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1.  Role of Lung Function Genes in the Development of Asthma 
PLoS ONE  2016;11(1):e0145832.
Although our previous GWAS failed to identify SNPs associated with pulmonary function at the level of genomewide significance, it did show that the heritability for FEV1/FVC was 41.6% in a Japanese population, suggesting that the heritability of pulmonary function traits can be explained by the additive effects of multiple common SNPs. In addition, our previous study indicated that pulmonary function genes identified in previous GWASs in non-Japanese populations accounted for 4.3% to 12.0% of the entire estimated heritability of FEV1/FVC in a Japanese population. Therefore, given that many loci with individual weak effects may contribute to asthma risk, in this study, we created a quantitative score of genetic load based on 16 SNPs implicated in lower lung function in both Japanese and non-Japanese populations. This genetic risk score (GRS) for lower FEV1/FVC was consistently associated with the onset of asthma (P = 9.6 × 10−4) in 2 independent Japanese populations as well as with the onset of COPD (P = 0.042). Clustering of asthma patients based on GRS levels indicated that an increased GRS may be responsible for the development of a particular phenotype of asthma characterized by early onset, atopy, and severer airflow obstruction.
PMCID: PMC4709100  PMID: 26752288
2.  CMTR1 is associated with increased asthma exacerbations in patients taking inhaled corticosteroids 
Inhaled corticosteroids (ICS) are the most effective controller medications for asthma, and variability in ICS response is associated with genetic variation. Despite ICS treatment, some patients with poor asthma control experience severe asthma exacerbations, defined as a hospitalization or emergency room visit. We hypothesized that some individuals may be at increased risk of asthma exacerbations, despite ICS use, due to genetic factors. A GWAS of 237,726 common, independent markers was conducted in 806 Caucasian asthmatic patients from two population‐based biobanks: BioVU, at Vanderbilt University Medical Center (VUMC) in Tennessee (369 patients), and Personalized Medicine Research Project (PMRP) at the Marshfield Clinic in Wisconsin (437 patients). Using a case–control study design, the association of each SNP locus with the outcome of asthma exacerbations (defined as asthma‐related emergency department visits or hospitalizations concurrent with oral corticosteroid use), was evaluated for each population by logistic regression analysis, adjusting for age, gender and the first four principal components. A meta‐analysis of the results was conducted. Validation of expression of selected candidate genes was determined by evaluating an independent microarray expression data set. Our study identified six novel SNPs associated with differential risk of asthma exacerbations (P < 10−05). The top GWAS result, rs2395672 in CMTR1, was associated with an increased risk of exacerbations in both populations (OR = 1.07, 95% CI 1.03–1.11; joint P = 2.3 × 10−06). Two SNPs (rs2395672 and rs279728) were associated with increased risk of exacerbations, while the remaining four SNPs (rs4271056, rs6467778, rs2691529, and rs9303988) were associated with decreased risk. Three SNPs (rs2395672, rs6467778, and rs2691529) were present in three genes: CMTR1, TRIM24 and MAGI2. The CMTR1 mRNA transcript was significantly differentially expressed in nasal lavage samples from asthmatics during acute exacerbations, suggesting potential involvement of this gene in the development of this phenotype. We show that genetic variability may contribute to asthma exacerbations in patients taking ICS. Furthermore, our studies implicate CMTR1 as a novel candidate gene with potential roles in the pathogenesis of asthma exacerbations.
PMCID: PMC4693729  PMID: 26734457
Asthma; GWAS; inhaled corticosteroids; EMR; exacerbations; pharmacogenomics
3.  Genome-Wide Association Study Identifies Novel Pharmacogenomic Loci For Therapeutic Response to Montelukast in Asthma 
PLoS ONE  2015;10(6):e0129385.
Genome-wide association study (GWAS) is a powerful tool to identify novel pharmacogenetic single nucleotide polymorphisms (SNPs). Leukotriene receptor antagonists (LTRAs) are a major class of asthma medications, and genetic factors contribute to variable responses to these drugs. We used GWAS to identify novel SNPs associated with the response to the LTRA, montelukast, in asthmatics.
Using genome-wide genotype and phenotypic data available from American Lung Association - Asthma Clinical Research Center (ALA-ACRC) cohorts, we evaluated 8-week change in FEV1 related to montelukast administration in a discovery population of 133 asthmatics. The top 200 SNPs from the discovery GWAS were then tested in 184 additional samples from two independent cohorts.
Twenty-eight SNP associations from the discovery GWAS were replicated. Of these, rs6475448 achieved genome-wide significance (combined P = 1.97 x 10-09), and subjects from all four studies who were homozygous for rs6475448 showed increased ΔFEV1 from baseline in response to montelukast.
Through GWAS, we identified a novel pharmacogenomic locus related to improved montelukast response in asthmatics.
PMCID: PMC4470685  PMID: 26083242
4.  Inhaled corticosteroid treatment modulates ZNF432 gene variant's effect on bronchodilator response in asthmatics 
Single nucleotide polymorphisms (SNPs) influence a patient's response to inhaled corticosteroids and β2-agonists, and the effect of treatment with inhaled corticosteroids is synergistic with the effect of β2-agonists. We hypothesized that use of inhaled corticosteroids could influence the effect of SNPs associated with bronchodilator response.
To assess whether, among asthma subjects, the association of SNPs with bronchodilator response is different between those treated with inhaled corticosteroids vs. those on placebo.
A genome-wide association analysis was conducted using 581 white subjects from the Childhood Asthma Management Program (CAMP). Using data for 449,540 SNPs, we conducted a gene by environment analysis in PLINK with inhaled corticosteroid treatment as the environmental exposure and bronchodilator response as the outcome measure. We attempted to replicate the top 12 SNPs in the Leukotriene Modifier Or Corticosteroid or Corticosteroid-Salmeterol (LOCCS) Trial.
The combined P-value for the CAMP and LOCCS populations was 4.81E-08 for rs3752120, which is located in the zinc finger protein gene ZNF432, and has unknown function.
Inhaled corticosteroids appear to modulate the association of bronchodilator response with variant(s) in the ZNF432 gene among adults and children with asthma.
Clinical Implications
Clinicians who treat asthma patients with inhaled corticosteroids should be aware that the patient's genetic makeup likely influences response as measured in lung function.
Capsule Summary
Our study suggests that inhaled corticosteroids could influence the effect of multiple SNPs associated with bronchodilator response across the genome.
PMCID: PMC3943570  PMID: 24280104
asthma; bronchodilator response; lung function; inhaled corticosteroids; single nucleotide polymorphisms; zinc finger proteins; ZNF432
5.  Genetic predictors associated with improvement of asthma symptoms in response to inhaled corticosteroids 
To date, genome-wide association studies (GWASs) of inhaled corticosteroid (ICS) response in asthmatic patients have focused primarily on lung function and exacerbations.
We hypothesized that GWAS analysis could identify novel genetic markers predicting a symptomatic response to ICSs.
We analyzed differences in asthma symptoms in response to ICSs in 124 white children from the Childhood Asthma Management Program (CAMP) trial using scores from diary cards. Of the 440,862 single nucleotide polymorphisms (SNPs) analyzed, the top 100 ranked SNPs were pursued for replication initially in subjects from the pediatric Childhood Asthma Research and Education trials (77 white children) and then in subjects from the adult Asthma Clinical Research Network (110 white adults) and Leukotriene Modifier or Corticosteroid or Corticosteroid-Salmeterol trials (110 white adults).
The lowest P value for GWAS analysis in the CAMP trial was 8.94 × 10−8 (rs2388639). Of the 60 SNPs available in the Childhood Asthma Research and Education Network trials, rs1558726 (combined P = 1.02 × 10−5), rs2388639 (combined P = 8.56 × 10−9), and rs10044254 (combined P = 9.16 × 10−8) independently replicated. However, these 3 SNPs were not additionally replicated in the adult asthmatic patients of the remaining trials. rs10044254 lies in the intronic region of F-box and leucine-rich repeat protein 7 (FBXL7) and is associated with decreased expression in immortalized B cells derived from CAMP participants.
We have identified a novel SNP, rs10044254, associated with both decreased expression of FBXL7 and improved symptomatic response to ICSs in 2 independent pediatric cohorts. Our results suggest that there might be a specific genetic mechanism regulating symptomatic response to ICSs in children that does not carry over to adults.
PMCID: PMC4112383  PMID: 24486069
Asthma; child; glucocorticoid; pharmacogenomics; polymorphism
7.  High density genotyping study identifies four new susceptibility loci for atopic dermatitis 
Nature genetics  2013;45(7):808-812.
Atopic dermatitis is a common inflammatory skin disease with a strong heritable component. Pathogenetic models consider keratinocyte differentiation defects and immune alterations as scaffolds1, and recent data indicate a role for autoreactivity in at least a subgroup of patients2. With filaggrin (FLG) a major locus causing a skin barrier deficiency was identified3. To better define risk variants and identify additional susceptibility loci, we densely genotyped 2,425 German cases and 5,449 controls using the Immunochip array, followed by replication in 7,196 cases and 15,480 controls from Germany, Ireland, Japan and China. We identified 4 new susceptibility loci for atopic dermatitis and replicated previous associations. This brings the number of atopic dermatitis risk loci reported in individuals of European ancestry to 11. We estimate that these susceptibility loci together account for 14.4% of the heritability for atopic dermatitis.
PMCID: PMC3797441  PMID: 23727859
8.  Genome-Wide Association Study for Levels of Total Serum IgE Identifies HLA-C in a Japanese Population 
PLoS ONE  2013;8(12):e80941.
Most of the previously reported loci for total immunoglobulin E (IgE) levels are related to Th2 cell-dependent pathways. We undertook a genome-wide association study (GWAS) to identify genetic loci responsible for IgE regulation. A total of 479,940 single nucleotide polymorphisms (SNPs) were tested for association with total serum IgE levels in 1180 Japanese adults. Fine-mapping with SNP imputation demonstrated 6 candidate regions: the PYHIN1/IFI16, MHC classes I and II, LEMD2, GRAMD1B, and chr13∶60576338 regions. Replication of these candidate loci in each region was assessed in 2 independent Japanese cohorts (n = 1110 and 1364, respectively). SNP rs3130941 in the HLA-C region was consistently associated with total IgE levels in 3 independent populations, and the meta-analysis yielded genome-wide significance (P = 1.07×10−10). Using our GWAS results, we also assessed the reproducibility of previously reported gene associations with total IgE levels. Nine of 32 candidate genes identified by a literature search were associated with total IgE levels after correction for multiple testing. Our findings demonstrate that SNPs in the HLA-C region are strongly associated with total serum IgE levels in the Japanese population and that some of the previously reported genetic associations are replicated across ethnic groups.
PMCID: PMC3851760  PMID: 24324648
9.  Genomewide Association between GLCCI1 and Response to Glucocorticoid Therapy in Asthma 
The New England journal of medicine  2011;365(13):1173-1183.
The response to treatment for asthma is characterized by wide interindividual variability, with a significant number of patients who have no response. We hypothesized that a genomewide association study would reveal novel pharmacogenetic determinants of the response to inhaled glucocorticoids.
We analyzed a small number of statistically powerful variants selected on the basis of a family-based screening algorithm from among 534,290 single-nucleotide polymorphisms (SNPs) to determine changes in lung function in response to inhaled glucocorticoids. A significant, replicated association was found, and we characterized its functional effects.
We identified a significant pharmacogenetic association at SNP rs37972, replicated in four independent populations totaling 935 persons (P = 0.0007), which maps to the glucocorticoid-induced transcript 1 gene (GLCCI1) and is in complete linkage disequilibrium (i.e., perfectly correlated) with rs37973. Both rs37972 and rs37973 are associated with decrements in GLCCI1 expression. In isolated cell systems, the rs37973 variant is associated with significantly decreased luciferase reporter activity. Pooled data from treatment trials indicate reduced lung function in response to inhaled glucocorticoids in subjects with the variant allele (P = 0.0007 for pooled data). Overall, the mean (± SE) increase in forced expiratory volume in 1 second in the treated subjects who were homozygous for the mutant rs37973 allele was only about one third of that seen in similarly treated subjects who were homozygous for the wild-type allele (3.2 ± 1.6% vs. 9.4 ± 1.1%), and their risk of a poor response was significantly higher (odds ratio, 2.36; 95% confidence interval, 1.27 to 4.41), with genotype accounting for about 6.6% of overall inhaled glucocorticoid response variability.
A functional GLCCI1 variant is associated with substantial decrements in the response to inhaled glucocorticoids in patients with asthma. (Funded by the National Institutes of Health and others; number, NCT00000575.)
PMCID: PMC3667396  PMID: 21991891
10.  Integration of Mouse and Human Genome-Wide Association Data Identifies KCNIP4 as an Asthma Gene 
PLoS ONE  2013;8(2):e56179.
Asthma is a common chronic respiratory disease characterized by airway hyperresponsiveness (AHR). The genetics of asthma have been widely studied in mouse and human, and homologous genomic regions have been associated with mouse AHR and human asthma-related phenotypes. Our goal was to identify asthma-related genes by integrating AHR associations in mouse with human genome-wide association study (GWAS) data. We used Efficient Mixed Model Association (EMMA) analysis to conduct a GWAS of baseline AHR measures from males and females of 31 mouse strains. Genes near or containing SNPs with EMMA p-values <0.001 were selected for further study in human GWAS. The results of the previously reported EVE consortium asthma GWAS meta-analysis consisting of 12,958 diverse North American subjects from 9 study centers were used to select a subset of homologous genes with evidence of association with asthma in humans. Following validation attempts in three human asthma GWAS (i.e., Sepracor/LOCCS/LODO/Illumina, GABRIEL, DAG) and two human AHR GWAS (i.e., SHARP, DAG), the Kv channel interacting protein 4 (KCNIP4) gene was identified as nominally associated with both asthma and AHR at a gene- and SNP-level. In EVE, the smallest KCNIP4 association was at rs6833065 (P-value 2.9e-04), while the strongest associations for Sepracor/LOCCS/LODO/Illumina, GABRIEL, DAG were 1.5e-03, 1.0e-03, 3.1e-03 at rs7664617, rs4697177, rs4696975, respectively. At a SNP level, the strongest association across all asthma GWAS was at rs4697177 (P-value 1.1e-04). The smallest P-values for association with AHR were 2.3e-03 at rs11947661 in SHARP and 2.1e-03 at rs402802 in DAG. Functional studies are required to validate the potential involvement of KCNIP4 in modulating asthma susceptibility and/or AHR. Our results suggest that a useful approach to identify genes associated with human asthma is to leverage mouse AHR association data.
PMCID: PMC3572953  PMID: 23457522
11.  Haplotypes with Copy Number and Single Nucleotide Polymorphisms in CYP2A6 Locus Are Associated with Smoking Quantity in a Japanese Population 
PLoS ONE  2012;7(9):e44507.
Smoking is a major public health problem, but the genetic factors associated with smoking behaviors are not fully elucidated. Here, we have conducted an integrated genome-wide association study to identify common copy number polymorphisms (CNPs) and single nucleotide polymorphisms (SNPs) associated with the number of cigarettes smoked per day (CPD) in Japanese smokers ( = 17,158). Our analysis identified a common CNP with a strong effect on CPD (rs8102683; ) in the 19q13 region, encompassing the CYP2A6 locus. After adjustment for the associated CNP, we found an additional associated SNP (rs11878604; ) located 30 kb downstream of the CYP2A6 gene. Imputation of the CYP2A6 locus revealed that haplotypes underlying the CNP and the SNP corresponded to classical, functional alleles of CYP2A6 gene that regulate nicotine metabolism and explained 2% of the phenotypic variance of CPD (ANOVA -test ). These haplotypes were also associated with smoking-related diseases, including lung cancer, chronic obstructive pulmonary disease and arteriosclerosis obliterans.
PMCID: PMC3458030  PMID: 23049750
12.  Genome-Wide Association Analysis in Asthma Subjects Identifies SPATS2L as a Novel Bronchodilator Response Gene 
PLoS Genetics  2012;8(7):e1002824.
Bronchodilator response (BDR) is an important asthma phenotype that measures reversibility of airway obstruction by comparing lung function (i.e. FEV1) before and after the administration of a short-acting β2-agonist, the most common rescue medications used for the treatment of asthma. BDR also serves as a test of β2-agonist efficacy. BDR is a complex trait that is partly under genetic control. A genome-wide association study (GWAS) of BDR, quantified as percent change in baseline FEV1 after administration of a β2-agonist, was performed with 1,644 non-Hispanic white asthmatic subjects from six drug clinical trials: CAMP, LOCCS, LODO, a medication trial conducted by Sepracor, CARE, and ACRN. Data for 469,884 single-nucleotide polymorphisms (SNPs) were used to measure the association of SNPs with BDR using a linear regression model, while adjusting for age, sex, and height. Replication of primary P-values was attempted in 501 white subjects from SARP and 550 white subjects from DAG. Experimental evidence supporting the top gene was obtained via siRNA knockdown and Western blotting analyses. The lowest overall combined P-value was 9.7E-07 for SNP rs295137, near the SPATS2L gene. Among subjects in the primary analysis, those with rs295137 TT genotype had a median BDR of 16.0 (IQR = [6.2, 32.4]), while those with CC or TC genotypes had a median BDR of 10.9 (IQR = [5.0, 22.2]). SPATS2L mRNA knockdown resulted in increased β2-adrenergic receptor levels. Our results suggest that SPATS2L may be an important regulator of β2-adrenergic receptor down-regulation and that there is promise in gaining a better understanding of the biological mechanisms of differential response to β2-agonists through GWAS.
Author Summary
Bronchodilator response (BDR) is an important asthma phenotype that measures reversibility of airway obstruction by comparing lung function before and after the administration of short-acting β2-agonists, common medications used for asthma treatment. We performed a genome-wide association study of BDR with 1,644 white asthmatic subjects from six drug clinical trials and attempted to replicate these findings in 1,051 white subjects from two independent cohorts. The most significant associated variant was near the SPATS2L gene. We knocked down SPATS2L mRNA in human airway smooth muscle cells and found that β2-adrenergic receptor levels increased, suggesting that SPATS2L may be a regulator of BDR. Our results highlight the promise of pursuing GWAS results that do not necessarily reach genome-wide significance and are an example of how results from pharmacogenetic GWAS can be studied functionally.
PMCID: PMC3390407  PMID: 22792082
13.  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 β2-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.
PMCID: PMC3159073  PMID: 20656951
asthma; TSLP; bronchial epithelial cells; combination therapy; genetic polymorphisms
14.  82 Association of Matrix Metalloproteinase-7 and -12 Genes polymorphisms With Asthma: A Case-Control Study of MMP-7 and -12 in a Japanese Population 
Genetic variants influencing lung function or immune system may be involved in the development of asthma and/or its symptoms. Matrix metalloproteinases (MMPs) contribute to both normal and pathological tissue remodeling and also act as regulatory molecules by processing cytokines or adhesion molecules. In animal models, growing evidences suggest that MMPs play important roles in asthma phenotypes. Some MMP genes (e.g. MMP-9 and MMP-12) have recently been shown to be associated with asthma in Caucasian populations. We investigated whether single nucleotide polymorphisms (SNPs) in MMP-7 and MMP-12 could affect the susceptibility to and clinical phenotypes of asthma in the Japanese population.
We conducted a case-control study between SNPs in MMP-7 and MMP-12 genes and asthma-related phenotypes using childhood and adult Japanese populations (653 childhood asthma patients and 423 controls, and 428 adult asthma patients and 646 controls, respectively). To investigate the effects of amino acid substitutions by SNPs on MMPs' enzymatic activity, MMP activity assays were performed using commercially available kits based on fluorescence resonance energy transfer (FRET) peptide. We also evaluated the effect of 3’UTR SNP in MMP-7 on its mRNA stability and the effect of SNP in MMP-12 on its antimicrobial activity.
We found that, in the Japanese population, SNPs of MMP-7 (rs10502001, G/A, Arg77His; rs14983, C/T, 3’UTR) (P = 0.006; odds ratio (OR), 1.46; 95% confidential interval (CI), 1.126-1.903) and MMP-12 (rs652438, A/G, Asn357Ser) (P = 0.015; OR, 1.60; 95% CI, 1.002-2.556) showed significant association with adult and childhood asthma, respectively. We also found that the SNP (rs652438) in MMP-12 was associated with severity in adult asthma (P = 0.010). Using supernatant from cultured HEK293 cells stably transfected with the pcDNA3.1(+)-MMP-7 or MMP-12 as MMP proteins, we evaluated activation kinetics, rate of proteolytic cleavage of FRET peptide, Michaelis constant, and substrate specificity of the enzyme. In this system, we couldn't detect the functional effects of amino acid substitutions by SNPs on the enzymatic activity.
Our association study suggested that genetic variants of MMP7 and MMP12 conferred risk for development of asthma in the Japanese population.
PMCID: PMC3512818
15.  ORAI1 Genetic Polymorphisms Associated with the Susceptibility of Atopic Dermatitis in Japanese and Taiwanese Populations 
PLoS ONE  2012;7(1):e29387.
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.
PMCID: PMC3258251  PMID: 22253717
16.  Variants of C-C Motif Chemokine 22 (CCL22) Are Associated with Susceptibility to Atopic Dermatitis: Case-Control Studies 
PLoS ONE  2011;6(11):e26987.
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 (1st population, 916 cases and 1,032 controls; 2nd 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.
PMCID: PMC3219642  PMID: 22125604
17.  Genome-Wide Association Study Identifies HLA-DP as a Susceptibility Gene for Pediatric Asthma in Asian Populations 
PLoS Genetics  2011;7(7):e1002170.
Asthma is a complex phenotype influenced by genetic and environmental factors. We conducted a genome-wide association study (GWAS) with 938 Japanese pediatric asthma patients and 2,376 controls. Single-nucleotide polymorphisms (SNPs) showing strong associations (P<1×10−8) in GWAS were further genotyped in an independent Japanese samples (818 cases and 1,032 controls) and in Korean samples (835 cases and 421 controls). SNP rs987870, located between HLA-DPA1 and HLA-DPB1, was consistently associated with pediatric asthma in 3 independent populations (Pcombined = 2.3×10−10, odds ratio [OR] = 1.40). HLA-DP allele analysis showed that DPA1*0201 and DPB1*0901, which were in strong linkage disequilibrium, were strongly associated with pediatric asthma (DPA1*0201: P = 5.5×10−10, OR = 1.52, and DPB1*0901: P = 2.0×10−7, OR = 1.49). Our findings show that genetic variants in the HLA-DP locus are associated with the risk of pediatric asthma in Asian populations.
Author Summary
Asthma is the most common chronic disorder in children, and asthma exacerbation is an important cause of childhood morbidity and hospitalization. Here, taking advantage of recent technological advances in human genetics, we performed a genome-wide association study and follow-up validation studies to identify genetic variants for asthma. By examining 6,428 Asians, we found rs987870 and HLA-DPA1*0201/DPB1*0901 were associated with pediatric asthma. The association signal was stretched in the region of HLA-DPB2, collagen, type XI, alpha 2 (COL11A2), and Retinoid X receptor beta (RXRB), but strong linkage disequilibrium in this region made it difficult to specifically identify causative variants. Interestingly, the SNP (or the HLA-DP allele) associated with pediatric asthma (Th-2 type immune diseases) in the present study confers protection against Th-1 type immune diseases, such as type 1 diabetes and rheumatoid arthritis. Therefore, the association results obtained in the present study could partially explain the inverse relationship between asthma and Th-1 type immune diseases and may lead to better understanding of Th-1/Th-2 immune diseases.
PMCID: PMC3140987  PMID: 21814517
18.  An interaction between Nrf2 polymorphisms and smoking status affects annual decline in FEV1: a longitudinal retrospective cohort study 
BMC Medical Genetics  2011;12:97.
An Nrf2-dependent response is a central protective mechanism against oxidative stress. We propose that particular genetic variants of the Nrf2 gene may be associated with a rapid forced expiratory volume in one second (FEV1) decline induced by cigarette smoking.
We conducted a retrospective cohort study of 915 Japanese from a general population. Values of annual decline in FEV1 were computed for each individual using a linear mixed-effect model. Multiple clinical characteristics were assessed to identify associations with annual FEV1 decline. Tag single-nucleotide polymorphisms (SNPs) in the Nrf2 gene (rs2001350, rs6726395, rs1962142, rs2364722) and one functional SNP (rs6721961) in the Nrf2 promoter region were genotyped to assess interactions between the Nrf2 polymorphisms and smoking status on annual FEV1 decline.
Annual FEV1 decline was associated with smoking behavior and inversely correlated with FEV1/FVC and FEV1 % predicted. The mean annual FEV1 declines in individuals with rs6726395 G/G, G/A, or A/A were 26.2, 22.3, and 20.8 mL/year, respectively, and differences in these means were statistically significant (pcorr = 0.016). We also found a significant interaction between rs6726395 genotype and smoking status on the FEV1 decline (p for interaction = 0.011). The haplotype rs2001350T/rs6726395A/rs1962142A/rs2364722A/rs6721961T was associated with lower annual decline in FEV1 (p = 0.004).
This study indicated that an Nrf2-dependent response to exogenous stimuli may affect annual FEV1 decline in the general population. It appears that the genetic influence of Nrf2 is modified by smoking status, suggesting the presence of a gene-environment interaction in accelerated decline in FEV1.
PMCID: PMC3160350  PMID: 21774808
19.  Identification of Nine Novel Loci Associated with White Blood Cell Subtypes in a Japanese Population 
PLoS Genetics  2011;7(6):e1002067.
White blood cells (WBCs) mediate immune systems and consist of various subtypes with distinct roles. Elucidation of the mechanism that regulates the counts of the WBC subtypes would provide useful insights into both the etiology of the immune system and disease pathogenesis. In this study, we report results of genome-wide association studies (GWAS) and a replication study for the counts of the 5 main WBC subtypes (neutrophils, lymphocytes, monocytes, basophils, and eosinophils) using 14,792 Japanese subjects enrolled in the BioBank Japan Project. We identified 12 significantly associated loci that satisfied the genome-wide significance threshold of P<5.0×10−8, of which 9 loci were novel (the CDK6 locus for the neutrophil count; the ITGA4, MLZE, STXBP6 loci, and the MHC region for the monocyte count; the SLC45A3-NUCKS1, GATA2, NAALAD2, ERG loci for the basophil count). We further evaluated associations in the identified loci using 15,600 subjects from Caucasian populations. These WBC subtype-related loci demonstrated a variety of patterns of pleiotropic associations within the WBC subtypes, or with total WBC count, platelet count, or red blood cell-related traits (n = 30,454), which suggests unique and common functional roles of these loci in the processes of hematopoiesis. This study should contribute to the understanding of the genetic backgrounds of the WBC subtypes and hematological traits.
Author Summary
White blood cells (WBCs) are blood cells that mediate immune systems and defend the body against foreign microorganisms. It is well known that WBCs consist of various subtypes of cells with distinct roles, although the genetic background of each of the WBC subtypes has yet to be examined. In this study, we report genome-wide association studies (GWAS) for the 5 main WBC subtypes (neutrophils, lymphocytes, monocytes, basophils, and eosinophils) using 14,792 Japanese subjects. We identified 12 significantly associated genetic loci, and 9 of them were novel. Evaluation of the associations of these identified loci in cohorts of Caucasian populations demonstrated both ethnically common and divergent genetic backgrounds of the WBC subtypes. These loci also indicated a variety of patterns of pleiotropic associations within the hematological traits, including the other WBC subtypes, total WBC count, platelet count, or red blood cell-related traits, which suggests unique and common functional roles of these loci in the processes of hematopoiesis.
PMCID: PMC3128095  PMID: 21738478
20.  Lower FEV1 in non-COPD, nonasthmatic subjects: association with smoking, annual decline in FEV1, total IgE levels, and TSLP genotypes 
Few studies have investigated the significance of decreased FEV1 in non-COPD, nonasthmatic healthy subjects. We hypothesized that a lower FEV1 in these subjects is a potential marker of an increased susceptibility to obstructive lung disease such as asthma and COPD. This was a cross-sectional analysis of 1505 Japanese adults. We divided the population of healthy adults with no respiratory diseases whose FEV1/FVC ratio was ≥70% (n = 1369) into 2 groups according to their prebronchodilator FEV1 (% predicted) measurements: <80% (n = 217) and ≥80% (n = 1152). We compared clinical data – including gender, age, smoking habits, total IgE levels, and annual decline of FEV1 – between these 2 groups. In addition, as our group recently found that TSLP variants are associated with asthma and reduced lung function, we assessed whether TSLP single nucleotide polymorphisms (SNPs) were associated with baseline lung function in non-COPD, nonasthmatic healthy subjects (n = 1368). Although about half of the subjects with lower FEV1 had never smoked, smoking was the main risk factor for the decreased FEV1 in non-COPD, nonasthmatic subjects. However, the subjects with lower FEV1 had a significantly higher annual decline in FEV1 independent of smoking status. Airflow obstruction was associated with increased levels of total serum IgE (P = 0.029) and with 2 functional TSLP SNPs (corrected P = 0.027–0.058 for FEV1% predicted, corrected P = 0.015–0.033 for FEV1/FVC). This study highlights the importance of early recognition of a decreased FEV1 in healthy subjects without evident pulmonary diseases because it predicts a rapid decline in FEV1 irrespective of smoking status. Our series of studies identified TSLP variants as a potential susceptibility locus to asthma and to lower lung function in non-COPD, nonasthmatic healthy subjects, which may support the contention that genetic determinants of lung function influence susceptibility to asthma.
PMCID: PMC3064418  PMID: 21468164
airflow obstruction; asthma; chronic obstructive pulmonary disease; pulmonary function test; thymic stromal lymphopoietin
21.  Large scale genotyping study for asthma in the Japanese population 
BMC Research Notes  2009;2:54.
Asthma is a complex phenotype that is influenced by both genetic and environmental factors. Genome-wide linkage and association studies have been performed to identify susceptibility genes for asthma. These studies identified new genes and pathways implicated in this disease, many of which were previously unknown.
To perform a large-scale genotyping study to identify asthma-susceptibility genes in the Japanese population.
We performed a large-scale, three-stage association study on 288 atopic asthmatics and 1032 controls, by using multiplex PCR-Invader assay methods at 82,935 single nucleotide polymorphisms (SNPs) (1st stage). SNPs that were strongly associated with asthma were further genotyped in samples from asthmatic families (216 families, 762 members, 2nd stage), 541 independent patients, and 744 controls (3rd stage).
SNPs located in the 5' region of PEX19 (rs2820421) were significantly associated with P < 0.05 through the 1st to the 3rd stage analyses; however, the P values did not reach statistically significant levels (combined, P = 3.8 × 10-5; statistically significant levels with Bonferroni correction, P = 6.57 × 10-7). SNPs on HPCAL1 (rs3771140) and on IL18R1 (rs3213733) were associated with asthma in the 1st and 2nd stage analyses, but the associations were not observed in the 3rd stage analysis.
No association attained genome-wide significance, but several loci for possible association emerged. Future studies are required to validate these results for the prevention and treatment of asthma.
PMCID: PMC2674055  PMID: 19335888
22.  Single nucleotide polymorphism-based genome-wide linkage analysis in Japanese atopic dermatitis families 
BMC Dermatology  2007;7:5.
Atopic dermatitis develops as a result of complex interactions between several genetic and environmental factors. To date, 4 genome-wide linkage studies of atopic dermatitis have been performed in Caucasian populations, however, similar studies have not been done in Asian populations. The aim of this study was to identify chromosome regions linked to atopic dermatitis in a Japanese population.
We used a high-density, single nucleotide polymorphism genotyping assay, the Illumina BeadArray Linkage Mapping Panel (version 4) comprising 5,861 single nucleotide polymorphisms, to perform a genome-wide linkage analysis of 77 Japanese families with 111 affected sib-pairs with atopic dermatitis.
We found suggestive evidence for linkage with 15q21 (LOD = 2.01, NPL = 2.87, P = .0012) and weak linkage to 1q24 (LOD = 1.26, NPL = 2.44, P = .008).
We report the first genome-wide linkage study of atopic dermatitis in an Asian population, and novel loci on chromosomes 15q21 and 1q24 linked to atopic dermatitis. Identification of novel causative genes for atopic dermatitis will advance our understanding of the pathogenesis of atopic dermatitis.
PMCID: PMC2082241  PMID: 17900373

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