Abstract
Adam33 is a member of a family of genes that encode membrane-anchored proteins with a disintegrin and a metalloprotease domain and that are primarily expressed in lung fibroblasts and bronchial smooth muscle. The human Adam33 gene is located on chromosome 20p13, a region that has been linked to asthma and bronchial hyperresponsiveness. Recently, the polymorphisms in Adam33 have been found to be associated with asthma. In this study, we performed polymorphism scanning of the entire genomic region, including the promoter region of Adam33, by direct sequencing. We identified 16 novel polymorphisms in the Adam33 gene. Among these novel polymorphisms, three polymorphisms (−2154G→A, −753T→A, and −330C→T) were found to be in the promoter region and one polymorphism (13491 G→A) was located in 3' untranslated region of the Adam33 gene.
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Introduction
The membrane-anchored proteins of the Adam gene family possess a unique domain structure, having a disintegrin and a metalloprotease domain (Stone et al. 1999; Wolfsberg et al. 1995a). Adam proteins also contain a signal sequence, prodomain, catalytic domain, cysteine-rich domain, epidermal-growth-factor-like domain, and a cytoplasmic domain (Black and White 1998; Primakoff and Myles 2000).
These domains of the Adam gene family have been suggested to be involved in adhesive interactions, cell fusion, and proteolysis and in numerous biological processes, including myoblast fusion, neurogenesis, sperm maturation, and protein-ectodomain shedding of cytokines and other cell surface proteins (Howard et al. 2000; Shi et al. 2000; Wolfsberg et al. 1995b). Over 30 Adam genes have been identified in various species. Many of the Adam genes show tissue-specific expression in testes and are thought to be involved in spermatogenesis and fertilization (Cerretti et al. 1999; van Huijsduijnen 1998; Wolfsberg and White 1996; Zhu et al. 1999). In addition, Kuzbanian (Adam10) has been shown to play an important role in neurogenesis and neural network formation (Hattori et al. 2000; Pan and Rubin 1997). Adam proteins have also been implicated in several disease processes, including Alzheimer's disease (Moss et al. 2001; Yamamoto et al. 1999).
Recently, the Adam33 gene has been identified and characterized in mouse and human (Gunn et al. 2002; Yoshinaka et al. 2002). It maps to human chromosome 20p13, encodes an open reading frame of 2442 bp consisting of 22 exons, and is expressed in lung fibroblasts, heart, and bronchial smooth muscle, but not in bronchial epithelial cells (Van Eerdewegh et al. 2002; Yoshinaka et al. 2002). Van Eerdewegh et al. (2002) have reported that the Adam33 gene is associated with asthma and bronchial hyperresponsiveness. In this study, we have scanned the entire Adam33 gene sequence, including the promoter region, by direct sequencing in order to detect polymorphic sites in Adam33.
Materials and method
DNA samples
Blood samples were obtained from 24 healthy Korean individuals. Genomic DNAs were extracted from leukocytes of peripheral blood by a standard phenol-chloroform method.
Polymerase chain reaction
Genomic regions of Adam33 containing the promoter region were partially amplified and sequenced by using the 32 primer pairs shown in Table 1. The polymerase chain reaction (PCR) was carried out in a 25-µl reaction volume containing 50 ng genomic DNA, 0.5 µM primers, 0.2 mM dNTP, 1.5 mM MgCl2, 10 mM TRIS-HCl (pH 8.3), and 1 U Ex Taq polymerase (TaKaRa). PCR was run for 30 cycles of denaturation at 95°C for 30 s, annealing at the melting temperature of each primer pair for 1 min, and extension at 72°C for 1 min on a GeneAmp PCR system 9700 thermocycler (Applied Biosystem).
Sequencing analysis
Both the sense and antisense strands of the PCR products were directly sequenced by using the same primers as those for the PCR amplification. PCR products purified by a PCR purification kit (Millipore, USA) were used as the template DNA for cycle sequencing. Sequence analysis was performed by BigDye Terminator cycle sequencing (Applied Biosystems) on an ABI 3700 Prism Automated DNA sequencer (Applied Biosystems) according to the manufacturer's instructions.
Results and discussion
To discover novel single-nucleotide polymorphisms (SNPs) in the Adam33 gene, we performed scanning by direct sequencing on genomic DNA samples isolated from 24 Korean healthy individuals. We found a total of 51 SNPs in the Adam33 gene, 16 SNPs of which were identified as novel polymophic sites (Fig. 1, Table 2). Among the novel SNPs, three SNPs (−2154G→A, which is a G to A substitution at position −2154 counting from the translation start site, −753T→A, and −330C→T) were located in the promoter region, five SNPs (419C→T, 517A→G, 1733A→G, 2062G→A, and 2272C→T) were located in intron 1, two SNPs (2291T→C and 4565G→A) were located in the intron 2, three SNPs (5065G→C, 5272C→T, and 6139G→C) were located in intron 3, and 10474C→T, 12136G→C, and 13491G→A were located in intron 16, intron 19, and the 3' untranslated region (UTR), respectively. Among the polymorphic sites located in introns, polymorphic site 12136G→C was located in intron 19 and showed a high allele frequency (42.5%). Van Eerdewegh et al. (2002) showed that some of the SNPs having significant association with asthma were located in the introns and 3'UTR of the Adam33 gene. Indeed, several SNPs located in intron 19, such as 12136G→C, were significantly associated with asthma (Van Eerdewegh et al. 2002). The SNPs identified in this study should provide important information for case-control association studies. Linkage disequilibrium (LD) coefficients between each SNP (381A→G, 1712G→C, 11434A→C, and 13506C→G) pair including our two novel SNPs (517A→G and 12136G→C) were calculated. We observed no strong LD (data not shown).
In many genes, SNPs located within promoter regions have been described as affecting gene transcription and causing inter-individual variations in gene production. We detected three novel SNPs in the promoter region of the Adam33 gene. Specifically, a G to A polymorphism at the position −2154 had a high allele frequency (29.2%). It would be interesting to analyze the allele frequency of these variances with large samples of patients with autoimmune and allergic diseases, since evidence for the involvement of Adam33 in asthma and bronchial hyperresponsiveness has been reported (Van Eerdewegh et al. 2002).
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Acknowledgements
This work was supported by a grant from the Korea Health 21 Research and Development Project of the Ministry of Health and Welfare (01-PJ3-PG6-01GN09-003).
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Chae, SC., Yoon, KH. & Chung, HT. Identification of novel polymorphisms in the Adam33 gene. J Hum Genet 48, 278–281 (2003). https://doi.org/10.1007/s10038-003-0019-1
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DOI: https://doi.org/10.1007/s10038-003-0019-1
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