A 2466bp fragment (from -2158 to +313) of the human CysLTR1 gene promoter was prepared by PCR using two haplotype forms (ht1 [C-A-A] and ht2 [T-C-G];
C-634T, A-475C, A-336G) of genomic DNA as a template, each of which was separately subcloned into an KpnI-XhoI site of the pGL3-Basic luciferase reporter vector (Promega Corp., Madison, WI, USA). A transfection experiment into Jurkat
cells (human T lymphocytes, No. 40152, KCLB, Korea) was undertaken nine times (27 in total) , A549 (human lung carcinoma cell, No. 10185, KCLB, Korea) (30 in total), and U937 cells (Human monocytes, 12 in total) .
Next, to performed luciferase activity, Jurkat cells, U937, and A549 cells were cultured in RPMI 1640 with 10% FBS (Gibco-BRL, Paisley, UK), 100units/ml penicillin, and 100ug/ml streptomycin at 37’C in a humidified atmosphere with 5%
CO2. The cells were grown in growth medium and seeded in six-well plates. At 24h after seeding, cells were harvested and washed with OPTI-MEMI reduced serum medium (Life Technology, USA). In each transfection, 2.5µg of the pGL3 constructs 1µg of pSV beta-galactosidase expression vector (Promega, Madison, WI) and 10ul lipofectamin (Life Technology, USA) were used. At 48h after transfection, cells were lyses and assayed for the firefly luciferase activity using luciferase assay system (Promega, Madison, WI) by according to the manufacturer’s instruction. Luciferase activity was normalized to beta-galactosidase activity, and the relative luciferase activity of the reporter constructs was expressed in relation to the activity of the pGL3-basic vector. Data are the mean values of three independent experiments, plus or minus the SD.
D. Nuclear extracts preparation and electrophoresis gel mobility shift assay
1. Nuclear extracts preparation
Nuclear extracts were prepared from THP-1 and U937cells that were untreated and treated with mitogens phorbol 12-myristate 13-acetate (PMA) and
10ng/ml IL-4 (Sigma Aldrich), as described by Schreiber et al (Schreiber and Schaffner, 1989) with some modification. Briefly, 5×106 cells were resuspended in 400 µl of cold hypo-osmotic buffer [10 mM Hepes /KOH, 2 mM MgCl2, 0.1mM EDTA, 10 mM KCl, 1 mM DTT (dithiothreitol), 1 mM PMSF and protease inhibitors (Roche Molecular Biochemicals), pH 7.9] and left on ice for 10 min.
Then, Nonidet P40 (0.007%) was added to the cell suspension that was mixed by vortex and centrifuged at 15000g for 30 sec. The supernatant containing the cytoplasmic proteins could be stored at −80℃. The pellets of nuclei were gently resuspended in 15 µl of cold saline buffer [50 mM Hepes/KOH, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% (v/v) glycerol, 1 mM DTT, 1 mM PMSF and protease inhibitors (Roche Molecular Biochemicals), pH 7.9] and left for 20 min on ice. After centrifugation at 15000g for 15 min, at 4℃, the supernatant containing nuclear proteins was stored at −80℃. Protein concentrations were measured with the Bradford protein assay kit from Bio-Rad.
2. Electrophretic mobility shift assay (EMSA)
To investigate whether the SNPs created nuclear transcription protein, we carried out EMSA. In brief, 5 µg of nuclear proteins was incubated for 30 min at room temperature (25℃) in a volume of 10 µl with 0.2 ng of labelled oligonucleotide probe in binding buffer [20 mM Hepes/ KOH (pH 7.9), 75 mM NaCl, 1 mM EDTA, 5%glycerol, 0.5 mM MgCl2 and 1 mM DTT] containing 2 µg of BSA and 400ng of poly (dI-dC) (Amersham Biosciences, Roosendael, The Netherlands). DNA–protein complexes were then resolved by electrophoresis on a
non-denaturing 6% (w/v) polyacrylamide gel for 2 h at 300 V in 0.5×TBE (2.5 mM Tris, 2.5 mM H3BO3 and 2 mM EDTA). The gels were then dried and auto-radio graphed on an IP-plate (Fuji photofilm Co., Ltd., Tokyo).The image produced by the FLA3000 scanner was analyzed using image-gauge ver.4 software (Fuji photofilm Co., Ltd., Tokyo). The sequences of the oligonucleotide probes were as follows: CysLTR1 C634T probe, 5’CTGCTATG TCCC[c/t]TTTCTCCAGAAG -3’; CysLTR1 A-475C probe, 5’-GAAA[a/c]AACAGTTTTAATCCAATGG-3’;CysLTR1 A-336G probe, 5’-AGAGAGAGAGAGA AAC[a/g]GGAGAGATG-3’, and NF-κB probe;GGTTACAAGGGACTTTCCGCTG-3 and 5’-TTGGCAGCGGAAAGTCCCTTGT-3’.To the determine the specificities of the DNA complexes, 1.75uM unlabeled probe was incubated with the nuclear extract for 20min before addition of the labeled probe. The oligonucleotide probes were labelled by infilling with the Klenow DNA polymerase ( Promega Corp., Madison, WI, USA).
3. Sequence mutation analysis
To determine whether these SNP of CysLTR1 created or losed a transcription factor binding site, sequences were submitted to the ‘Signal Scan’ online program (www.bimas.cit.nih.gov/molbio/signal) and compared with mammalian transcription factor binding sequences in the transcription factors (TRANSFAC) database. There was no transcription factor binding site matched on ‘Signal Scan’
online program and TRANSFAC data base.
E. CysLTR1 promoter deletion constructs and luciferase reporter assay
Region of interest were amplified from above 2466bp plasmid by PCR other primers containing consensus sequence for SacI and SmaI restriction enzymes using pfu polymerase (Solgent), under the following pairs of primers were used:
B567 construct: (C1) 5’- ACGGAGCTC TCCCCACC TACAAATAGTCTAATG-3’/(R1) CGGCCCG GGCGCTGCTAACTTCAAGGTCCA-3’; B981:(C2) 5’-ACGGAGCTCCACATACCAGACTGCTATG-3’/(R1); B1570: (C3) 5’- ACGG AGCTCACTGTAGCTCAAAATGGCTG-3’/(R1); B808: (C2)/(R2) 5’- CGGCCC GGGATTCACAGAGTGCCTGCTC-3’. PCR products were gel purified with agarose gel purification kit (iNtRON, Korea), cut with the restriction enzymes (Takara, Shuzo, Japan) and ligated into pGL3 basic vector using T4 DNA ligase (Elpis Biotech, Korea). All constructs were verified by sequencing. Plasmid DNAs were prepared from these construct using the Endo Free plasmid Maxi kit (Qiagen) and quantities and integrity by UV spectrometry and gel loading.
A549 cells were grown in growth medium and seeded in 12-well plates. In each transfection, 1µg of the plasmid constructs 0.5µg of pSV beta-galactosidase expression vector (Promega, Madison, WI) and 4ul lipofectamin (Life Technology) were used. At 48h after transfection, cells were lysed and assayed for the firefly luciferase activity (Promega Madison, WI) by according to the manufacturer’s instruction. Luciferase activity was normalized compared to beta-galactosidase activity, and the relative luciferase activity of the reporter constructs was expressed in relation to the activity of the pGL3-basic vector. Data are the mean values of
three independent experiments, plus or minus the SD.
F. Pharmacological treatments and reporter gene assay
A549 cells were cultured in RPMI 1640 medium supplemented with 10% heat -inactivated FBS (Gibco/ BRL), 100 U/ml penicillin, 25 mM Hepes, and 100 mg/ml streptomycin in a humidified atmosphere with 5% carbon dioxide (CO2) at 37°C. Cells were cultured overnight in complete media before changing to fresh serum free media containing IL-4 (10ng/ml) (Sigma-Aldrich, USA). MK-571 and dexamethasone (all from Sigma-Aldrich, USA) were dissolved in dimethylsulfoxide (DMSO) and absolute ethanol. Final concentrations of DMSO / ethanol added to cells were <0.1% and this had no effect on any of the responses (data not shown). The day before each experiment, cells were seeded into 12 well and 6 well tissue culture dishes the density of 1×105 cells. For the evaluation of dexametasone and MK-571, the cells were incubated with various concentrations of dexamethasone and MK-571 for 24hr before the stimulation by IL-4 (10ng/ml).
Transient transfections were performed using the lipofectamine reagent (Invitrogen) according to the manufacturer’s suggested protocol. To correct for the different transfection efficiencies of the various luciferase construct, the pSV beta-galactosidase expression vector plasmid (Promega, Madison, WI) was co-transfected with the CysLTR1 Promoter luciferase construct into the cells. Briefly, 1×105 cells (per wells) were seeded into 12 well tissue culture plates before the day of transfection in RPMI1640 medium plus 10% FBS. For each well 1µ g CysLTR1 promoter luciferase construct and 0.5ug pSV beta-galactosidase plasmid was
co-transfected into the cells in and 4ul lipofectamine (Gibco BRL) into the cells in 1ml serum free medium. After 5h of transfection, 1 ml medium containing 20%
FBS was added. And the cells were further incubated for a total transfection time of 48h. After at 48h, the RPMI 1640 medium was replaced. Then serum free medium, stimulant, stimulant plus drugs (dexamethasne and MK-571) were added to the transfected cells. Luciferase activity was measured using the firefly luciferase reporter assay system (Promega Madison, WI). Briefly, 100ul of lysis buffer was added to each well and plates were agitated for 15min at room temperature. 20ul of supernatant was added to 100ul luciferase assay reagent and light unit measured for 10s at three times on DLR-Ready. The promoter activity was calculated as luciferase activity / beta-galactosidase activity, and expressed as fold of increase relative to the activity of the pomoterless pGL3-basic vector.
In experiments where the effect of stimulant (IL-4, 10ng/ml), dexamethasone, and MK-571(CysLTR1 specific antagonist) on luciferase activities were studied, both the control vector-transfected (PGL3 basic vector) and promoter construct-transfected cells were treated with the corresponding drug in culture medium containing 2% FBS for the designated periods time before doing the beta-galactosidase and luciferase assay. The pharmacological reagents used in this study were purchased from the Sigma-Aldrich Corp. (Oakville, Ontario, Canada). Data are presented as the mean values of three independent experiments, plus or minus the SD.
G. CysLTR1 expression by RT-PCR, Real-time PCR and Flowcytometry
1. RT-PCR analysis for CysLTR1 mRNA
Total RNA was extracted with the use of Easy-Blue reagent (iNtRON Biotechnology, Korea) from A549 cells cultured in 6-well tissue culture plates.
The 4ug of total RNA was converted to cDNA by the reverse transcribe using the MMLV-reverse transcriptase (Promega, Madison, WI) according to the manufacturer’s suggested protocol. After incubation at 42°C for 90min, the samples were heated for 10min at 72°C to terminate the reactions and were stored at -20°C until used. PCR was performed with DNA Engine thermal cycler (MJ research, Inc., Biozym) for 32 cycles consisting of 2 min denaturation at 94°C, 30 s annealing at 55°C, and 30 s extensions at 72°C, followed by a final 10min extension at 72°C. CysLTR1 was amplified with the primers derived from the cDNA sequence for CysLTR1: 5’-CTTTGTGCTCTAT GTCCTCA-3’ as sense and 5’ CACGACCATGATCATTCCTA-3’ as antisense. Samples were subjected to parallel amplification of the constitutively expressed, housekeeping gene, β-actin using the following primers: 5’- TCCTTCTGCATCCTGTCG GCA -3’ as sense and 5’-CAAGAGATGGCCACGGC TGCT-3’as antisense. A 10ul aliquot from each PCR was allowed to migrate by electrophoresis in a 1% agarose gel. The CysLTR1 amplified fragment contained 600 bps. The gel was then colored with ethidium bromide and photographed under UV transillumination. No PCR products were obtained when reverse transcriptase was omitted, indicating that there was no DNA contamination.
2. Real-time PCR analysis for CysLTR1 mRNA
The realtime PCR were carried out on the on ABI Prism 7500 sequence detection system (Applied Biosystems), using the SYBR green dyes (Molecular probe), according to the recommendations of the vendors. The primer combination of CysLTR1 gene were as followed: 5’- TGACCGCTGCCTTTTTAGTC-3’ as sense and 5’ GAGAGGGTCAAAGCAACAATTG-3’as anti-sense, this amplified fragment contained 192bps. PCR protocol was applied: 15min hot start at 95℃, followed by 40 cycles of denaturation (30s at 94℃), annealing (30s at 60℃) and synthesis (30s at 72℃, total volume of 20ul). For melting curve analysis, the temperature was elevated slowly from 60℃ to 95℃. Data were acquired and analyzed with the ABI 7500 prism software, the amplication kinetics were recorded as sigmoid progress curves, for which the fluorescence was plotted against the number of amplication cycles. The threshold cycle number (CT) was used to define the initial amount for each template. CT was the first cycle, for which a detectable fluorescence signal was observed and relative gene expression was calculated as a fold induction compared with control.
3. CysLTR1 expression by flow cytometry
The expression of CysLTR1 in A549 cells and IL-4 primed A549 cells was assessed using a polyclonal anti-CysLTR1 antibody (Cayman) directed against the carboxyl-terminal portion of the receptor. The antibody was raised against a peptide corresponding to amino acids 318–337 of the C terminus of human CysLTR1. For flow cytometry studies, A549 cells were washed with PBS and fixed with 2% paraformaldehyde for 15 min at room temperature followed by
permeabilization with 0.1% Triton X-100 for an additional 15 min at room temperature. Cells were resuspended with PBS/2% BSA and labeled for 30 min at 4°C with anti-CysLTR1 antibody (or with control, non-pertinent Ab). Cells were then washed with cold PBS and incubated for 30 min at 4°C with FITC-conjugated goat anti-rabbit IgG (SantaCruiz). Finally, cells were washed again and resuspended in PBS before single-color immunofluorescence analysis of 10000 cells was performed on a FACS vantage (BD). A 1/1000 dilution of the anti-CysLTR1 antiserum was used in all flowcytometry studies.
H. Statistical analysis
Genotype–phenotype association between CysLTR1 SNPs and asthma-associated phenotypes was analyzed with the SPSS version 11 package (SPSS Inc., Chicago, IL, USA). The biallelic SNPs were coded into two (dominant, recessive model) or three (additive model) classes and analyzed categorically relative to the most common homozygous genotype for each SNP. Bivariate analysis used analysis of variance (ANOVA) or t-tests to compare continuous outcomes across the levels of each genotype and w2 tests and calculation of odds ratios (ORs) with 95% confidence intervals (CIs) on contingency tables when comparing genotype to categorical variables. Among control subjects, Hardy–Weinberg equilibrium was tested at each SNP locus by a w2 goodness-of-fit test. Pairwise linkage disequilibrium (LD) between SNP loci was measured using both the absolute value of Lewontin’s D0 and r2 (Hedrick PW, 1987). Haplotypes of the CysLTR1 gene were analysed using Haploview v2.0 (Barrett JC and Daly, 2005).
III. RESULTS
A. Identification of three genetic polymorphism in CysLTR1 gene
We investigated a genetic variation in 5’ upstream region of CysLTR1 gene in 43 normal healthy controls (NCs) by direct sequencing and found 3 SNPs in CysLTR1 gene promoter. The transcription start site of the human CysLTR1 transcript was determined by 5’-RACE and three polymorphic sites: C-634T, A-475C, A-336G were identified from the transcriptional start site.
Next, we investigated genetic association study of three genetic polymorphisms of CysLTR1 in three groups of study subjects classified as AIA (n=105), ATA (n=111), and NCs (n=125) by direct sequencing, in which the 2466bp promoter region of CysLTR1 gene was included. Nucleotide position on (+1) is transcription start site determined by 5’-RACE and the sequence is shown in Fig. 2. A graphic overview of 3 SNPs identified in relation to the exon / intron structure of human CysLTR1 gene is shown in Fig. 3A. The 3 SNPs were in strong linkage disequilibrium (LD) with each other and the linkage disequilibrium coefficients (1D’1) and r2 among the SNPs were calculatedfor each SNP (Fig. 3B).
B. Genetic association study of CysLTR1 genetic polymorphism
1. Clinical characteristics of the study subjects
One hundred and five ASA-intolerant asthmatics, 111 ASA-tolerant asthmatics and 125 normal healthy control subjects were genotyped at the three CysLTR1 SNPs,
1 CATTTGGGAA TGGGTGAATC ACTGCCTTCC CCATTGCTCA AGAACAGAAA ATTAGCTAAT 61 GAAAGAAACA GGTAGGGGAG CACAAAGAAG AACCACAGGA AACACCCTCT CTGCCACAGC 121 TATTTCGGTG CCAACTTGAA GCTCAAGGAT TGAAGGGGGC CTGCCCCTCC ACGCCTGTGG 181 GTATTTCTCA TCAGGTGGTA CGAGAGACTG AGAAAAGAAA TGACACAGAG ACAAAGTATA 241 GAGAAAGAAC AGTGGGCCCA GGGGACCGGC AGCCTCAGCA TGTGAGGACC TGCACCAGCG 301 CTGGTCTCTG AGTTCCCTCA GTATTTATTG ATCATTATTT TTACTATCTT GGCAAGGGGA 361 GTGTAGCAGA GCAACAGGTG GGGAGAAGGT CAGCAGGAAA ACGTGAGCAA AGGATCTGTA 421 TCATGCATAA ATTCAAGGAA AGGTACTGTG CCTGGATGTG CATGTAGGCC AGATTTATGT 481 TTCACTTTAT ACAAACACCT CAGTGTAGCA AAGAGTAACA GAGCAGTATT TCTGCCAGCA 541 TATCTCGCCT CTAGCCACAG GGCGGTTTTA TCCTATCTCA GAATAGAAAG AATGGGAATG 601 GTTGGCTTTA CAGGAGACAT TCCATTCCCA GGGAGGAGCA GGAGACAGAA GCCTTCCTCT 661 TATCTCAACT GCAAAGAGAC CTCCCTCTTT CACTACTCCT CCTCAGCAGA GACCCTTTAC 721 GGGTGTTGTG CTGGGGTATG GTATCAAAGC AGAAACAATT TTTCCTGGTA CAGATCAAAA 781 TGGAATTTCT TGTGTCTTCC TTTTCTACAT AGACACAGTA ACAATCTGAT CTCTCTTATC 841 CCCACAAAGG ATCTAGCTCC TTTCTCTTTT CTTCATTTCC TGAGTATGAA TTGCCCTTCC 901 ACTGTAGCTC AAAATGGCTG TAACATTTCA GACTGGAGGA AAAGTGATAA TTCACTCTGT 961 GTCTCACTTA TAAATTTTCC TGTAGATATT TGGGGATCCA AACTCAGTTG AAGAAATACT 1021 AGTTTGAATG TCAGTTTAGT TACTAAAACT CCCTAGGAGA GGATTAGAAA GTCTAGTCTT 1081 GAATCCAAAT GTTAACATCC CTTCAAATGT GGTTGGATAG CCTTTTCCTC CAACTATCAC 1141 CATCACCACC ACAATCCAGA ACTCTGTGAA AAGAGATAAA GATTCAAGGG CCTTAAAGCG 1201 ATTGTCCCAG GTTGGTTGTG AGGTAATTCT TGTACCTAAA GATACAAAAT TTGAGTAAAT 1261 GACTTTGTTC CTACAGCAGG TAGCAAGCCA GAAGGCTACT GCTGGGCTCA TATTTTACAA 1321 GTACCTAAAA CTAGTTGTTA GTTATGGGGC ATGTTTACAA AGTGTTAACA TCAACATATT 1381 TATGCCCATC TCCATATAGA ATCTTCTTAA TATAACTCAA GATAATTGAT GTAACAAATT 1441 CACATCATGA TCTTACCAAT TTGGTTTTTA ACATCAAAGT GCTGCCCCAG GCTTCAATCA 1501 GCACATACCA GACTGCTATG TCCCTTTTCT CCAGAAGCCA TATTGAGCAC CCCACAGCTC (* C-634 T)
1561 TAACACCTCA GAGCCTAGCC AAAGGCCAAG AACACTTGCC TGATCCCCCA AATTTCCAAC 1621 ACAGGACCAT TGACAGCAAG CCCAGTCATT CCAGATCATC TTCAGTGGGA CAAAAAAAGA 1681 AAACAACAGT TTTAATCCAA TGGAGGCAAT TTATTGTTAT GAAGATTTCA GGAAACAAAG (* A-475C)
1741 CTCAAAAAGG AACCAGAAAG GAAAAGGCTT TTTAGAATGT GGATAGAGCC ACAAGTGTCA 1801 TTAAGGAGAG AGAGAGAGAA ACGGAGAGAT GAAATTTATG TTACTTAAGA TCAAGTTTCA (* A-336 G)
1861 CATAATGCCA GTTATATTAG CATATACTGG CAGGATTATC TTTCCCCACC TACAAATAGT 1921 CTAATGACCT CACAGTCACA GAAATCACAG AGAACTAAGC TGAAGAGAGA ACACTCGTCC 1981 CTGCTTCCCA TCTTAGAGCA GCTGAATAAT TTCCTGAGAA TTCTATTCCT GAAGCTAGGA 2041 AGAAAAGTTT ATTTATACAT ACACGCAACC TGCAAGTCTC CAGTTTCTAT TCTTCCTTCC 2101 TCTTTGACCC TTCCCCTCCC CCACTTTGCA CCAGAGAAGT CAGACTCCGG GAGTGCTTTA (5’ RACE/*+1) 2161 ACAGTTTGAA GGCTAATCTG AAAGAGGAAG AAGAATCTGT ATATCTGTAT ATATTGGCTA ( EXON I)
2221 GCAAATGTGC CCTGCTCTCT CCCCTCTTAA AAATAGCAGC AACCCATCTT TGCAAAGAAG 2281 CTTGCCTATA GAGCAGGCAC TCTGTGAATG GACTGTGCTT TTACGACCCT ACAGGGTATC 2341 AAGATACTGT GCAGCTCGCC AACAAGGATT AATTGCAAGG ACTGGTAGAT CGAATTTACT 2401 GAAGACTTGG AGCTTGCTTC TGAGAACAAA CGCAAAAGGA CAGTAAACTG TGGACCTTGA 2461 AGTTAGCAGCG
Fig. 2. 5’-RACE sequence and 3 SNPs location on the 2466bp fragment of CysLTR1gene. Transcription start site as denoted as +1 found from the 5’-RACE and presented in bold letter and under line (_). Sequences of three genetic variants (C-634T, A-475C, and A-336G) in promoter region were represented as aterisk (*).
A
B
r2 lD’l C-634T A-475C A-336G
C-634T 1 1
A-475C 0.9 1
A-336G 1 0.9
Fig. 3. Gene map and linkage disequilibrium (LD) coefficients in cysteinyl leukotrienes receptor 1 (CysLTR1). A. Gene map and 3 SNPs in CysLTR1 on chromosome Xq13-q21. Coding exons are marked by black blocks and 5’-and 3’- untranslated regions by white blocks. Transcription start site was denoted as nucleotide +1. (B), Linkage disequilibrium coefficient (lD’l) of CysLTR1 SNPs.
Exon 1 2 3 4 5
+1+1
+1+1 (5(5(5(5’ RACE) RACE) RACE) RACE)
***
C-634T, A-475C, and A-336G. The clinical characteristics of the study subjects are summarized in Table 1. There were no significant differences in the FEV1 % predicted, total IgE level, asthma duration and airway hyperresponsiveness to PC20 methacholine between the AIA and ATA groups (p=0.37, 0.20, 0.09, 0.22, respectively). There were no significant difference in mean age, prevalence of sex, atopy status among three groups (p>0.05). Between the AIA and ATA groups, there were significant differences in percent in fall of FEV1 during lysine-aspirin bronchoprovocation test (p<0.001). There were significant difference in the prevalence of nasal polyps between AIA and ATA, this results suggest that AIA patient (48%) were found to have significantly higher frequencies of nasal polyps than ATA patients (6.7%, p=0.003).
As the CysLTR1 gene is on the X chromosome, the distribution of the CysLTR1 promoter genotypes within male subjects deviated from Hardy-Weinberg equilibrium (p<0.01). All analyses were stratified by sex.
2. Genotype distribution of CysLTR1 3 SNPs
Next, we investigated a genetic association study of three genetic polymorphisms CysLTR1 in three groups of study subjects classified as AIA (n=105), ATA (n=111), and NCs (n=125). Allele and genotype frequencies of the 3 SNPs within the phenotypic groups are described in Table 2. Allele frequencies (q) were determined for each polymorphism. Male AIA patients were found to
have significantly higher frequencies of the minor alleles (T, C,G) of the CysLTR1 Table 1. Clinical characteristics of the study subjects
P value
AIA: ASA-intolerant asthma; NC: normal controls; ATA: ASA-tolerant asthma. N: number of patients;
NA: not applicable; NS: not significant; M: male. Data are expressed as the mean ±SD
The methacholine provocative concentration producing a 20% fall in forced expiratory volume in 1 second (PC20-FEV1) was calculated as an index of bronchial hyperresponsiveness (BHR).
promoter SNPs than male control subjects (p=0.031 for AIA vs. NC; p=0.022 for AIA vs. ATA). In contrast, there were no significant differences in allele and genotype frequencies among the three groups within female subjects (p>0.05).
Haplotypes of CysLTR1 gene were constructed by Bayesian algorithm with genotyped SNPs (Table 2) and 2 common haplotypes (frequency > 5%) were analyzed. Among males, AIA patients had a lower frequency of major haplotype, ht1 [C-A-A] (p=0.031, OR=0.36, 95%CI: 0.14~0.90 for AIA vs. NC; p=0.022, OR=0.34, 95%CI: 0.14~0.85 for AIA vs. ATA), and higher frequency of minor haplotype, ht2 [T-C-G] (p=0.031, OR=2.42, 95%CI: 0.98~5.98 in AIA vs. NC; p=0.031, (OR=2.57, 95%CI: 1.04~6.31 in AIA vs. ATA), than those in male control groups. Significant differences in the frequency of ht1 and ht2 were not found in female subjects. In addition, there were no significant differences in the haplotype distributions in female subjects by three alternative analysis models (co-dominant, dominant and recessive models).
3. Association analysis of AIA-associated quantitative phenotypes
Initially, AIA related phenotypes such as atopy, serum total IgE level, initial baseline FEV1 % predicted value and PC20 methacholine were evaluated for any association with the CysLTR1 C-634T SNP which is a tagging SNP of the 3 SNPs (Table 3).
In female AIA patients, with mutant genotypes of CysLTR1 promoter polymorphisms showed higher total IgE levels (p=0.003), while no significant associations were found in male subjects. No significant associations were found between CysLTR1 polymorphisms and other phenotypes such as airway
Table 2. Genotype frequencies of the three SNPs in CysLTR1 promoter
*Each P value was calculated with co-dominant, dominant and recessive models. q: minor allele frequency; AIA: ASA-intolerant asthma; NC: normal controls; ATA: ASA-tolerant asthma; N:
number of patients; NS: not significant. Values in bold indicate significant p value.
* C-634T is a tagging SNP of the three SNPs in CysLTR1 promoter. Significant associations (P<0.01) are printed in bold. P values are genotype-specific means ± SD.
Table 3. Association of CysLTR1 C-634T with atopy, total IgE, asthma duration, PC20 methacholine, FEV1 and fall of FEV1.
Gender Genotype Age Atopy
Log IgE (IU/ml)
Asthma duration (year)
PC20
methacholine (mg/ml)
FEV1 (%)
Fall of FEV1 (%)
Male C 43.16 ± 12.9 11(57.9%) 2.26 ± 0.63 8.4 ± 6.1 4.56 ± 8.78 84. 85 ± 25.1 20.3 ± 10.4 T 36.1 ± 12.2 11(55.0%) 2.34 ± 0.57 5.5 ± 4.2 8.81 ± 15.3 76.79 ± 23.8 21.5 ± 13.0
p 0.087 0.86 0.670 0.095 0.268 0.309 0.769
Female CC + CT 47.7± 12.9 22(40.7%) 2.08 ± 0.47 6.5 ± 6.1 4.01 ± 7.05 86.7 ± 20.2 23.2 ± 11.7 TT 42.6 ± 13.7 9(75%) 2.56 ± 0.55 7.2 ± 5.6 3.76 ± 4.83 77.3 ± 28.5 19.6 ± 7.1
p 0.226 0.031 0.003 0.716 0.929 0.182 0.438
hyperresponsiveness to metahcholine and pulmonary function in both sexes.
4. Effect of 3 SNPs on transcriptional activity
To explore the functional significance of the CysLTR1 promoter polymorphisms at the molecular level, promoter fragments containing all 2466bp CysLTR1 promoter haplotypes ht1 [C-A-A] and ht2 [T-C-G] were cloned into 5’
of the luciferase reporter gene within pGL3 basic vector. To perform this experiment, we screened several cell lines such as A549 cells, Hela cells, HL-60 cells, HUVEC, THP-1 cells, HEK-293T cells, U937 cells, Jurkat cells, and COS-7
of the luciferase reporter gene within pGL3 basic vector. To perform this experiment, we screened several cell lines such as A549 cells, Hela cells, HL-60 cells, HUVEC, THP-1 cells, HEK-293T cells, U937 cells, Jurkat cells, and COS-7