© 2003 American Thoracic Society
The Promoter Polymorphism in the Eosinophil Cationic Protein Gene and Its Influence on the Serum Eosinophil Cationic Protein LevelDepartments of Medical Genetics and Immunology, Institute of Basic Medical Sciences; Department of Pediatrics, Institute of Clinical Medicine; Institute of Biological Sciences, University of Tsukuba, Tsukuba; and Akiba Hospital, Mito-City, Ibaraki, Japan Correspondence and requests for reprints should be addressed to Emiko Noguchi, M.D., Ph.D., Department of Medical Genetics, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki-ken, 305-8575, Japan. E-mail: enoguchi{at}md.tsukuba.ac.jp
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.
Key Words: asthma • promoter • luciferase assay
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.
Subjects 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 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.
Molecular Method
Plasmid Construction
Transient Transfections
Electrophoretic Mobility Shift Assay
Statistics
Polymorphisms and TDT 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) . The frequencies of the parental genotypes for the -393T, -38A, and 124Thr alleles were 0.08, 0.20, and 0.25, respectively, and did not deviate from those expected under the Hardy–Weinberg equilibrium (p > 0.1). Significant linkage disequilibrium was observed among these three polymorphisms (D = 0.016, D' = 1.0, p = 0.0007 between -393C/T and -38C/A; D = 0.018, D' = 0.91, p = 0.0004 between -393C/T and 124Arg/Thr; and D = 0.038, D' = 0.77, p < 0.0001 between -38C/A and 124Arg/Thr).
Table 1 shows the clinical characteristics of the families with asthma. TDT analyses showed that no ECP allele was transmitted preferentially to asthma-affected children (p > 0.1, Table 2) .
Serum ECP levels of children with asthma with each ECP genotype are shown in Table 3 . Serum ECP levels of probands with asthma with the -393C/T and -393T/T genotypes tended to be lower than the level in probands with the -393C/C genotype (p = 0.022), and this trend was stronger when all children with asthma (probands and their siblings with asthma) were analyzed (p = 0.004). Of 192 children with asthma, only one subject possessed the -393T/T genotype. His serum ECP levels were undetectable (< 2.0 µg/L) on three separate measurements, although his peripheral blood eosinophil counts and EDN level were elevated (800/µl for the blood eosinophil counts and 160 µg/L for the serum EDN level). The child's parents and his sister who had asthma were all heterozygous for the -393C/T polymorphism, and the ECP level for his sister was 10 µg/L. Stepwise analysis revealed that both the -393C/T variant and eosinophil counts were independently and significantly associated with serum ECP levels (p = 0.009, R2 = 0.083 for eosinophil counts, and p < 0.0001, R2 = 0.276 for -393C/T polymorphism). The total variance explained by these parameters was 35.9%. No associations were observed between serum ECP levels and either genotype of the -38C/A or Arg124Thr polymorphism (p > 0.1).
Reporter Assay and Electrophoretic Mobility Shift Assay 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 . Constructs with -393T showed reduced promoter activity compared with that of the wild-type constructs (-393C).
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 , lane 1, band a) was observed with wild-type oligonucleotide (-393C). This band was also observed with mutant-type oligonucleotide (-393T), but the intensity of the band was much weaker than that of the wild-type (Figure 3, lane 2). This DNA–protein complex (band a) was efficiently competed with C/EBP-specific cold oligonucletides (Figure 3, lane 3). Band a was supershifted by incubation of nuclear extract with C/EBP-  antibody (Figure 3, lane 4, band b), confirming that C/EBP proteins bind directly to the region around the -393C/T polymorphism. In the transactivation assay, the C/EBP expression vector was cotransfected with the -393C luciferase construct or the -393T luciferase construct. As shown in Figure 4
, cotransfection of C/EBP with the -393C construct yielded a fivefold increase in transcription activity, whereas cotransfection with the -393T construct yielded only a twofold increase, suggesting that C/EBP influences the transcription of ECP through the region around the -393C/T polymorphic site.
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.
This study was supported by Grant-in-Aid for Scientific Research on Priority Areas "Medical Genome Science" from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Received in original form April 7, 2002; accepted in final form August 8, 2002
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ABSTRACT
INTRODUCTION
-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 
