Association study of matrix metalloproteinase-12 gene polymorphisms and asthma in a Japanese population.

BACKGROUND: Matrix metalloproteinase 12 gene (MMP12) has been shown to be associated with asthma in a Caucasian population. In this study, we investigate whether single-nucleotide polymorphisms (SNPs) of MMP12 are associated with a risk for asthma in a Japanese population. METHODS: We tested for an association between SNPs in MMP12 and asthma, including its severity, in a Japanese population (630 pediatric and 417 adult patients with atopic asthma and 336 children and 632 adults as controls). The rs652438 A and G variants (N357S) were generated by site-directed mutagenesis and an assay with artificial peptide substrates was used to compare two types of MMP12 activity. The effect of MMP12 inhibition with MMP12-specific small interfering RNA (siRNA) on chemokine secretion from airway epithelial cells was also tested in vitro. RESULTS: N357S showed a p value <0.05 for childhood and combined (adult plus childhood) asthma in the dominant model [odds ratio (OR) 1.60, 95% confidence interval (CI) 1.00-2.56, p = 0.047; OR 1.40, 95% CI 1.04-1.89, p = 0.028, respectively]. This risk variant is associated with asthma severity in adult patients. In the functional assay, the minor-allele enzyme showed significantly lower activity than the major-allele enzyme. MMP12-specific siRNA suppressed IP-10 secretion from airway epithelial cells upon stimulation with IFN-ß. CONCLUSIONS: Our results suggest that MMP12 confers susceptibility to asthma and is associated with asthma severity in a Japanese population. MMP12 may be associated with asthma through inappropriate attraction of leukocytes to the inflamed tissue.

The fractional exhaled nitric oxide and serum high sensitivity C-reactive protein levels in cough variant asthma and typical bronchial asthma.

BACKGROUND: Fractional exhaled nitric oxide (FeNO) is known to be a good marker of airway eosinophilic inflammation in bronchial asthma. Recently, serum high sensitivity C-reactive protein (hs-CRP) has been shown to be also useful to detect the airway inflammation. METHODS: Newly diagnosed 90 cough variant asthma and 92 bronchial asthma patients were enrolled. FeNO, serum hs-CRP, pulmonary function tests, bronchial hyperresponsiveness, IgE and sputum eosinophils ratio were compared. Ninety healthy control subjects were set for FeNO and serum hs-CRP normal range reference. We have compared the clinical utilities of FeNO and serum hs-CRP to differentiate bronchial asthma and cough variant asthma. RESULTS: FeNO was significantly higher in bronchial asthma (92.6 ± 85.5ppb) than in cough variant asthma (35.6 ± 43.3; p < 0.001) and both were significantly higher than normal range (18.0 ± 6.4, p < 0.001, respectively), and in differentiating between the two groups showed a sensitivity of 0.69 and a specificity of 0.73 at the cutoff value of 28 ppb. Serum hs-CRP did not differ significantly between bronchial asthma (723 ± 1162ng/ml) and cough variant asthma (558 ± 758) even if both were significantly higher than normal range (345 ± 401, p < 0.01 and p < 0.05 respectively). CONCLUSIONS: FeNO is more useful than serum hs-CRP in differentiating patients with bronchial asthma from those with cough variant asthma, and healthy persons.

[Persistence rate for clinic visit in children with asthma after initiating controller therapy].

OBJECT: To investigate the actual condition of the persistence rate for clinic visit in children with asthma requiring controller medication. METHODS: Seventy-three asthma children (0 to 15 years of age) who initiated controller medication were enrolled this study. Patients and their parents were educated about this disease (mechanism of asthma, airway inflammation, and necessity of asthma medication) and instructed to visit clinics regularly every 30 days. We prospectively monitored visit rate of the patients for 180 days. RESULTS: Mean persistence rate for clinic visit of all patients was gradually decreased, 90.4% on the 30 day, 82.2% on the 60 day, 78.1% on the 90 day, 67.1% on the 120 day and 38.4% on the 180 day, respectively. The persistence rate on the 180 day of 2 to 5 years old patients was significantly lower than that of < 2 years old patients and the rate on the 60 days or more after in 6 to 15 years old patients was lower than that of < 2 years old patients. It was also significantly lower in patients with moderate and severe persistent asthma than in mild persistent patients. CONCLUSION: Despite of the investigation in the setting of asthma specialty clinic, the persistence rate for clinic visit was decreased with time, especially in 6 to 15 years old or moderate to severe asthmatic patients. In order to improve the situation, it is necessary to analyze the cause of poor adherence and make a concrete action plan.

Thymic stromal lymphopoietin gene promoter polymorphisms are associated with susceptibility to bronchial asthma.

Thymic stromal lymphopoietin (TSLP) triggers dendritic cell--mediated T helper (Th) 2 inflammatory responses. A single-nucleotide polymorphism (SNP), rs3806933, in the promoter region of the TSLP gene creates a binding site for the transcription factor activating protein (AP)-1. The variant enhances AP-1 binding to the regulatory element, and increases the promoter--reporter activity of TSLP in response to polyinosinic-polycytidylic acid (poly[I:C]) stimulation in normal human bronchial epithelium (NHBE). We investigated whether polymorphisms including the SNP rs3806933 could affect the susceptibility to and clinical phenotypes of bronchial asthma. We selected three representative (i.e., Tag) SNPs and conducted association studies of the TSLP gene, using two independent populations (639 patients with childhood atopic asthma and 838 control subjects, and 641 patients with adult asthma and 376 control subjects, respectively). We further examined the effects of corticosteroids and a long-acting ß(2)-agonist (salmeterol) on the expression levels of the TSLP gene in response to poly(I:C) in NHBE. We found that the promoter polymorphisms rs3806933 and rs2289276 were significantly associated with disease susceptibility in both childhood atopic and adult asthma. The functional SNP rs3806933 was associated with asthma (meta-analysis, P = 0.000056; odds ratio, 1.29; 95% confidence interval, 1.14-1.47). A genotype of rs2289278 was correlated with pulmonary function. Moreover, the induction of TSLP mRNA and protein expression induced by poly(I:C) in NHBE was synergistically impaired by a corticosteroid and salmeterol. TSLP variants are significantly associated with bronchial asthma and pulmonary function. Thus, TSLP may serve as a therapeutic target molecule for combination therapy.

[Examination of the inspiratory flow rates of COPD patients in Japanese medical facilities].

UNLABELLED: Chronic obstructive pulmonary disease (COPD) is treated primarily with inhalation therapy. However, as many COPD patients are elderly, whether or not patients inhale dry powder at an adequate inspiratory flow rate requires investigation. We therefore conducted a multicenter investigation of the effects of height, body weight, age, disease severity, and other factors on inspiratory flow rate in COPD patients. METHODS: We measured inspiratory flow rates and forced expiratory volume in the first second (FEV1) with the In-Check Dial (Clement Clarke) fitted with a discus adapter in 175 COPD patients of 40 years old and over (mean age 71.3, men 89.1%, women 10.9%). RESULTS: A total of 98.9% of the patients had an inspiratory flow rate exceeding 30L/minute. Two patients had a rate of 30L/minute or less. The mean inspiratory flow rate was 98 +/- 30L/minute overall, 104 +/- 27L/minute in mild COPD, 109 +/- 28L/minute in moderate COPD, 91 +/- 26L/minute in severe COPD, 71 +/- 24L/minute in very severe COPD, and 83 +/- 28L/minute in patients 75 years and older. Inspiratory flow rates significantly correlated with body mass index and FEV1. CONCLUSION: Most elderly patients with COPD maintained a sufficient inspiratory flow rate when inhaling dry powder.

Replication of genetic association studies in asthma and related phenotypes.

In asthma genetics, the association of highly replicated susceptibility genes lacks consistency across populations. To identify genuine associations, we investigated the reproducibility of the 23 most promising asthma and asthma-related candidate genes in a moderately sized sample from the Japanese population. We compared the frequency of 33 polymorphisms in unrelated cases and controls and tested for their association with asthma, atopy and serum total IgE levels using allele frequency, codominant, dominant and recessive genotype models. On the basis of the consistency of our findings with previous meta-analyses and large replication studies, IL13, TNF, ADAM33, IL4RA and TBXA2R might represent common major asthma and asthma-related trait genes. Individual gene assessment was extended to the interactions between two polymorphisms using our original method. Interactions between TBXA2R and ADAM33, and IL4RA and C3 were suggested to increase the risk for childhood and all asthma (adult and childhood asthma combined). The confirmation of previously reported associations between gene polymorphisms and phenotypes was problematic when as few as several hundred samples per group were used. Stratification of the subjects by environmental factors or other confounding factors may be necessary to improve the sensitivity and reliability of association results.

A functional polymorphism in IL-18 is associated with severity of bronchial asthma.

RATIONALE: IL-18 is a unique cytokine that enhances innate immunity and both Th1- and Th2-driven immune responses. Recent murine and human genetic studies have shown its role in the pathogenesis of asthma. OBJECTIVES: We conducted an association study in a Japanese population to discover variants of IL-18 that might have an effect on asthma susceptibility and/or progression and conducted functional analyses of the related variants. METHODS: The IL-18 gene locus was resequenced in 48 human chromosomes. Asthma severity was determined according to the 2002 Global Initiative for Asthma Guidelines. Association and haplotype analyses were performed using 1,172 subjects. MEASUREMENTS AND MAIN RESULTS: Although no polymorphisms differed significantly in frequency between the control and adult asthma groups, rs5744247 C>G was significantly associated with the severity of adult asthma (steps 1, 2 vs. steps 3, 4; P = 0.0034). We also found a positive association with a haplotype (P = 0.0026). By in vitro functional analyses, the rs5744247 variant was found to increase enhancer-reporter activity of the IL-18 gene in bronchial epithelial cells. Expression levels of IL-18 in response to LPS stimulation in monocytes were significantly greater in subjects homozygous for the susceptibility G allele at rs5744247 C>G. Furthermore, we found a significant correlation between the serum IL-18 level and the genotype of rs5744247 (P = 0.031). CONCLUSIONS: Although the association results need to be replicated by other studies, IL-18 variants are significantly associated with asthma severity, and the rs5744247 variant reflects higher transcriptional activity and higher expression of IL-18 in LPS-stimulated monocytes and a higher serum IL-18 level.

Associations of functional NLRP3 polymorphisms with susceptibility to food-induced anaphylaxis and aspirin-induced asthma.

BACKGROUND: NLR family, pyrin domain containing 3 (NLRP3), controls the activity of inflammatory caspase-1 by forming inflammasomes, which leads to cleavage of the procytokines IL-1beta and IL-18. Recent studies have shown associations of human NLRP3 polymorphisms with susceptibility to various inflammatory diseases; however, the association with allergic diseases remains unclear. OBJECTIVE: We sought to examine whether NLRP3 polymorphisms are associated with susceptibility to food allergy, food-induced anaphylaxis, and aspirin-induced asthma (AIA). METHODS: We selected 15 tag single nucleotide polymorphisms (SNPs) of NLRP3 and conducted association analyses of NLRP3 using 574 and 1279 samples for food allergy and AIA, respectively. We further performed functional analyses of the susceptible SNPs. RESULTS: Two NLRP3 SNPs (rs4612666 and rs10754558) were significantly associated with susceptibility to food-induced anaphylaxis (P = .00086 and P = .00068, respectively). The NLRP3 haplotype of the 2 SNPs also showed a significant association (P = .000098). We could confirm the association with susceptibility to another hypersensitivity phenotype, AIA (rs4612666, P = .0096). Functional analysis revealed that the risk alleles of rs4612666 and rs10754558 increased the enhancer activity of NLRP3 expression and NLRP3 mRNA stability, respectively. CONCLUSION: Our results indicate that the NLRP3 SNPs might play an important role in the development of food-induced anaphylaxis and AIA in a gain-of-function manner. Further research on the NLRP3 inflammasome will contribute to the development of novel diagnostic and therapeutic methods for food-induced anaphylaxis and AIA.

Functional analysis of the thymic stromal lymphopoietin variants in human bronchial epithelial cells.

Thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that triggers dendritic cell-mediated T helper (Th)2 inflammatory responses, and is implicated in the pathogenesis of allergic diseases in humans. Two TSLP splice variants have been reported. To find functional genetic variants that might contribute to disease, we conducted analyses of single nucleotide polymorphisms (SNPs) of the TSLP gene in human bronchial epithelial cells. We surveyed SNPs on the TSLP gene by sequencing genomic DNA from 36 subjects, and characterized the linkage disequilibrium of the gene. We examined whether the SNPs have functional effects on mRNA expression or protein production using real-time PCR, reporter gene analysis, and enzyme-linked immunosorbent assay. We identified a total of 23 polymorphisms in the TSLP gene. The long form of TSLP, which is associated with allergic inflammation, was highly induced by poly(I:C) (double-stranded RNA) stimulation in normal human bronchial epithelial cells (NHBE) (P = 0.0060). The SNP rs3806933 (-847C > T) in the promoter region of long-form TSLP was found to create a binding site for the transcription factor activating protein (AP)-1, and in vitro functional analyses demonstrated that the SNP enhanced AP-1 binding to the regulatory element. The functional variant increased promoter-reporter activity of long-form TSLP in response to poly(I:C) stimulation in NHBE. Functional genetic polymorphism of the TSLP gene appears to contribute to Th2-polarized immunity through higher TSLP production by bronchial epithelial cells in response to viral respiratory infections.

Effects of alendronate on bone mineral density and bone metabolic markers in postmenopausal asthmatic women treated with inhaled corticosteroids.

We have recently shown that long-term use of inhaled corticosteroids decreases bone mineral density (BMD) of the lumbar spine in postmenopausal asthmatic women. The present study aimed to evaluate the efficacy of alendronate in comparison with that of alfacalcidol (1-alpha-hydroxyvitamin D(3)) for the treatment of BMD reduction in postmenopausal asthmatic patients who had inhaled corticosteroid therapy without regular use of systemic corticosteroids. Twenty-eight postmenopausal asthmatic patients with BMD T score of -1.0 or less were randomized to receive alendronate (5 mg/d) or alfacalcidol (1 microg/d). Bone mineral density was determined at baseline and 12 months after the treatment, and biochemical markers of bone metabolism were measured at baseline and after 6 and 12 months of treatment. The mean (+/-SD) BMD values at the lumbar spine, the total hip, and the Ward's triangle significantly increased by 4.9 +/- 4.5% (P = .0005), 2.4 +/- 2.2% (P = .0005), and 3.6 +/- 5.2% (P = .02) at 12 months in the alendronate group, whereas the corresponding values did not significantly change in the alfacalcidol group. In the alendronate group, urinary N-telopeptide (NTx), serum osteocalcin, and serum alkaline phosphatase concentrations significantly decreased, and serum intact parathyroid (PTH) level significantly increased, from baseline at both 6 and 12 months. In the alfacalcidol group, urinary NTx showed modest but significant decrease, although the extent of the change was smaller than that in the alendronate group. We concluded that alendronate was effective to improve reduced BMD in postmenopausal asthmatic patients on inhaled corticosteroid therapy through the mechanism of inhibiting bone resorption.

Natural history and risk factors of obstructive changes over a 10-year period in severe asthma.

BACKGROUND: The clinical features, physiology, and pathology of severe asthma are poorly understood. Recently, the forced vital capacity (FVC) has been shown to be reduced in severe asthma compared to mild asthma, possibly due to air trapping. However, the natural history and risk factors of obstructive change for such asthmatic patients have not been fully elucidated. METHODS: We examined the data of a retrospective analysis of lung function changes over a 10-year period in 54 severe asthma patients. RESULTS: The faster obstructive changes detected by FEV(1) (forced expiratory volume in one second) were accompanied by excessive loss of FVC (r = 0.85, p < 0.0001) and the reduction in FVC was 1.2 times larger than the FEV(1) change. Age, baseline FVC, exacerbation rate and oral corticosteroids use showed significantly negative correlations with the rate of annual change in FVC. CONCLUSIONS: These data indicate that the decline in FVC is more evident than FEV(1) in severe asthma, suggesting that small airway susceptibility may be the cause of rapid disease progression. Aging, exacerbations of asthma, and use of systemic corticosteroids are related to excess FVC decline, particularly if FVC is still normal.

ORAI1 genetic polymorphisms associated with the susceptibility of atopic dermatitis in Japanese and Taiwanese populations.

Atopic dermatitis is a chronic inflammatory skin disease. Multiple genetic and environmental factors are thought to be responsible for susceptibility to AD. In this study, we collected 2,478 DNA samples including 209 AD patients and 729 control subjects from Taiwanese population and 513 AD patients and 1027 control subject from Japanese population for sequencing and genotyping ORAI1. A total of 14 genetic variants including 3 novel single-nucleotide polymorphisms (SNPs) in the ORAI1 gene were identified. Our results indicated that a non-synonymous SNP (rs3741596, Ser218Gly) associated with the susceptibility of AD in the Japanese population but not in the Taiwanese population. However, there is another SNP of ORAI1 (rs3741595) associated with the risk of AD in the Taiwanese population but not in the Japanese population. Taken together, our results indicated that genetic polymorphisms of ORAI1 are very likely to be involved in the susceptibility of AD.

Genome-wide association study identifies eight new susceptibility loci for atopic dermatitis in the Japanese population.

Atopic dermatitis is a common inflammatory skin disease caused by interaction of genetic and environmental factors. On the basis of data from a genome-wide association study (GWAS) and a validation study comprising a total of 3,328 subjects with atopic dermatitis and 14,992 controls in the Japanese population, we report here 8 new susceptibility loci: IL1RL1-IL18R1-IL18RAP (P(combined) = 8.36 × 10(-18)), the major histocompatibility complex (MHC) region (P = 8.38 × 10(-20)), OR10A3-NLRP10 (P = 1.54 × 10(-22)), GLB1 (P = 2.77 × 10(-16)), CCDC80 (P = 1.56 × 10(-19)), CARD11 (P = 7.83 × 10(-9)), ZNF365 (P = 5.85 × 10(-20)) and CYP24A1-PFDN4 (P = 1.65 × 10(-8)). We also replicated the associations of the FLG, C11orf30, TMEM232-SLC25A46, TNFRSF6B-ZGPAT, OVOL1, ACTL9 and KIF3A-IL13 loci that were previously reported in GWAS of European and Chinese individuals and a meta-analysis of GWAS for atopic dermatitis. These findings advance the understanding of the genetic basis of atopic dermatitis.

Variants of C-C motif chemokine 22 (CCL22) are associated with susceptibility to atopic dermatitis: case-control studies.

Atopic dermatitis (AD) is a common inflammatory skin disease caused by multiple genetic and environmental factors. AD is characterized by the local infiltration of T helper type 2 (Th2) cells. Recent clinical studies have shown important roles of the Th2 chemokines, CCL22 and CCL17 in the pathogenesis of AD. To investigate whether polymorphisms of the CCL22 gene affect the susceptibility to AD, we conducted association studies and functional studies of the related variants. We first resequenced the CCL22 gene and found a total of 39 SNPs. We selected seven tag SNPs in the CCL22 gene, and conducted association studies using two independent Japanese populations (1(st) population, 916 cases and 1,032 controls; 2(nd) population 1,034 cases and 1,004 controls). After the association results were combined by inverse variance method, we observed a significant association at rs4359426 (meta-analysis, combined P = 9.6×10⁻6; OR, 0.74; 95% CI, 0.65-0.85). Functional analysis revealed that the risk allele of rs4359426 contributed to higher expression levels of CCL22 mRNA. We further examined the allelic differences in the binding of nuclear proteins by electrophoretic mobility shift assay. The signal intensity of the DNA-protein complex derived from the G allele of rs223821, which was in absolute LD with rs4359426, was higher than that from the A allele. Although further functional analyses are needed, it is likely that related variants play a role in susceptibility to AD in a gain-of-function manner. Our findings provide a new insight into the etiology and pathogenesis of AD.

Genome-wide association study identifies three new susceptibility loci for adult asthma in the Japanese population.

Bronchial asthma is a common inflammatory disease caused by the interaction of genetic and environmental factors. Through a genome-wide association study and a replication study consisting of a total of 7,171 individuals with adult asthma (cases) and 27,912 controls in the Japanese population, we identified five loci associated with susceptibility to adult asthma. In addition to the major histocompatibility complex and TSLP-WDR36 loci previously reported, we identified three additional loci: a USP38-GAB1 locus on chromosome 4q31 (combined P = 1.87 × 10(-12)), a locus on chromosome 10p14 (P = 1.79 × 10(-15)) and a gene-rich region on chromosome 12q13 (P = 2.33 × 10(-13)). We observed the most significant association with adult asthma at rs404860 in the major histocompatiblity complex region (P = 4.07 × 10(-23)), which is close to rs2070600, a SNP previously reported for association with FEV(1)/FVC in genome-wide association studies for lung function. Our findings offer a better understanding of the genetic contribution to asthma susceptibility.

Asthma is an independent risk for elevation of plasma C-reactive protein levels.

BACKGROUND: Chronic inflammation of the airways plays a major role in the pathogenesis of asthma. Although C-reactive protein (CRP) is now an established circulating marker for cardiovascular diseases, it remains unclear whether asthma is an independent risk for increased plasma CRP. METHODS: In Study 1, we evaluated CRP levels in 329 asthmatic patients and 1684 non-asthmatic subjects. Inhaled corticosteroids were regularly used by 308 asthmatics. In Study 2, the effects of inhaled corticosteroids on CRP levels were examined in 64 corticosteroid-naive asthmatic patients. RESULTS: In Study 1, plasma CRP levels were associated with body mass index (R=0.349, P<0.0001) and age (R=0.111, P<0.0001) in all study subjects, but were higher in patients with asthma, hypertension, diabetes and/or dyslipidemia than in those without these disorders. Multivariate regression analysis identified body mass index, age and asthma, but not hypertension, diabetes or dyslipidemia, as independently associated with an elevation of CRP levels. In Study 2, treatment of 64 corticosteroid-naive asthmatic patients with inhaled corticosteroids for 3 months significantly reduced plasma CRP levels. CONCLUSIONS: Asthma is a disorder associated with increased plasma CRP levels independent of various other factors. Treatment with inhaled corticosteroids was associated with a significant reduction in plasma CRP levels, which may reflect their clinical effect on the inflammatory process within airways.