Abstract
Asthma is characterized by reversible airway obstruction and airway inflammation. Serum levels of eosinophil cationic protein (ECP) might reflect eosinophilic airway inflammation and asthma activity. However, serum ECP levels are not elevated in some patients with asthma, even when they are symptomatic. In this study, we screened for polymorphisms in the ECP gene and analyzed association between these polymorphisms and asthma and serum ECP levels in 137 Japanese families identified through children with asthma. We identified three polymorphisms (−393C/T, −38C/A, and 124Arg/Thr) in human ECP. We did not find associations between these polymorphisms and asthma by the transmission disequilibrium test. However, we found that serum ECP levels in subjects with the −393T allele were significantly lower than those in subjects with the −393C allele. A reporter construct with the −393T allele showed significantly lower promoter activity than one with the −393C allele. Gel shift assay revealed that C/EBP proteins can bind the −393C/T polymorphic site. These data indicate that C/EBP proteins play an important role in the regulation of ECP and that a significant amount of the variance in baseline serum ECP levels may be explained by the −393C/T polymorphism. Although ECP polymorphisms are not likely to be involved in the development of asthma, measurement of ECP levels for the assessment of asthma activity may be improved when done in combination with genotyping of the −393C/T polymorphism.
Asthma is one of the most common childhood diseases in developed countries, and it has been increasing in frequency (1, 2). Asthma is characterized by reversible airway obstruction, bronchial hyperresponsiveness, and airway inflammation, and eosinophilic inflammation of the lung is a factor underlying these abnormalities (3). Serum levels of eosinophil cationic protein (ECP), which is produced by activated eosinophils, reflect the degree of activation of the circulating eosinophil pool in the body (4). Therefore, serum ECP levels might reflect ongoing eosinophilc airway inflammation. However, serum ECP levels are not elevated in some patients with asthma, even when they are symptomatic (5).
ECP is a member of the eosinophil-associated RNase family, and the gene is located on human chromosome 14q11.2. The eosinophil peroxidase and eosinophil-derived neurotoxin (EDN) genes are highly homologous to ECP across the promoter, exons, and introns. Transcription from the ECP promoter has been analyzed (6), and a consensus binding site for the NFAT-1 transcription factor in intron 1 of the gene plays a crucial role in enhancing expression (6). In addition, a C/EBP binding site in the EDN promoter regulates the transcription of EDN (7). These findings lead us to speculate that genetic polymorphisms that influence production of ECP exist in the human ECP gene.
In the present study, we screened for mutations in the 5′-flanking region, coding regions, and intron 1 of the ECP gene in patients with asthma. We found three nuclear variants in the ECP gene and conducted an association study in Japanese families identified through children with asthma.
The probands were children with atopic asthma who visited the Pediatric Allergy Clinic of the University Hospital of Tsukuba. Criteria used in the recruitment of subjects with asthma are described in an online supplement. A full verbal and written explanation of the study was given to all family members interviewed, and 137 families (466 individuals) gave informed consent and participated in the study. This study was approved by the Committee of Ethics of the University of Tsukuba (Japan).
Blood samples for ECP measurement were collected into SST tubes (Becton Dickinson, Mountain View, CA), stored 1 hour at room temperature, and centrifuged at 1300 × g at 4°C for 10 minutes. The sera were stored at −20°C until they were measured with the ECP radioimmunoassay kit (Pharmacia Diagnostics, Uppsala, Sweden). The detection limit of the kit is 2.0 μg/L. The samples were obtained from individuals with asthma when they were in a stable condition. One hundred and sixty-eight samples from the family members (70 children with asthma and 98 parents) mentioned previously were used for ECP measurements.
The number of blood eosinophils was counted with a Coulter MAXM (Coulter Corp., Miami, FL) by multiplying the total white blood cell count (106/L) by the percentage of eosinophils. Blood eosinophil counts were available for 65 children with asthma.
DNA was extracted from peripheral blood leukocytes. Two exons and the 5′-flanking region of the ECP gene were amplified from the genomic DNAs of 32 unrelated people with asthma. Additional details on the method for genotyping polymorphisms detected in this study are provided in the online supplement.
A fragment of the ECP promoter from nucleotides −534 to −1 relative to the translation initiation site (−267 to +277 relative to transcription initiation site) was amplified with primers 5′-GAAGATCTTCCACCCAGAGTCCAGATCC-3′ and 5′-GAAGATCTTCGTTTCCTGTAAGAAAAGAAGAGAAG-3′ from DNAs of subjects with genotypes −393C/C and −393T/T. Polymerase chain reaction products were digested with BslII overnight at 37°C and then subcloned into BslII-digested pGL3-Basic Vector (Promega, Madison, WI).
For promoter assay, HL-60 clone 15 (American Type Culture Collection, Rockville, MD) and U937 (American Type Culture Collection) were electroporated with the plasmid construct containing the ECP promoter fragment. For transactivation assay, K562 cells (Cell Resource Center for Biomedical Research, Tohoku University, Miyagi, Japan) were transfected with the test construct together with a pcDNA3-based vector (Invitrogen Corp., Carlsbad, CA) containing the cDNA encoding the putative activation domains of rat C/EBP-α. Additional details on the method for transient transfections are provided in the online supplement.
Nuclear extracts of U937 cells were prepared as described previously (8). Oligonucleotide sequences (nucleotides −403 to −383 relative to the translation initiation site) were −393C-sense, 5′-AGGATGATTGCACAAGTGGAC-3′; −393C-antisense, 5′-GTCCACTTGTGCAATCATCCT-3′; −393T-sense, 5′-AGGATGATTGTACAAGTGGAC-3′; and −393T-antisense, 5′-GTCCACTTGTACAATCATCCT-3′. Double-stranded oligonucleotides were radiolabeled with γ-32P-dATP with T4 polynucleotide kinase (Takara Bio, Otsu, Japan) and incubated with U937 nuclear extract on ice for 30 minutes. Oligonucletides used as C/EBP competitor were 5′-CTAGGGCTTGCGCAATCTATATTCG-3′ (sense) and 5′-CGAATATAGATTGCGCAAGCCCTAG-3 (antisense) (Geneka Biotechnology Inc., Montreal, Canada). For supershift assay, U937 nuclear extracts were preincubated with C/EBP α antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 30 minutes at room temperature before adding the radiolabeled probe. DNA–protein complexes were separated by electrophoresis on 5% polyacrylamide gels and visualized by autoradiography.
Linkage disequilibrium and an allelic transmission disequilibrium were assessed with the SIB-PAIR program version 0.99.9 (9). One proband was selected from each family for transmission disequilibrium test (TDT) analysis, and 137 trios were analyzed for TDT. Associations between serum ECP levels and genotypes were analyzed by the Wilcoxon test with JMP software version 4 (SAS Institute, Cary, NC). p Values for associations between serum ECP levels and genotypes were corrected for multiple comparisons, and p values less than 0.0083 was considered statistically significant. Because blood eosinophil counts are known to correlate with serum ECP levels, we performed forward stepwise regression analysis to determine whether a ECP polymorphism combined with eosinophil counts contributed to the variation in ECP levels. Serum ECP levels and eosinophil counts were log-transformed before analysis because their distributions were skewed.
We identified one polymorphism in the promoter region (−393C/T), one polymorphism in intron 1 (−38C/A), and one missense polymorphism in exon 2 (124Arg/Thr) of the ECP gene (Figure 1)
Figure 1. Locations of polymorphisms in ECP and sequence alignment between the EDN and ECP genes. Numbers indicate positions relative to the translation initiation site. (a) C/EBP binding site in the EDN promoter as reported by Baltus and colleagues (9). (b) NFAT-1 consensus binding site reported by Handen and Rosenberg (8).
[More] [Minimize]No. of Families | 137 |
|---|---|
| No. of affected children | 192 |
| Mean age, yr (SD) | 11.0 (4.4) |
| Mean log [total IgE], IU/ml (SD) | 2.67 (0.64) |
| Mean Df-RAST IgE, UA/ml | 64 |
| Percent of log [total IgE] > +1 SD | 90 |
| Percent of atopy* | 100 |
| No. of parents | 274 |
| Mean age, yr (SD) | 41.1 (5.5) |
| Mean log [total IgE], IU/ml (SD) | 2.12 (0.69) |
| Mean Df-RAST IgE, UA/ml | 8.6 |
| Percent of log [total IgE] > +1 SD | 37 |
| Percent of atopy* | 43 |
Polymorphism | Allele | Transmitted | Not Transmitted | p Value |
|---|---|---|---|---|
| −393C/T | T | 30 | 31 | 0.90 |
| −38A/C | A | 59 | 48 | 0.33 |
| 124Arg/Thr | Thr | 67 | 61 | 0.66 |
Serum ECP levels of children with asthma with each ECP genotype are shown in Table 3
Polymorphism | Subjects | n | Genotype and Serum Level ± SD (Frequencies) | p Value | ||||
|---|---|---|---|---|---|---|---|---|
| CC | CT | TT | ||||||
| −393C/T | Proband | 45 | 32.1 ± 2.8 (0.87) | 10.2 ± 10.2 (0.11) | 2.1 (0.02) | 0.022* | ||
| All asthmatic children | 70 | 32.7 ± 2.0 (0.89) | 15.9 ± 5.9 (0.10) | 2.1 (0.01) | 0.004† | |||
| Genotype | A/A | A/C | C/C | |||||
| −38A/C | Proband | 45 | 44.6 ± 9.1 (0.09) | 31.4 ± 4.4 (0.38) | 26.5 ± 3.7 (0.53) | 0.23 | ||
| All asthmatic children | 70 | 43.2 ± 7.3 (0.07) | 32.7 ± 3.4 (0.33) | 28.0 ± 2.5 (0.60) | 0.19 | |||
| Genotype | Arg/Arg | Arg/Thr | Thr/Thr | |||||
| Arg124Thr | Proband | 45 | 28.6 ± 3.5 (0.65) | 32.5 ± 4.7 (0.35) | – | 0.52 | ||
| All asthmatic children | 70 | 31.2 ± 2.6 (0.59) | 28.5 ± 3.3 (0.37) | 41.0 ± 9.6 (0.04) | 0.25 | |||
We performed a reporter assay to determine whether there was a functional difference between the −393C and −393T alleles. We generated luciferase reporter gene constructs that contained the region of ECP from nucleotides −534 to −1 and differed only at position −393 (C versus T). The promoter activity of each construct was analyzed in a myelomonocytic cell line (U937) and an eosinophilic cell line (HL60 clone 15). Firefly luciferase activity normalized to renilla luciferase activity is shown in Figure 2
Figure 2. Reporter gene activity of constructs transfected into human hematopoietic cells lines (U937 and HL-60 clone 15). Firefly luciferase activity from each experimental construct was normalized to renilla luciferase activity from cotransfected pRL-TK plasmid (internal control plasmid) and expressed as relative luciferase activity. Bars indicate the mean value of three independent experiments; error bars represent ± 1 SD.
[More] [Minimize]The C/EBP family plays an important role in the regulation of EDN (7) (Figure 1), which has approximately 90% sequence homology with ECP. Computer analysis of transcription factor binding sites (TFSEARCH, available at http://www.cbrc.jp/research/db/TFSEARCHJ.html) (10) revealed that the −393C/T polymorphism is located within a putative C/EBP binding site and that the −393C-to-T change would eliminate this C/EBP binding site. To examine the possible role of C/EBP binding in the regulation of ECP expression, electrophoretic mobility shift assay and a transactivation assay were performed. In Electrophoretic Mobility Shift Assay with U937 nuclear extracts, a band with retarded mobility (Figure 3
Figure 3. Electrophoretic mobility shift assay of ECP promoter fragments. ECP promoter fragments with either −393C or −393T were synthesized and used as DNA probes. Assays were done in the presence (lane 3, −393C) and absence (lane 1, −393C; lane 2, −393T) of cold C/EBP-specific oligonucleotides. In a supershift assay, U937 nuclear extracts were incubated with C/EBP-α (lane 4) antibody before adding the −393C probe.
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Figure 4. Transactivation assay of the ECP promoter with C/EBP-α. K562 cells were transfected with the −393C or −393T luciferase construct together with expression plasmid encoding rat C/EBP-α. Luciferase activity is presented normalized as fold inductions compared with those cotransfected with empty vectors. Bars indicate the mean value of three independent experiments; error bars represent ± 1 SD.
[More] [Minimize]In the present study, we identified three polymorphisms in ECP. Although these polymorphisms are unlikely to be associated with the development of asthma, a promoter polymorphism, −393C/T, is associated with serum ECP levels, suggesting that serum levels of ECP are influenced genetically to some extent.
Zhang and Rosenberg (11) sequenced 2417 bp of the ECP and EDN genes and identified seven mutations in human ECP (−399T/C, −393C/T, −43C/T, −38C/A, 124Arg/Thr, 499G/C, and 577A/T) in various ethnic groups including an Asian population. We sequenced 1569 bp of the ECP gene and found three polymorphisms (−393C/T, −38C/A, and 124Arg/Thr) in our Japanese population. According to Zhang and Rosenberg (11), the −399T/C and −43C/T variants are rare and not found in the Asian population. Frequencies of the 499G/C and 577A/T alleles in the Asian population in their study were 0.16 and 0.04, respectively. Therefore, our failure to detect these variants in our population may be due to ethnic differences and the low frequencies of these variant alleles in our population.
ECP has approximately 90% nucleotide sequence homology with EDN. An EDN promoter chloramphenicol acetyltransferase assay showed that activation of the EDN promoter by C/EBP proteins is regulated by the C/EBP binding site centered at −124 bp upstream of the transcription initiation site (7). This region in the EDN promoter corresponds to the area around the −393C/T polymorphism in ECP (Figure 1). Therefore, our current findings that C/EBP regulates expression of ECP through interactions around the −393C/T polymorphism confirm the importance of this binding site in expression of the eosinophil-associated RNase family.
ECP is produced by activated eosinophil. Therefore, it has been suggested that ECP might be useful as a marker of inflammation in asthma (4, 12). However, serum ECP levels are not elevated in some patients with asthma, even when they are symptomatic (5). Associations between serum ECP levels and the −393C/T polymorphism and cis-elements around −393C/T in ECP may provide insights into this phenomenon. The frequency of the −393T allele in the Japanese is 0.08; therefore, the proportion of subjects with the −393T/T genotype is predicted to be 0.0064. Among 137 probands, only one patient with asthma possessed the −393T/T genotype, and serum ECP levels in this patient were low (< 2.0 μg/L) in three separate measurements. ECP levels in subjects with the −393C/T genotype are also lower than those in subjects with the −393C/C genotype. Our electrophoretic mobility shift assay assay showed that, although the intensity was weaker than that of the wild-type, −393T could bind C/EBP proteins. However, our luciferase and transactivation assays clearly showed that the −393T allele in comparison with the wild-type allele has lower promoter activity. Although the −393C/T polymorphism is not common, a significant amount of the variance in baseline serum ECP levels can be explained by this polymorphism. Therefore, our data suggest that measurement of serum ECP levels for assessment of asthma activity would be improved when used in combination with genotyping of the −393C/T polymorphism.
The authors thank Dr. Satoko Nakahara and Dr. Tetsuo Nogami for collecting samples.
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