Skip Navigation


Human Molecular Genetics Advance Access originally published online on September 2, 2004
Human Molecular Genetics 2004 13(21):2691-2697; doi:10.1093/hmg/ddh279
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
13/21/2691    most recent
ddh279v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (33)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Huang, J.-L.
Right arrow Articles by Huang, S.-K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huang, J.-L.
Right arrow Articles by Huang, S.-K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics, Vol. 13, No. 21 © Oxford University Press 2004; all rights reserved

Sequence variants of the gene encoding chemoattractant receptor expressed on Th2 cells (CRTH2) are associated with asthma and differentially influence mRNA stability

Jing-Long Huang1,{dagger}, Pei-Song Gao2,{dagger}, Rasika A. Mathias3,4,{dagger}, Tsung-Chieh Yao1, Li-Chen Chen1, Ming-Ling Kuo5, Shih-Chang Hsu2, Beverly Plunkett2, Alkis Togias2, Kathleen C. Barnes2, Cristiana Stellato2, Terri H. Beaty3 and Shau-Ku Huang2,*

1The Division of Allergy, Asthma and Rheumatology, Department of Pediatrics, Chang Gung Children's Hospital, 2Johns Hopkins Asthma and Allergy Center, Johns Hopkins University School of Medicine, 3Department of Epidemiology, Johns Hopkins University Bloomberg School of Public Health, 4Inherited Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Baltimore, MD, USA and 5The Department of Microbiology and Immunology, Graduate Institute of Basic Sciences, Chang Gung University, Taoyuan, Taiwan

Received April 12, 2004; Accepted August 23, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The gene, CRTH2, encoding a receptor for prostaglandin D2 (PGD2), is located within the peak linkage region for asthma on chromosome (Chr.) 11q reported in African American families. Family-based analysis of asthma and two common SNPs [G1544C and G1651A (rs545659)] in the 3'-untranslated region of CRTH2 showed significant evidence of linkage in the presence of disequilibrium for the 1651G allele (P=0.003) of SNP rs545659. Haplotype analysis yielded additional evidence of linkage disequilibrium for the 1544G–1651G haplotype (P<0.001). Population-based case–control analyses were conducted in two independent populations, and demonstrated significant association of the 1544G–1651G haplotype with asthma in an African American population (P=0.004), and in a population of Chinese children (P<0.001). Moreover, in the Chinese children the frequency of the 1651G allele in near-fatal asthmatics was significantly higher than mild-to-moderate asthmatics (P=0.001) and normal controls (P<0.001). The 1651G allele of SNP re545659 was also associated with a higher degree of bronchial hyperresponsiveness (P<0.027). Transcriptional pulsing experiments showed that the 1544G–1651G haplotype confers a significantly higher level of reporter mRNA stability, when compared with a non-transmitted haplotype (1544C–1651A), suggesting that the CRTH2 gene on Chr. 11q is a strong candidate gene for asthma.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Susceptibility genes for asthma, a chronic and often debilitating inflammatory disease, remain to be defined. Genetic mapping and candidate gene analyses have revealed suggestive evidence for linkage to a number of different chromosomal regions and for association with several candidate genes (reviewed in 1). Evidence for a genetic contribution to risk for fatal or near-fatal asthma has also been suggested (2,3), but the genetic basis is still unclear. These results have been generated mostly from studies of Caucasian populations. Previously, a genome-wide scan and fine-mapping analyses provided evidence of linkage to chromosome (Chr.) 11q in African Americans, but not in Caucasian Americans, with a peak NPL score of 4.38 at marker D11S1337 (4,5). This peak linkage region, spanning ~5 Mb between the peak marker (D11S1337) and a centrometric marker (D11S1313), is known to be gene rich and includes the CRTH2 gene.

The human CRTH2 gene encodes a G protein-coupled chemoattractant receptor selectively expressed on Th2 cells, basophils and eosinophils (6–8), and is a novel receptor for prostaglandin (PGD2), an important mediator in the regulation of inflammatory responses. In addition, CRTH2 has been implicated in the regulation of allergic inflammation (9). To examine whether sequence variation in the CRTH2 gene confers risk for developing asthma, family- and population-based association analyses were performed in 91 African American families (51 multiplex families and 40 asthmatic case–parent trios), from which the original evidence of linkage was generated (4), in African American control subjects and in a population of Chinese children. In this report, we provide evidence for the importance of CRTH2 in influencing risk, and that the sequence variants in the 3'-untranslated region (3'-UTR) region of the CRTH2 gene may underlie the association with the expression and severity of asthma.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although initial sequencing analysis for SNP discovery found no variants in the coding region sequence of the CRTH2 gene, two common SNPs [a G to C and a G to A substitution at positions 1544 and 1651 (based on GenBank sequence NM_004778), respectively (10)] were identified in the 3'-UTR region. SNP G1651A was identical to a variant previously found in silico and deposited in NCBI SNP database (dbSNP ID: rs545659), whereas SNP G1544C has not been reported. Two additional SNPs at very low frequency were identified in the intron region, which have not been reported in the SNP database (T179G and C197A, respectively; based on GenBank sequence AF118265). The two common SNPs in the 3'-UTR region were, therefore, chosen for subsequent genetic and functional analyses. Using genotype information on 92 unrelated founders in the African American family data, D' (11) was 0.591 between the 1544 and 1651 (rs545659) SNPs, indicating significant linkage disequilibrium (LD) between these two SNPs.

Family-based association was first conducted on all trios derived from 91 African American families (51 multiplex families and 40 trios) for each of the CRTH2 SNPs using both conventional transmission disequilibrium test (TDT) and the family-based association test (FBAT) statistics (Table 1). The results showed that the 1651G allele of SNP rs545659 gave the strongest evidence against the null hypothesis of no linkage or no disequilibrium (P=0.003), although significant evidence was also observed for the 1544G (P=0.005). The strongest evidence, however, was observed for the haplotype G–G across both SNPs (P<0.001 with the TDT). Results obtained from allelic tests using all possible trios were consistent with FBAT, which considers dependence among family members (when there are multiple affected in a family) and is the appropriate test of association in a region with prior demonstrated linkage, although as expected, the latter were more conservative.


View this table:
[in this window]
[in a new window]
  		Table 1. Analysis of linkage in the presence of disequilibrium among African American families
 
To further investigate the genetic association of those two CRTH2 variants with risk of asthma, a case–control analysis was performed using 82 unrelated individuals (asthmatic probands) chosen from the African American families and 78 normal African American volunteers. Allele and genotype frequencies of both SNPs in asthmatic cases and control subjects are shown in Table 2. The genotype distributions for the two polymorphisms tested were found to be in Hardy–Weinberg equilibrium. Although the frequency of the 1544G allele was similar in case and control subjects (78.7 and 78.8%, respectively), the frequencies of the 1651G allele of SNP rs545659 and the homozygous GG genotype were significantly higher among asthmatic cases compared with controls [odds ratio (OR): 2.529, P=0.005 and OR: 2.680, P=0.008 for G allele and GG genotype, respectively]. Further, in the population of Chinese children, the frequencies of the 1651G allele and 1651 GG genotype of SNP 545659 were also significantly associated with asthma compared with non-asthmatic–non-atopic controls (Table 2; OR: 3.690, P<0.001 and OR: 4.057, P<0.001, respectively), whereas suggestive evidence was obtained for the SNP 1544C allele (P=0.043).


View this table:
[in this window]
[in a new window]
  		Table 2. Association analysis in the African American and Chinese case–control data
 
Haplotypes were constructed for both cases and unrelated controls. As shown in Table 2, the ‘omnibus test’ to detect overall differences in haplotype frequency profiles between the case and control groups was statistically significant (P=0.006) in African Americans. The results also showed that the 1544G–1651G haplotype was significantly higher among cases than controls (P=0.004). In contrast, the 1544G–1651A haplotype was more frequent among controls (P=0.001). Significantly, the frequency of the 1544G–1651G haplotype was also significantly higher in cases than controls (P<0.001) among Chinese children. In contrast, the 1544G–1651A haplotype is more frequent in the controls (P<0.001). These results suggest, therefore, that 1651G allele of SNP rs545659 may contribute to the risk of asthma in these two populations.

When asthma severity was analyzed, the frequencies of the 1651G allele of SNP rs545659 in near-fatal (90.9%; P<0.001) and mild-to-moderate asthmatics (73.6%; P<0.001) were both significantly higher than in non-asthmatic–non-atopic controls (50.9%; Table 3), whereas the frequency of the 1651G allele of SNP rs545659 in the near-fatal asthma group was significantly higher than that found among mild-to-moderate asthma group (90.9% versus 73.6%, P=0.001). It is also noted that the 1544C allele is relatively more frequent in mild-to-moderate asthmatics (10.4%), but not in near-fatal asthmatics, than in control subjects (3.4%, P=0.030; Table 3).


View this table:
[in this window]
[in a new window]
  		Table 3. Allele frequencies for CRTH2 polymorphisms in children with varying asthma severity
 
We also investigated the impact of these sequence variants on several asthma phenotypes for asthmatic children where relevant data were available. A significantly lower PC20 was seen among carriers of the 1651G allele of SNP rs545659 compared with those homozygous for the 1651A allele (2.63±2.74 versus 0.84±1.05 mg/ml, P=0.027; Table 4). No significant difference in the allele frequency of SNP 1651G was found when the total serum IgE, specific IgE levels and blood eosinophil counts were analyzed (Table 4).


View this table:
[in this window]
[in a new window]
  		Table 4. Association of SNP 1651GG genotype with blood eosinophil counts, atopy, total serum IgE levels, and bronchial hyperresponsiveness
 
Regulation of mRNA turnover by the 3'-UTR sequences is now recognized as an important step for controlling the fate of cytoplasmic mRNA and consequently gene expression. To examine a potential functional difference of 3'-UTR sequence variants of the CRTH2 gene, a recently developed ‘transcriptional pulsing’ strategy (12) was used, in which a Tet-regulated promoter system was included to achieve a transient burst of reporter gene transcription in NIH3T3 B2A2 cells constitutively expressing Tet-controlled transactivator (tTA). The decay of reporter transcript in the absence or presence of 3'-UTR variant sequences was then kinetically measured in three duplicated experiments and compared after the addition of Tet to stop the transcription.

The results demonstrated a relatively faster decay rate for a reporter construct containing the 1544C–1651A haplotype, and inclusion of this 1544G–1651G haplotype sequence showed a higher level of reporter mRNA stability over the course of 5 h after the addition of Tet (Fig. 1). The reporter construct without CRTH2 3'-UTR sequence variants showed similar decay kinetics to those seen for 1544G–1651G haplotype. These results suggest, therefore, that the 3'-UTR sequence of the CRTH2 gene may contain a destabilizing motif, and the 1544G–1651G haplotype could affect the mRNA stability of the transcript.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 1. Transcription pulsing analysis. (A) NIH 3T3 B2A2 cells constitutively expressing tTA were co-transfected with the construct pSV{alpha}1/GAPDH as a control and each of the reporter constructs, pTet-BBB, pTet-CRTH2GG and pTet-CRTH2CA. Twenty-four hours after transfection, the cells were cultured without Tet to induce transcription, and 3 h later treated with 500 ng/ml of Tet to stop the transcription. Total RNAs were extracted at different time intervals as indicated, subjected to northern blotting analysis as described in Materials and Methods, and the relative level of the reporter (ß-globin) transcript in each preparation was normalized to that for {alpha}-globin. (B) The graph demonstrates the mean±SEM of the values obtained from three independent experiments. The half-life of mRNA (t1/2) was calculated as the time required for a given transcript to decrease to 50% of its initial abundance. The change in mRNA half-life between ptet-CRTH2GG and ptet-CRTH2AC was significant: *P<0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The importance of CRTH2 on inflammatory cell activation and recruitment has been demonstrated (6–8,13). PGD2 selectively induces, via CRTH2, chemotactic activity in Th2 cells, eosinophils, basophils and type 2 cytotoxic CD8+ T lymphocytes (9). Moreover, significantly increased PGD2 levels have also been seen in the bronchoalveolar lavage fluid following antigen challenge in asthmatic subjects (14). In the current study, both the multiplex families and case–parent trios showed evidence for linkage and association for the 1651G allele and 1544G–1651G haplotype of the CRTH2 gene when analyzing the asthma phenotype. This allelic association was further substantiated in a case–control analysis of two independent populations. These results, together with the physical location of CRTH2 on Chr. 11q12–13, where evidence for susceptibility genes for asthma and asthma-associated traits have been demonstrated by several independent studies (4; reviewed in 1), support the concept that CRTH2 is an important candidate for influencing risk to asthma.

Although we recognize that our study is limited in sample size, it does not appear to be underpowered when testing for associations between CRTH2 and asthma. Our study has yielded significant evidence of association (P<0.001 for haplotype analysis in the Chinese case–control study). This argues against the observed association between SNPs in CRTH2 and asthma being simply a type I error despite the multiple testing done here. Between 9 (African American data) and 18 (Chinese data) tests were carried out as part of our case–control analyses. Even with the stringent Bonferroni correction, our findings would be considered statistically significant.

At this time, we cannot exclude a possibility of LD between these two CRTH2 variants and other neighboring genes without an additional fine-mapping analysis of SNPs throughout the region. However, it is noted that CRTH2 (GPR44) is the only gene with known function within a 100 kb flanking region. In addition, considering the importance of the CRTH2 gene in allergic response, the evidence for association of those CRTH2 variants with asthma and the functional difference between haplotypes showing excess transmission to asthmatic children is significant in and of itself. It is worth noting that the sequence variants have been identified in two additional candidate genes on chromosome 11, a promoter SNP (T-109C) in the FCER1B gene encoding the high affinity IgE receptor ß subunit (15), and a non-coding region SNP (G38A) (16) of the gene encoding Clara cell 10 kD protein (CC10), an immunomodulator (17), both of which have been shown to be associated with atopic phenotypes, leading to an increased risk of asthma (16,18). However, no evidence for linkage in the presence of disequilibrium was obtained for these two SNPs in these African American families (data not shown).

The mechanisms underlying the involvement of the CRTH2 gene in the pathogenesis of asthma and modulation of its severity, including near-fatal attacks, remain to be identified. In a population of Chinese children, the frequency of the 1651G allele of SNP rs545659 among near-fatal asthmatics (90.9%) was significantly higher than that for mild-to-moderate asthmatics (73.6%, OR: 3.582, P=0.001), and was also associated with a higher degree of bronchial hyperresponsiveness (P=0.027). These results suggest that CRTH2 polymorphisms may influence the severity of asthma, and may serve as a genetic risk factor for life-threatening asthma attacks in Chinese children. In addition, the results from our study could provide an important basis for identifying high-risk individuals, preferably in early childhood, when they could be targeted for more rigorous education and specific preventative measures.

Using a reporter system, we presented evidence that the 1544G–1651G haplotype confers a higher level of reporter mRNA stability, raising the possibility that higher levels of CRTH2 expression, and potentially its responsiveness to its ligand, PGD2, could be the biological basis for the observed association with asthma. It is thus tempting to speculate that 1544G–1651G haplotype sequences may be involved in posttranscriptional modification favoring a more stable transcript, through modulation of the binding of nuclear factors to these particular sequence variants or neighboring elements. At present, the nature of any putative binding factor(s) to this region remains undefined. Therefore, a better understanding of the mechanism of post-transcriptional regulation exerted by the 3'-UTR sequence variants of CRTH2 gene awaits further investigation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Study populations
The study protocols were approved by the Institution Review Board of Johns Hopkins University School of Medicine and by the Ethics Committee of Chang Gung Children's Hospital. Informed consent was obtained from each of the study subjects.

African American population.
A total of 51 multiplex African American families recruited via an asthmatic sib-pair and 40 African American asthmatic case–parent trios were included (5). The ascertainment scheme for both families recruited via an asthmatic sib-pair and asthmatic probands in case–parent trios has been described in detail elsewhere (4). In the case–control design, 82 unrelated individuals were selected from among asthmatic probands of 51 multiplex African American families and 31 case–parent trios. Control subjects were recruited from 78 normal African American volunteers (55 men, 27 women; aged 42.8±10.2) with no history of atopic diseases.

Chinese children population.
A total of 197 unrelated Chinese children (111 males and 86 females) were enrolled in the study (mean age, 7.2±3.5 years). Subjects were divided into three groups: (1) those who had experienced near-fatal asthma attacks (N=44) with a history of hospital admission requiring intubations and ventilation for acute exacerbation of asthma symptoms or had developed hypercapnic respiratory failure during an acute asthmatic episode with a PaCO2>45 mm Hg. All subjects had reversible airflow obstruction; (2) mild-to-moderate asthmatics (N=94) who had never experienced a fatal or near-fatal asthma episode, and were not taking oral steroids. The diagnosis and classification of the clinical severity of asthma was made according to the Global Initiative for Asthma (GINA) guidelines National Heart, Lung and Blood Institute/World Health Organization Workshop on the Global Strategy for Asthma), and was made according to the clinical features before treatment and lung function over a 12-month period; (3) age-matched normal controls (N=59), who were selected from children visiting the Chang Gung Children's Hospital for treatment of non-allergic and non-immunological diseases, and met the following criteria: no symptoms or history of allergic diseases; and total serum IgE levels below the general population mean for their ages. There was no significant difference in the mean ages or sex ratios among the three groups of study subjects. In addition, no significant differences were found between the near-fatal asthma and mild-to-moderate asthma groups for total serum IgE levels or the percentage of atopic subjects. Most asthmatic children were atopic, including 90.0% in the near-fatal group and 90.4% in the mild-to-moderate group.

Total and allergen-specific IgE, blood eosinophil counts and bronchial hyperresponsivenes
The levels of serum total and specific IgE to common aeroallergens were determined by the use of fluorescent enzyme immunoassay (AutoCAP System; Pharmacia Diagnostics AB, Uppsala, Sweden). Children were considered atopic if they had at least one positive result for allergen-specific IgE to aeroallergens common in Taiwan, including Dermatophagoides pteronyssinus, D. farinae, cockroach, cat and dog dander, and Candida albicans. The absolute peripheral-blood eosinophil counts were measured through the use of a Sysmex SE-9000 Automated Hematology Analyzer (TOA Medical Electronics Co., Kobe, Japan).

Bronchial challenge tests were performed as previously described (12). Methacholine was administered using an aerosol provocation system, with a fixed output (APS Jaeger) in escalating doses (0.075, 0.156, 0.312, 0.625, 1.25, 2.5, 5.0, 10.0 and 25.0 mg/ml). Lung function was measured 1 min after each dose at an interval of 5 min. At each dose, the subject performed five successive full-capacity inspirations, with nebulization triggered by inspiration. Each inhalation was 0.5 s in duration. An inhalation unit was defined as one inhalation of 1 mg/ml methacholine. The PC20 was interpolated, using a provocative concentration of methacholine to produce a 20% fall in FEV1 on the concentration–FEV1 response curve. The challenge was terminated prior to completion of the dosing schedule if FEV1 declined by 20% or more from the reference value after saline inhalation. To aid recovery from induced bronchoconstriction, fenoterol was administered via inhalation when necessary. The methacholine challenge was performed in asthmatic patients who had no symptoms for at least 2 weeks prior. These children had not received any anti-asthmatic medication, including oral ß-2 agonists, theophylline, steroids or antihistamines for at least 7 days, and inhaled ß-2 agonists were withheld for at least 12 h before the challenge test.

Genotyping
Genomic DNAs from African American subjects were genotyped by single-stranded conformation polymorphism (SSCP) (20) or in some cases, PCR–restriction fragment length polymorphism (PCR–RFLP) assay using the PCR primers: 5'-AGCAGTGCTTCTCAAACCTTG and 5'-CTAGAGTGGGAAGTCTATCAT. The underlined sequence was exchanged to incorporate the polymorphic site. After the PCR reaction, the amplified products were digested using restriction endonuclease BssSI (New England Biolabs, Beverly, MA, USA) for both SNPs, G1544C and G1651A (rs545659). For quality control, sequence-confirmed samples were included in each assay, and samples from these two assays were randomly selected for additional sequence confirmation. For the Chinese children population, the genomic DNAs of all 197 individuals was genotyped by direct sequencing of PCR fragments encompassing SNPs G1544C and G1651A, with primers (forward: 5'-AGCAGTGCTTCTCAAACCTTG and reverse: 5'-CACTGCCCTAGAGTGGGAAG).

Data analysis
Haplotype generation.
In addition to analyses carried out at each individual SNP, haplotypes across the two SNPs of the CRTH2 gene were also examined. Haplotypes were constructed using expectation–maximization (EM) algorithm for both family (21) and case–control (22) data. In the family study, data from all genotyped individuals were used to construct haplotypes without assuming linkage equilibrium. The algorithm determines the best solution of haplotypic states most likely for these data, and returns the most likely haplotype state based on the family as a whole. For the case–control data, group-wise haplotype frequencies were generated using the EM algorithm, and analyses were carried out as described in the following sections.

Family-based analyses.
Two tests of association were implemented: the convention TDT as first described by Spielman et al. (23) and the FBAT as described by Rabinowitz and Laird (24). Although both analyses are family-based tests of association, the TDT tests the composite null hypothesis of no linkage or no disequilibrium, and is subject to an inflated type I error rate when multiple affected offspring are used. In the situation of testing for association in a region previously yielding evidences for linkage with the trait, the simple TDT can have inflated type I errors. The FBAT statistic with the empirical variance–covariance estimator adjusts for any correlation in marker genotypes among relatives, and thus provides an unbiased test for association in the presence of linkage (25).

Case–control analyses.
In the case–control design, allele and genotype frequencies, OR and Chi-square tests were analyzed using the SPSS computer program (version 10). Haplotype frequencies and Chi-square values were calculated for this case–control analysis of each individual haplotype versus all others and empiric significance was assessed using 10 000 permutations of the observed data. The omnibus test was performed to detect differences in overall haplotype frequency profiles between asthmatics and controls (22). Total serum IgE levels, blood eosinophil counts and PC20 values between different groups were compared by means of the Mann–Whitney's U-test or the Kruskal–Wallis test, as appropriate. For all test results, P<0.05 were considered statistically significant.

Transcription pulsing experiment
The Tet-regulated reporter construct, pTet-BBB, was kindly provided by Dr Ann-Bin Shyu, University of Texas at Houston, and the protocol for transcription pulsing experiment has been described previously (12). The reporter constructs were generated by insertion of a 500 bp genomic DNA sequence containing either a transmitted (1544G–1651G) or a non-transmitted (1544C–1651A) haplotype into an unique BglII site in the 3'-UTR of the rabbit ß-globin gene (reporter) of the pTet-BBB vector. Co-transfections of tTA-expressing NIH 3T3 B2A2 cells were performed with pSV{alpha}1/GAPDH as a control and each of the reporter constructs, pTet-BBB, pTet-CRTH2GG and pTet-CRTH2CA. The cells were washed extensively 24 h after transfection and cultured for an additional 3 h in the absence of Tet, followed by the addition of 500 ng/ml of Tet to stop the transcription. Total RNAs were extracted at different time intervals, and equivalent amounts of RNA (5 µg per lane) were size-fractionated in 1% agarose gels containing 0.66 M formaldehyde, transferred onto membranes and cross-linked by exposure to UV light. The membranes were then hybridized with {alpha}- and ß-globin cDNA probes (12) using Primer-a Gene® labeling system in the presence of [{alpha}-32p]dATP. Signals were visualized and quantified with densitometric analysis (Molecular Dynamics, Sunnyvale, CA, USA). The relative level of the reporter (ß-globin) transcript from each condition was normalized to {alpha}-globin by calculating the ß-globin/{alpha}-globin ratio for each sample. The half-life of mRNA (t1/2) was calculated as the time required for a given transcript to decrease to 50% of its initial abundance. Comparison between pTet-CRTH2GG and pTet-CRTH2AC were analyzed in three independent experiments using SPSS program with independent simple t-test (SPSS Inc., Chicago, IL, USA).


   ACKNOWLEDGEMENTS
 
We thank all those who have participated in the Collaborative Studies on Genetics of Asthma (CSGA). We also thank Maria Stockton, Xiaodong Li and Eva Ehrlich for technical assistance. This work was supported, in part, by the National Science Institute, Taiwan, Grant No. NSC90-2314-B182A-163, NIH grants, HL-49612, AI-52468 and by Philip Morris USA Inc.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224-6801, USA. Tel: +1 4105502006; Fax: +1 4105502527; Email: skhuang{at}jhmi.edu

{dagger} The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Hakonarson, H. and Wjst, M. (2001) Current concepts on the genetics of asthma. Curr. Opin. Pediatr., 13, 267–277.[CrossRef][Web of Science][Medline]

  2. Sandford, A.J., Changani, T., Zhu, S., Weir, T.D., Bai, T.R. and Spinelli, J.J. (2000) Polymorphisms in the IL-4, IL-4RA and FCERIB genes and asthma severity. J. Allergy Clin. Immunol., 106, 135–140.[CrossRef][Web of Science][Medline]

  3. Yao, T.C., Kuo, M.L., See, L.C., Chen, L.C., Yan, D.C., Ou, L.S., Shaw, C.K. and Huang, J.L. (2003) RANTES promoter polymorphism: a genetic risk factor for near-fatal asthma in Chinese children. J. Allergy Clin. Immunol., 111, 1285–1292.[CrossRef][Web of Science][Medline]

  4. Xu, J., Meyers, D.A., Ober, C., Blumenthal, M.N., Mellen, B., Barnes, K.C., King, R.A., Lester, L.A., Howard, T.D., Solway, J. et al. (2001) Genomewide screen and identification of gene–gene interactions for asthma-susceptibility loci in three U.S. populations: collaborative study on the genetics of asthma. Am. J. Hum. Genet., 68, 1437–1446.[CrossRef][Web of Science][Medline]

  5. Huang, S.K., Mathias, R.A., Ehrlich, E., Plunkett, B., Liu, X., Cutting, G.R., Wang, X.J., Li, X.D., Togias, A., Barnes, K.C. et al. (2003) Evidence for asthma susceptibility genes on chromosome 11 in an African-American population. Hum. Genet., 113, 71–75.[CrossRef][Web of Science][Medline]

  6. Nagata, K., Hirai, H., Tanaka, K., Ogawa, K., Aso, T., Sugamura, K., Nakamura, M. and Takano, S. (1999) CRTH2, an orphan receptor of T-helper-2-cells, is expressed on basophils and eosinophils and responds to mast cell-derived factor(s). FEBS Lett., 459, 195–199.[CrossRef][Web of Science][Medline]

  7. Nagata, K., Tanaka, K., Ogawa, K., Kemmotsu, K., Imai, T., Yoshie, O., Abe, H., Tada, K., Nakamura, M., Sugamura, K. and Takano, S. (1999) Selective expression of a novel surface molecule by human Th2 cells in vivo. J. Immunol., 162, 1278–1286.[Abstract/Free Full Text]

  8. Cosmi, L., Annunziato, F., Galli, M.I.G., Maggi, R.M.E., Nagata, K. and Romagnani, S. (2000) CRTH2 is the most reliable marker for the detection of circulating human type 2 Th and type 2 T cytotoxic cells in health and disease. Eur. J. Immunol., 30, 2972–2979.[CrossRef][Web of Science][Medline]

  9. Hirai, H., Tanaka, K., Yoshie, O., Ogawa, K., Kenmotsu, K., Takamori, Y., Ichimasa, M., Sugamura, K., Nakamura, M., Takano, S. and Nagata, K. (2001) Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2. J. Exp. Med., 193, 255–261.[Abstract/Free Full Text]

  10. Hsu, S.C., Chen, L.C., Kuo, M.L., Huang, J.L. and Huang, S.K. (2002) Novel SNPs in a candidate gene, CRTH2, for allergic diseases. Genes Immun., 3, 114–116.[CrossRef][Web of Science][Medline]

  11. Lewontin, R.C. (1964) The interaction of selection and linkage. I. General considerations; heterotic models. Genetics, 49, 49–67.[Free Full Text]

  12. Xu, N.H., Loflin P., Chen, C.Y. and Shyu, A.B. (1998) A broader role for AU-rich element-mediated mRNA turnover revealed by a new transcriptional pulse strategy. Nucl. Acids Res., 26, 558–565.[Abstract/Free Full Text]

  13. Iwasaki, M., Nagata, K., Takano, S., Takahashi, K., Ishii, N. and Ikezawa, Z. (2002) Association of a new-type prostaglandin D2 receptor CRTH2 with circulating T helper 2 cells in patients with atopic dermatitis. J. Invest. Dermatol., 119, 609–616.[CrossRef][Web of Science][Medline]

  14. Murry, J.J., Tonnel, A.B., Brash, A.R., Roberts, L.J., Gosset, P., Workman, R., Capron, A. and Oates, J.A. (1983) Release of prostaglandin D2 into human airways during acute antigen challenge. N. Engl. J. Med., 315, 800–804.

  15. Hizawa, N., Yamaguchi, E., Jinushi, E., Konno, S., Kawakami, Y. and Nishimura, M. (2001) Increased total serum IgE levels in patients with asthma and promoter polymorphisms at CTLA4 and FCER1B. J. Allergy Clin. Immunol., 108, 74–79.[CrossRef][Web of Science][Medline]

  16. Laing, I.A., Goldblatt, J., Eber, E., Rye, P.J., Gibson, N.A., Palmer, L.J., Burton, P.R. and LeSouef, P. (1997) A common polymorphism of the CC16 gene is associated with an increased risk of asthma. Eur. Respir. J., 10, 302S.

  17. Chen, L.C., Zhang, Z., Myers, A.C. and Huang, S.K. (2001) Altered pulmonary eosinophilic inflammation in mice deficient for Clara cell secretory 10-kDa protein. J. Immunol. Cutting Edge, 267, 3025–3028.

  18. Sengler, C., Heinzmann, A., Jerkic, S.P., Haider, A., Sommerfeld, C., Niggemann, B., Lau, S., Forster, J., Schuster, A., Kamin, W. et al. (2003) Clara cell protein 16 (CC16) gene polymorphism influences the degree of airway responsiveness in asthmatic children. J. Allergy Clin. Immunol., 111, 515–519.[CrossRef][Web of Science][Medline]

  19. Hunag, J.L., Wang, S.Y. and Hsieh, K.H. (1991) Effect of short-term exposure to low levels of SO2 and NOx on pulmonary function and methacholine and allergen bronchial sensitivities in asthmatic children. Arch. Environ. Health., 46, 296–299.[Web of Science][Medline]

  20. Nickel, R.G., Casolaro, V., Wahn, U., Beyer, K., Barnes, K.C., Plunkett, B.S., Freidhoff, L.R., Sengler, C., Plitt, J.R., Schleimer, R.P. et al. (2000) Atopic dermatitis is associated with a functional mutation in the promoter of the CC chemokine RANTES. J. Immunol., 164, 1612–1616.[Abstract/Free Full Text]

  21. Rohde, K. and Fuerst, R. (2001) Haplotyping and estimation of haplotype frequencies for closely linked biallelic multilocus genetic phenotypes including nuclear family information. Hum. Mutat., 17, 289–295.[CrossRef][Web of Science][Medline]

  22. Fallin, D. and Schork, N.J. (2000) Accuracy of haplotype frequency estimation for biallelic loci, via the expectation–maximization algorithm for unphased diploid genotype data. Am. J. Hum. Genet., 67, 947–959.[CrossRef][Web of Science][Medline]

  23. Spielman, R.S., McGinnis, R.E. and Ewens, W.J. (1993) Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM). Am. J. Hum. Genet., 52, 506–516.[Web of Science][Medline]

  24. Rabinowitz, D. and Laird, N. (2000) A unified approach to adjusting association tests for population admixture with arbitrary pedigree structure and arbitrary missing marker information. Hum. Heredity, 50, 211–223.[CrossRef][Web of Science][Medline]

  25. Lake, S.L., Blacker, D. and Laird, N.M. (2000) Family-based tests of association in the presence of linkage. Am. J. Hum. Genet., 67, 1515–1525.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 BloodHome page
U. De Fanis, F. Mori, R. J. Kurnat, W. K. Lee, M. Bova, N. F. Adkinson, and V. Casolaro
GATA3 up-regulation associated with surface expression of CD294/CRTH2: a unique feature of human Th cells
Blood, May 15, 2007; 109(10): 4343 - 4350.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
K. C. Barnes, A. V. Grant, N. N. Hansel, P. Gao, and G. M. Dunston
African Americans with Asthma: Genetic Insights
Proceedings of the ATS, January 1, 2007; 4(1): 58 - 68.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
L. Schaeffer, H. Gohlke, M. Muller, I. M. Heid, L. J. Palmer, I. Kompauer, H. Demmelmair, T. Illig, B. Koletzko, and J. Heinrich
Common genetic variants of the FADS1 FADS2 gene cluster and their reconstructed haplotypes are associated with the fatty acid composition in phospholipids
Hum. Mol. Genet., June 1, 2006; 15(11): 1745 - 1756.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. N. Hata, T. P. Lybrand, and R. M. Breyer
Identification of Determinants of Ligand Binding Affinity and Selectivity in the Prostaglandin D2 Receptor CRTH2
J. Biol. Chem., September 16, 2005; 280(37): 32442 - 32451.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
N Gaukrodger, B M Mayosi, H Imrie, P Avery, M Baker, J M C Connell, H Watkins, M Farrall, and B Keavney
A rare variant of the leptin gene has large effects on blood pressure and carotid intima-medial thickness: a study of 1428 individuals in 248 families
J. Med. Genet., June 1, 2005; 42(6): 474 - 478.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
13/21/2691    most recent
ddh279v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (33)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Huang, J.-L.
Right arrow Articles by Huang, S.-K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huang, J.-L.
Right arrow Articles by Huang, S.-K.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?