response groups was then tested prospectively in a new cohort of patients. A gene candidate (vanin-1 [VNN1]) that consistently distinguished good versus poor response phenotypes was further studied in an experimental asthma model, and VNN1 promoter methylation was measured by means of bisulfite pyrosequencing in patients. RESULTS: VNN1 mRNA expression changes were associated with systemic corticosteroid treatment response in children with acute asthma, and VNN1 was required for optimal response to corticosteroid treatment in an experimental asthma model. A CpG site within the VNN1 promoter was differentially methylated between good versus poor treatment response groups, and methylation at this site correlated with VNN1 mRNA expression. CONCLUSIONS: We have identified a biological basis for poor corticosteroid treatment response that can be used to distinguish a subgroup of asthmatic children who respond poorly to systemic corticosteroid treatment. VNN1 contributes to corticosteroid responsiveness, and changes in VNN1 nasal epithelial mRNA expression and VNN1 promoter methylation might be clinically useful biomarkers of treatment response in asthmatic children.

Epistasis between serine protease inhibitor Kazal-type 5 (SPINK5) and thymic stromal lymphopoietin (TSLP) genes contributes to childhood asthma.

BACKGROUND: Epithelial genes have previously been associated with asthma but only explain a small fraction of heritability. In part, this might be due to epistasis, which is often not considered. OBJECTIVE: We sought to determine independent and epistatic associations between filaggrin (FLG), serine protease inhibitor Kazal-type 5 (SPINK5), and thymic stromal lymphopoietin (TSLP) gene variants and childhood asthma. METHODS: Using a candidate gene approach, we genotyped 29 variants in FLG, SPINK5, and TSLP in asthmatic, allergic, and nonallergic nonasthmatic white and black children participating in the well-phenotyped Greater Cincinnati Pediatric Clinic Repository. Associations with asthma were also assessed in 6 replication populations. RESULTS: We observed independent associations of variants in SPINK5 (P = .003) and TSLP (P = .006) with childhood asthma; a SPINK5 single nucleotide polymorphism was replicated. In subjects with 1 or more SPINK5 risk alleles, the absence of the TSLP protective minor alleles was associated with a significant increase in asthma (67% vs 53%, P = .0017). In contrast, the presence or absence of TSLP minor alleles did not affect asthma risk in subjects without the SPINK5 risk alleles. The SPINK5 and TSLP epistasis was replicated in a black population (P = .036) who did not display independent association with variants in these genes. CONCLUSIONS: Our results support epistasis between SPINK5 and TSLP, which contributes to childhood asthma. These findings emphasize the importance of using biology to inform analyses to identify genetic susceptibility to complex diseases. The results from our study have clinical relevance and support that the therapeutic effects of anti-TSLP therapy in asthmatic patients might be dependent on SPINK5 genotype.

IL-13 receptor α2 contributes to development of experimental allergic asthma.

BACKGROUND: IL-13 receptor α2 (IL-13Rα2) binds IL-13 with high affinity and modulates IL-13 responses. There are soluble and membrane forms of IL-13Rα2 generated by alternative splicing in mice, but human subjects express only the membrane form of IL-13Rα2 (memIL-13Rα2). OBJECTIVE: We determined the role of memIL-13Rα2 in the development of allergic inflammation in mouse models of asthma. METHODS: IL-13Rα2-deficient and memIL-13Rα2 lung epithelium-specific transgenic mice were challenged with house dust mite (HDM). Airway hyperresponsiveness (AHR) and inflammation were assessed based on the airway pressure-time index, bronchoalveolar lavage (BAL) cell counts, and lung histology. Mucus production was determined by means of periodic acid-Schiff staining of lung sections, Western blot analysis of chloride channel calcium activated 3 (CLCA3) expression in lung homogenates, and ELISA of Muc5ac in BAL fluid. The expression of cytokines and chemokines was determined by using RT-quantitative PCR. RESULTS: In IL-13Rα2-deficient mice AHR and airway inflammation were attenuated compared with levels seen in wild-type mice after HDM challenge. Lung epithelial overexpression of memIL-13Rα2 in the IL-13Rα2-deficient mice reconstituted AHR and inflammation to levels similar to those observed in HDM-challenged wild-type mice. Mucus production was attenuated in lungs from HDM-treated IL-13Rα2-deficient mice, whereas lung epithelial overexpression of memIL-13Rα2 increased mucus production. Lung epithelial overexpression of memIL-13Rα2 had no effect on levels of the soluble form of IL-13Rα2 in serum or BAL fluid and did not affect IL-13-dependent signal transducer and activator of transcription 6 activation in the lungs. CONCLUSION: These data collectively support a distinct role for memIL-13Rα2 in the lung and suggest that memIL-13Rα2 might contribute to allergic inflammation.

Genetic variation in small proline rich protein 2B as a predictor for asthma among children with eczema.

BACKGROUND: Small proline rich protein 2B (SPRR2B) is a skin and lung epithelial protein associated with allergic inflammation in mice that has not been evaluated in human atopic diseases. OBJECTIVE: To determine whether single-nucleotide polymorphisms (SNPs) in SPRR2B are associated with childhood eczema and with the phenotype of childhood eczema combined with asthma. METHODS: Genotyping for SPRR2B and filaggrin (FLG) was performed in 2 independent populations: the Cincinnati Childhood Allergy & Air Pollution Study (CCAAPS; N = 762; birth-age, 4 years) and the Greater Cincinnati Pediatric Clinical Repository (GCPCR; N = 1152; ages 5-10 years). Eczema and eczema plus asthma were clinical outcomes based on parental report and clinician's diagnosis. Genetic analyses were restricted to whites and adjusted for sex in both cohorts and adjusted for environmental covariates in CCAAPS. RESULTS: Variants in SPRR2B were not significantly associated with eczema in either cohort after Bonferroni adjustment. Children from both cohorts with the CC genotype of the SPRR2B rs6693927 SNP were at 4 times the risk for eczema plus asthma (adjusted odds ratio, 4.1; 95% confidence interval, 1.5-10.9; P = .005 in CCAAPS; and adjusted odds ratio, 4.0; 95% confidence interval, 1.8-9.1; P < .001 in the GCPCR), however. SNPs in SPRR2B were not in strong linkage disequilibrium with the R501X and del2282 FLG mutations, and these findings were independent of FLG. CONCLUSIONS: An SNP in SPRR2B was predictive of asthma among white children with eczema from 2 independent populations. SPRR2B polymorphisms may serve as important predictive markers for the combined eczema plus asthma phenotype.

Downregulation of glutathione S-transferase pi in asthma contributes to enhanced oxidative stress.

BACKGROUND: Glutathione S-transferase pi (GSTPi) is the predominant redox regulator in the lung. Although evidence implicates an important role for GSTPi in asthma, the mechanism for this has remained elusive. OBJECTIVES: We sought to determine how GSTPi is regulated in asthma and to elucidate its role in maintaining redox homeostasis. METHODS: We elucidated the regulation of GSTPi in children with asthma and used murine models of asthma to determine the role of GSTPi in redox homeostasis. RESULTS: Our findings demonstrate that GSTPi transcript levels are markedly downregulated in allergen- and IL-13-treated murine models of asthma through signal transducer and activator of transcription 6-dependent and independent pathways. Nuclear factor erythroid 2-related factor 2 was also downregulated in these models. The decrease in GSTPi expression was associated with decreased total glutathione S-transferase activity in the lungs of mice. Examination of cystine intermediates uncovered a functional role for GSTPi in regulating cysteine oxidation, whereby GSTPi-deficient mice exhibited increased oxidative stress (increase in percentage cystine) compared with wild-type mice after allergen challenge. GSTPi expression was similarly downregulated in children with asthma. CONCLUSIONS: These data collectively suggest that downregulation of GSTPi after allergen challenge might contribute to the asthma phenotype because of disruption of redox homeostasis and increased oxidative stress. Furthermore, GSTPi might be an important therapeutic target for asthma, and evaluation of GSTPi expression might prove beneficial in identifying patients who would benefit from therapy targeting this pathway.

A nonredundant role for mouse Serpinb3a in the induction of mucus production in asthma.

BACKGROUND: Asthma is a major public health burden worldwide. Studies from our group and others have demonstrated that SERPINB3 and SERPINB4 are induced in patients with asthma; however, their mechanistic role in asthma has yet to be determined. OBJECTIVE: To evaluate the role of Serpin3a, the murine homolog of SERPINB3 and SERPINB4, in asthma. METHODS: We studied wild-type Balb/c and Serpinb3a-null mice in house dust mite or IL-13-induced asthma models and evaluated airway hyperresponsiveness, inflammation, and goblet cell hyperplasia. RESULTS: Airway hyperresponsiveness and goblet cell hyperplasia were markedly attenuated in the Serpinb3a-null mice compared with the wild-type mice after allergen challenge, with minimal effects on inflammation. Expression of sterile alpha motif pointed domain containing v-ets avian erythroblastosis virus E26 oncogene homolog transcription factor (SPDEF), a transcription factor that mediates goblet cell hyperplasia, was decreased in the absence of Serpinb3a. IL-13-treated Serpinb3a-null mice showed attenuated airway hyperresponsiveness, inflammation, and mucus production. CONCLUSION: Excessive mucus production and mucus plugging are key pathologic features of asthma, yet the mechanisms responsible for mucus production are not well understood. Our data reveal a novel nonredundant role for Serpinb3a in mediating mucus production through regulation of SPDEF expression. This pathway may be used to target mucus hypersecretion effectively.

Intrinsically defective skin barrier function in children with atopic dermatitis correlates with disease severity.

BACKGROUND: Recent genetic evidence supports that an underlying defect in skin barrier function contributes to the pathogenesis of atopic dermatitis (AD). The integrity of the skin barrier can be assessed objectively by measuring transepidermal water loss (TEWL). Previous investigations of TEWL as a biomarker of skin barrier function have been limited by small sample size, and studies including African American subjects are lacking. OBJECTIVE: We sought to determine whether children with AD have inherently altered skin barrier function by comparing TEWL as a measure of skin barrier function in African American and white children with AD with that in control subjects without AD. METHODS: TEWL was measured on nonlesional normal-appearing skin at 4 sites (the volar forearm, dorsal arm, lower leg, and cheek) in (1) children with AD (cases), (2) children with asthma or allergic rhinitis but without AD (allergic control subjects), and (3) nonatopic control subjects. AD severity was assessed by using the objective SCORAD index. RESULTS: TEWL was increased in children with AD compared with that seen in both control groups at most of the anatomic sites tested (P < .05). TEWL also correlated with objective SCORAD score. The presence of allergic sensitization or other allergic conditions did not affect TEWL among children with AD. TEWL was higher in white than in African American children. CONCLUSION: Skin barrier function as assessed by TEWL is intrinsically compromised in children with AD but not in children with other allergic conditions. The magnitude of skin barrier dysfunction correlates with AD disease severity.

SERPINB3/B4 contributes to early inflammation and barrier dysfunction in an experimental murine model of atopic dermatitis.

Serine proteases are critical for epidermal barrier homeostasis, and their aberrant expression and/or activity is associated with chronic skin diseases. Elevated levels of the serine protease inhibitors SERPINB3 and SERPINB4 are seen in patients with atopic dermatitis and psoriasis. However, their mechanistic role in the skin is unknown. To evaluate the contribution of Serpinb3a (mouse homolog of SERPINB3 and SERPINB4) in atopic dermatitis, we examined the effect of topical Aspergillus fumigatus extract exposure in wild-type and Serpinb3a-null mice on transepidermal water loss (TEWL), sensitization, and inflammation. Allergen exposure induced Serpinb3a expression in the skin, along with increased TEWL, epidermal thickness, and skin inflammation, all of which were attenuated in the absence of Serpinb3a. Attenuated TEWL correlated with decreased expression of the pro-inflammatory marker S100A8. Silencing of SERPINB3/B4 in human keratinocytes decreased S100A8 expression, supporting a role for SERPINB3/B4 in the initiation of the acute inflammatory response. RNA-seq analysis following allergen exposure identified a network of pro-inflammatory genes induced in wild-type mice that was absent in Serpinb3a-null mice. In conclusion, Serpinb3a deficiency attenuates barrier dysfunction and the early inflammatory response following cutaneous allergen exposure, supporting a role for Serpinb3a (mice) and SERPINB3/B4 (humans) early in atopic dermatitis.

Functional variant in the autophagy-related 5 gene promotor is associated with childhood asthma.

RATIONALE AND OBJECTIVE: Autophagy is a cellular process directed at eliminating or recycling cellular proteins. Recently, the autophagy pathway has been implicated in immune dysfunction, the pathogenesis of inflammatory disorders, and response to viral infection. Associations between two genes in the autophagy pathway, ATG5 and ATG7, with childhood asthma were investigated. METHODS: Using genetic and experimental approaches, we examined the association of 13 HapMap-derived tagging SNPs in ATG5 and ATG7 with childhood asthma in 312 asthmatic and 246 non-allergic control children. We confirmed our findings by using independent cohorts and imputation analysis. Finally, we evaluated the functional relevance of a disease associated SNP. MEASUREMENTS AND MAIN RESULTS: We demonstrated that ATG5 single nucleotide polymorphisms rs12201458 and rs510432 were associated with asthma (p = 0.00085 and 0.0025, respectively). In three independent cohorts, additional variants in ATG5 in the same LD block were associated with asthma (p<0.05). We found that rs510432 was functionally relevant and conferred significantly increased promotor activity. Furthermore, Atg5 expression was increased in nasal epithelium of acute asthmatics compared to stable asthmatics and non-asthmatic controls. CONCLUSION: Genetic variants in ATG5, including a functional promotor variant, are associated with childhood asthma. These results provide novel evidence for a role for ATG5 in childhood asthma.

The Greater Cincinnati Pediatric Clinic Repository: A Novel Framework for Childhood Asthma and Allergy Research.

BACKGROUND: Allergic disorders, including asthma, allergic rhinitis, atopic dermatitis, eosinophilic esophagitis, and food allergy, are a major global health burden. The study and management of allergic disorders is complicated by the considerable heterogeneity in both the presentation and natural history of these disorders. Biorepositories serve as an excellent source of data and biospecimens for delineating subphenotypes of allergic disorders, but such resources are lacking. METHODS: In order to define subphenotypes of allergic disease accurately, we established an infrastructure to link and efficiently utilize clinical and epidemiologic data with biospecimens into a single biorepository called the Greater Cincinnati Pediatric Clinic Repository (GCPCR). Children with allergic disorders as well as healthy controls are followed longitudinally at hospital clinic, emergency department, and inpatient visits. Subjects' asthma, allergy, and skin symptoms; past medical, family, social, diet, and environmental histories; physical activity; medication adherence; perceived quality of life; and demographics are ascertained. DNA is collected from all participants, and other biospecimens such as blood, hair, and nasal epithelial cells are collected on a subset. RESULTS: To date, the GCPCR has 6,317 predominantly Caucasian and African American participants, and 93% have banked DNA. This large sample size supports adequately powered genetic, epidemiologic, environmental, and health disparities studies of childhood allergic diseases. CONCLUSIONS: The GCPCR is a unique biorepository that is continuously evaluated and refined to achieve and maintain rigorous clinical phenotype and biological data. Development of similar disease-specific repositories using common data elements is necessary to enable studies across multiple populations of comprehensively phenotyped patients.

Differences in candidate gene association between European ancestry and African American asthmatic children.

BACKGROUND: Candidate gene case-control studies have identified several single nucleotide polymorphisms (SNPs) that are associated with asthma susceptibility. Most of these studies have been restricted to evaluations of specific SNPs within a single gene and within populations from European ancestry. Recently, there is increasing interest in understanding racial differences in genetic risk associated with childhood asthma. Our aim was to compare association patterns of asthma candidate genes between children of European and African ancestry. METHODOLOGY/PRINCIPAL FINDINGS: Using a custom-designed Illumina SNP array, we genotyped 1,485 children within the Greater Cincinnati Pediatric Clinic Repository and Cincinnati Genomic Control Cohort for 259 SNPs in 28 genes and evaluated their associations with asthma. We identified 14 SNPs located in 6 genes that were significantly associated (p-values <0.05) with childhood asthma in African Americans. Among Caucasians, 13 SNPs in 5 genes were associated with childhood asthma. Two SNPs in IL4 were associated with asthma in both races (p-values <0.05). Gene-gene interaction studies identified race specific sets of genes that best discriminate between asthmatic children and non-allergic controls. CONCLUSIONS/SIGNIFICANCE: We identified IL4 as having a role in asthma susceptibility in both African American and Caucasian children. However, while IL4 SNPs were associated with asthma in asthmatic children with European and African ancestry, the relative contributions of the most replicated asthma-associated SNPs varied by ancestry. These data provides valuable insights into the pathways that may predispose to asthma in individuals with European vs. African ancestry.

Identification of KIF3A as a novel candidate gene for childhood asthma using RNA expression and population allelic frequencies differences.

BACKGROUND: Asthma is a chronic inflammatory disease with a strong genetic predisposition. A major challenge for candidate gene association studies in asthma is the selection of biologically relevant genes. METHODOLOGY/PRINCIPAL FINDINGS: Using epithelial RNA expression arrays, HapMap allele frequency variation, and the literature, we identified six possible candidate susceptibility genes for childhood asthma including ADCY2, DNAH5, KIF3A, PDE4B, PLAU, SPRR2B. To evaluate these genes, we compared the genotypes of 194 predominantly tagging SNPs in 790 asthmatic, allergic and non-allergic children. We found that SNPs in all six genes were nominally associated with asthma (p<0.05) in our discovery cohort and in three independent cohorts at either the SNP or gene level (p<0.05). Further, we determined that our selection approach was superior to random selection of genes either differentially expressed in asthmatics compared to controls (p = 0.0049) or selected based on the literature alone (p = 0.0049), substantiating the validity of our gene selection approach. Importantly, we observed that 7 of 9 SNPs in the KIF3A gene more than doubled the odds of asthma (OR = 2.3, p<0.0001) and increased the odds of allergic disease (OR = 1.8, p<0.008). Our data indicate that KIF3A rs7737031 (T-allele) has an asthma population attributable risk of 18.5%. The association between KIF3A rs7737031 and asthma was validated in 3 independent populations, further substantiating the validity of our gene selection approach. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that KIF3A, a member of the kinesin superfamily of microtubule associated motors that are important in the transport of protein complexes within cilia, is a novel candidate gene for childhood asthma. Polymorphisms in KIF3A may in part be responsible for poor mucus and/or allergen clearance from the airways. Furthermore, our study provides a promising framework for the identification and evaluation of novel candidate susceptibility genes.

Genetic and environmental risk factors for childhood eczema development and allergic sensitization in the CCAAPS cohort.

Eczema is very common and increasing in prevalence. Prospective studies investigating environmental and genetic risk factors for eczema in a birth cohort are lacking. We evaluated risk factors that may promote development of childhood eczema in the Cincinnati Childhood Allergy and Air Pollution Study (CCAAPS) birth cohort (n=762) of infants with at least one atopic parent. Objective environmental exposure data were available for each participant. At annual physical examinations, children underwent skin prick tests (SPTs), eczema was diagnosed by a clinician, and DNA was collected. Among Caucasian children, 39% developed eczema by age 3. Children with a pet dog were significantly less likely to have eczema at age one (odds ratio (OR)=0.62, 95% confidence interval (CI): 0.40-0.97) or at both ages 2 and 3 (OR=0.54, 95% CI: 0.30-0.97). This finding was most significant among children carrying the CD14-159C/T CC genotype. Carriers of the CD14-159C/T and IL4Ralpha I75V single-nucleotide polymorphisms (SNPs) had an increased risk of eczema at both ages 2 and 3 (OR=3.44, 95% CI: 1.56-7.57), especially among children who were SPT+. These results provide new insights into the pathogenesis of eczema in high-risk children and support a protective role for early exposure to dog, especially among those carrying the CD14-159C/T SNP. The results also demonstrate a susceptibility effect of the combination of CD14 and IL4Ralpha SNPs with eczema.

Functional effect of the R110Q IL13 genetic variant alone and in combination with IL4RA genetic variants.

BACKGROUND: IL-13 is a key mediator of allergic asthma. IL-13 mediates its effects via its receptor, a heterodimer composed of IL-4R alpha and IL-13R alpha1. Polymorphic variants of both IL-13 and IL-4R alpha have been shown to be associated with atopy. OBJECTIVE: We examined the functional consequences of the Q110 IL-13 variant in vitro and in vivo to determine whether it displays enhanced functional activity compared with R110 IL-13, both in the context of I50Q551 IL-4R alpha and of the atopy-associated variant V50R551 IL-4R alpha. METHODS: We used a mouse cell line stably expressing human IL-4R alpha and IL-13R alpha1 that readily responds to human IL-4 and IL-13. For in vivo analyses, we used BALB/c mice. RESULTS: The Q110 IL-13 variant displayed significantly increased activity compared with R110 IL-13. Furthermore, mice treated with Q110 IL-13 variant displayed increased airways hyperresponsiveness relative to R110 IL-13. We then examined the functional consequences of Q110 IL-13 variant in combination with an atopy-associated variant of its receptor, IL-4R alpha (V50R551). Q110 IL-13 variant had increased activity on these cells as well, and, strikingly, the effect was greater than that observed in cells expressing I50Q551 IL-4R alpha. CONCLUSION: Either Q110 IL-13 variant or V50R551 IL-4R alpha variant has enhanced function alone, but the 2 together have a synergistic effect on IL-13-dependent gene induction. Our data demonstrate the importance of relatively small individual differences in gene products from common single nucleotide polymorphisms that may result in larger combined differences. Furthermore, a relatively modest change in function from a single nucleotide polymorphism can result in an important biological difference in vivo.

Differential expression of type IV collagen isoforms in rat glomerular endothelial and mesangial cells.

Type IV collagen, which is encoded by six genetically distinct alpha-chains (alpha 1-alpha 6), is a major component of the kidney glomerulus. The alpha 1(IV) and alpha 2(IV) chains are present predominantly in the mesangial matrix, whereas the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains are localized almost exclusively to the glomerular basement membrane (GBM). Thickening of the GBM and expansion of the mesangial matrix are believed to contribute to the pathogenesis of diabetic nephropathy. In the present study, we evaluated the expression of alpha 1(IV), alpha 3(IV), and alpha 5(IV) chains in rat glomerular endothelial (GEndC) and mesangial cells (GMC). Under physiological concentrations of glucose (5 mM), alpha 1(IV) and alpha 5(IV) chains were detectable in GMCs, with an obvious absence of alpha 3(IV) chain. All three isoforms tested were present in GEndCs. At diabetic concentrations of glucose (25 mM), alpha 1(IV) was up-regulated in GMCs, whereas expression level of alpha 1(IV) remained unaltered in GEndCs. The alpha 3(IV) and alpha 5(IV) chains were up-regulated in GEndCs, but remained unchanged in GMCs under diabetic glucose concentrations (25 mM). Collectively, our results demonstrate that GMC might contribute to mesangial matrix expansion, mediated by alpha 1(IV) collagen, while GEndC might contribute to thickening of GBM, mediated by alpha 3(IV) collagen, in patients with diabetic nephropathy.

Analysis of the life cycle of stat6. Continuous cycling of STAT6 is required for IL-4 signaling.

Signal transducer and activator of transcription (Stat)6 is a transcription factor important for the development of Th2 cells and regulation of gene expression by IL-4 and IL-13. It is known that Stat6 is rapidly activated in response to IL-4; however, the fate of activated Stat6 is less clear. We examined the fate of activated Stat6 and found that during continuous exposure to IL-4, Stat6 activity was sustained for 72 h and that the maintenance of a constant level of activated Stat6 did not require new protein synthesis. In contrast, when cells were pulsed with IL-4 and then incubated in the absence of IL-4, the half-life of Stat6 phosphorylation and DNA binding activity was less than 1 h. Stat6 did not accumulate in the nucleus, and protein degradation did not play a major role in the disappearance of activated Stat6. Inhibition of kinase activity by staurosporine or the JAK inhibitor, AG490, revealed that maintenance of Stat6 activation in the continuous presence of IL-4 required ongoing phosphorylation of latent cytoplasmic Stat6 molecules. Cells treated with an inhibitor of nuclear export, leptomycin B, were unable to maintain Stat6 activation. Thus, the maintenance of Stat6 activation requires a constant cycle of activation, deactivation, nuclear export, and reactivation.

V75R576 IL-4 receptor alpha is associated with allergic asthma and enhanced IL-4 receptor function.

Asthma is a complex polygenic disease. Many studies have implicated the importance of IL-4R alpha in the development of allergic inflammation and its gene has been implicated in the genetics of asthma and atopy. In this study, we examined the functional consequences of two of the human IL-4R alpha allelic variants that have been found to associate with asthma and atopy. We examined the effects of each variant alone and in combination on IL-4-dependent gene induction. We found that neither the Q576R nor the I75V variants affected IL-4-dependent CD23 expression. However, the combination of V75R576 resulted in expression of an IL-4R alpha with enhanced sensitivity to IL-4. We next examined the genetics of five of the known IL-4R alpha allelic variants in asthmatic and nonatopic populations. Strikingly, the association of V75/R576 with atopic asthma was greater than either allele alone and the association of R576 with atopic asthma was dependent on the coexistence of V75. A haplotype analysis revealed a single IL-4R alpha haplotype that was associated with allergic asthma, VACRS, further confirming the importance of the V75 and R576 combination in the genetics of asthma. This is the first report demonstrating that a functional alteration in IL-4R alpha requires the coexistence of two naturally occurring single nucleotide polymorphisms (snps) in combination; neither snp alone is sufficient. These data illustrate the importance of studying snps in combination, because the functional significance of a given snp may only be evident in a specific setting of additional snps in the same or different genes.