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Ameloblastin gene polymorphisms in healthy Japanese
PEDIATRIC DENTAL JOURNAL 15(1): 58–63, 2005 58
Ameloblastin gene polymorphisms in healthy Japanese Seikou Shintani*1, Mitsuhiko Kobata*1, Satoru Toyosawa*2, Yoshiko Tanaka*1, Chiaki Takeuchi*1 and Takashi Ooshima*1 *1 Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry 1-8 Yamada-oka, Suita, Osaka 565-0871, JAPAN *2 Department of Oral Pathology, Osaka University Graduate School of Dentistry 1-8 Yamada-oka, Suita, Osaka 565-0871, JAPAN
Abstract Ameloblastin is one of the extracellular matrix proteins in tooth enamel and may be responsible for autosomal amelogenesis imperfecta (AI), since it plays a significant role in enamel crystal growth. We investigated polymorphisms of the human ameloblastin gene by polymerase chain reaction, DNA sequencing and single-strand conformational polymorphism (SSCP) analysis using genomic DNA from 50 Japanese subjects with sound dentition. One single sequential trinucleotide deletion and 3 single-nucleotide polymorphisms (SNPs) were identified in the translated region. The nucleotide deletion results in the lack of an amino acid residue and 2 of the SNPs cause nonsynonymous substitutions of amino acid residues. These results provide important background information for the investigation of autosomal AI in Japanese patients.
Introduction Ameloblastin1), also known as amelin2) or sheathlin3), is one of the non-amelogenin proteins present in tooth enamel. In situ hybridization and immunohistological studies have shown that ameloblastin is present in the enamel prism sheath4) and principally expressed by enamel-forming ameloblast cells5,6). Hence, it is considered to play an important role in the control of enamel crystal growth and determination of the prismatic structure7). The human ameloblastin gene has been cloned from tooth germs8) and shown to be located on chromosome 4q13.29). Amelogenesis imperfecta (AI), a hereditary disorder that causes abnormalities in the quantity and/or quality of dental enamel, is generally classified into 14 distinct subtypes based on the mode of inheritance and clinical manifestation10). Further, it is generally classified into 2 forms, autosomal and X-linked, based on inheritance. The Received on September 7, 2004 Accepted on November 29, 2004
58
Key words Ameloblastin, Amelogenesis imperfecta, Gene polymorphisms, Japanese
autosomal forms of AI are the most prevalent, representing over 95% of all reported cases, and genetically heterogeneous11). Four distinct mutations of the human enamelin gene and one of kallikrein 4, which cause autosomal AI, have recently been reported12–17), while mutations in the X locus of the amelogenin gene is considered to cause the defects seen in X-linked AI patients. The ameloblastin gene, which is closely linked to the locus of enamelin, is also thought to be responsible for autosomal AI, though its linkage to the disease has not been proven9). Hereditary diseases are generally caused by mutations in functional genes, including substitutions or deletions, however, mutations do not always induce a pathogenic condition. Such non-pathogenic mutations are called polymorphisms and are either neutral or functionally significant18). Thus, nucleotide mutations found in ameloblastin genes may lead to not only pathogenic mutations causing autosomal AI, but also nonpathogenic polymorphisms. To evaluate whether the ameloblastin gene is responsible for AI, it is necessary to investigate polymorphisms of ameloblastin
AMELOBLASTIN POLYMORPHISMS IN JAPANESE
59
Fig. 1 Schematic representation of the ameloblastin gene showing relative positions of the oligonucleotide primers used for PCR and DNA sequencing. Boxes show exons and arrow heads indicate primer orientations.
Table 1 Primer sequences used to amplify the coding region of the ameloblastin gene Primers names
Exon numbers
AMB-26 AMB-53 AMB-47 AMB-54 AMB-55 AMB-56 AMB-57 AMB-58 AMB-71 AMB-48 AMB-60 AMB-36 AMB-61 AMB-62 AMB-63 AMB-66 AMB-67 AMB-68
1 1 2 2 3 3 4 4 5 5 6 6 7, 8, 9 7, 8, 9 10, 11, 12 10, 11, 12 13 13
Nucleotide sequences 5⬘-ATCTTGGTTGGCATCATCAGGC-3⬘ 5⬘-ATTCAGCTACTGGTGACAAATGAAGG-3⬘ 5⬘-CGGTGGTTTTTGTAAGAGCAGAGACT-3⬘ 5⬘-TTAAATCAAGTGAGTCTATGCGTGGAC-3⬘ 5⬘-GGGCATTGAAGGAAGTTTTGTACCA-3⬘ 5⬘-GCTAGGAAAACTGAGAAGCACACGATTA-3⬘ 5⬘-TTCCACCTTTCAGTGATGATTTGTGTC-3⬘ 5⬘-CACTACCACCACCATGAATACTTGCA-3⬘ 5⬘-AAGGAAAAGGAAAACCAAATATAACCAATG-3⬘ 5⬘-TGTTAATGCTAGGACTTGGCTGTTTCT-3⬘ 5⬘-TGGTGCTTGCTATGTAAACTCAACTTC-3⬘ 5⬘-GGTTAGCTGGTGATTCTGATCTG-3⬘ 5⬘-CACTTTGTCTATTTTGTTTATTTTTTGACTGA-3⬘ 5⬘-TTGCAAGACAGTGTCTCATTGAGA-3⬘ 5⬘-ACTGTTATGGGGATGTGCCTGTGAG-3⬘ 5⬘-AAGTGACTGTTCTTCCCTGGCCACT-3⬘ 5⬘-GGTATAGTTAATAGCATGTGATGATGGCA-3⬘ 5⬘-TTGAAAGCAAGAAGGGGACCTACACT-3⬘
from non-AI individuals and compare them with those from autosomal AI patients. In the present study, we investigated the presence of nucleotide polymorphisms of the ameloblastin gene in Japanese subjects who did not show signs of AI, as a method of screening for the mutations causing autosomal AI.
Orientation sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense
Product size (bp) 164 310 204 243 300 431 481 774 776
Materials and methods
DNA extraction kit (Qiagen, Hilden, Germany). All subjects were healthy and had sound dentition, and gave their written informed consent prior to the study. All procedures were carried out in full compliance with the Japanese Public Health Service and Osaka University Health Guidelines Involving Human Subjects, and were approved by our Institute Review Board.
Sample population and genomic DNA preparation Venous blood (10 ml) was collected using acidcitrate dextrose as an anticoagulant from 50 Japanese volunteers (28 males and 22 females, 25 to 55 years old) and genomic DNA was isolated using a Qiagen
PCR amplification Isolated genomic DNA (⬍1g) was amplified by a PCR procedure in 50l of PCR buffer using a TaKaRa LA PCR kit, (version 2.1, TAKARA, Otsu, Shiga, Japan), following adjustment as recommended by the manufacturer with a GeneAmp 2400 thermal cycler
60
Shintani, S., Kobata, M., Toyosawa, S. et al.
Table 2 Allelic frequencies of polymorphisms in the translated region of ameloblastin genes from 50 normal subjects Exon
Position
7 12 13 13
Codon 180 Codon 255 Codon 329 Codon 441
Polymorphism GGA (Gly) vs. ***(deletion) GCC (Ala) vs. GTC (Val) CCG (Pro) vs. CTG (Leu) GCA (Ala) vs. GCG (Ala)
Allelic frequencies
Mårdh Kärrman et al. (2001) & NCBI data
0.81 vs. 0.19 0.97 vs. 0.03 0.98 vs. 0.02 0.91 vs. 0.09
— 0.974 vs. 0.026 — 0.925 vs. 0.075
(Applied Biosystems, Mihama, Japan). The program consisted of 3 minutes of initial denaturation at 95°C followed by 35 amplification cycles. Each cycle consisted of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute, and the reaction was completed by a primer extension step for 10 minutes at 72°C. PCR products were evaluated under UV light following 1.5% agarose gel electrophoresis and staining with ethidium bromide. The primers were based on the 5⬘ and 3⬘ sequences flanking the exons (Fig. 1 and Table 1). Single-strand conformational polymorphism (SSCP) analysis by silver staining SSCP is the scanning method in common use and based on the differences in electrophoretic migration between mutant and wild-type DNAs. It can detect and map only a subset of mutations to an accuracy of Ⳳ200 bp. Amplified DNA segments from exons 1, 2, 3, 4, 5, 6, and 7 to 9 from 30 of the subjects were analyzed by SSCP. Approximately 10 ng of each PCR product was heat-denatured in the presence of 95% deionized formamide and electrophoresed at 18°C using a GeneGel Excel 12.5/24 Kit (Amersham Pharmacia Biotech), with a GenePhor Electrophoresis Unit (Amersham Pharmacia Biotech). DNA fragments were visualized using a DNA Silver Staining Kit (Amersham Pharmacia Biotech). DNA Sequencing The remaining PCR products were prepared using a Centricon 100 spin column (Millipore, Bedford, Mass, USA) for the DNA sequencing templates. The products were then analyzed using an automated DNA sequencer model 373 (Applied Biosystems, Buckinghamshire, UK) with a DYEnamic ET Terminator Cycle Sequencing kit (Amersham Pharmacia Biotech). Aberrant PCR products by SSCP were also sequenced.
(A)
(B)
Fig. 2 Molecular study of exon 7 to 9 of the ameloblastin gene in Japanese by PCR-SSCP. (A) control subject, (B) subject whose codon 180 is deleted.
Results One single sequential trinucleotide deletion and 3 single nucleotide polymorphisms (SNPs) were detected in the translated region of the ameloblastin gene in the Japanese subjects (Table 2). Further, a trinucleotide deletion at codon 180 in exon 7 resulted in the lack of an amino acid residue (Fig. 2). Among the 3 SNPs, 2 at codon 225 in exon 12 and codon 329 in exon 13 caused nonsynonymous substitutions of amino acid residues, while the other, at codon 441 in exon 13, was a synonymous substitution (Fig. 3). All polymorphisms were observed in a heterozygous state. The remaining nucleotide sequences of the translated regions were completely identical and all of the splice sites showed consensus splice sequences, with all donor sites containing ‘GT’ and all acceptor sites containing ‘AG’.
AMELOBLASTIN POLYMORPHISMS IN JAPANESE
Fig. 3 Nucleotide and translated amino acid sequences of the human ameloblastin gene. Amino acid residues are given in the IUPAC-IUB single letter code. The signal peptide is underlined with a dotted line and vertical lines indicate exon borders. Numbering shows codon positions. The polymorphic sites are indicated by the boxes. The allelic nucleotides and amino acids show beneath the consensuses.
61
62
Shintani, S., Kobata, M., Toyosawa, S. et al.
Discussion Nucleotide sequence polymorphisms in Swedish subjects were previously described19) and deposited in GenBank (accession no. AF209780, AY009121009124). The most common sequence in those was the portion coding for the N-terminal region of the protein and was completely identical with that in the present Japanese subjects, whereas the remaining part differed by several single nucleotide substitutions (2 nonsynonymous substitutions in exon 6, 1 nonsynonymous and 1 synonymous substitution in exon 7, and 6 nonsynonymous and 1 synonymous substitutions in exon 13). The trinucleotide deletion in exon 7 and 2 SNPs in exon 13 are reported here for the first time. Further, 2 nonsynonymous SNPs were previously reported in the Swedish subjects19), of which 1 was present in the Japanese subjects (codon 255 in exon 12), and the allele frequencies between the Japanese and Swedish subjects were very similar (3% vs. 2.6%). The other was not detected (codon 354 in exon 13) in the Japanese subjects. The trinucleotide deletion found in exon 7 caused a single glycine deletion, however, it is apparent that the deletion does not have a relationship to either autosomal dominant or recessive AI, because of the high allele frequency (19%). The human ameloblastin gene has 2 extra exons (exon 8 and 9), which are absent in other reported vertebrates20,21) that represent an internal sequence duplication of exon 78). The functional role of repetitive polypeptides has not been clarified, however, an advantage may be that exon 8 or 9 compensates for exon 7 when the part of the ameloblastin protein associated with exon 7 lacks its natural functions due to mutation. The ameloblastin gene is thought to be a good candidate for involvement in autosomal AI because of its role in amelogenesis. Our findings provide useful information for further investigation of the relationship between the ameloblastin gene and autosomal AI in Japanese.
3)
4)
5)
6)
7)
8)
9) 10)
11)
12)
13)
References 1) Krebsbach, P.H., Lee, S.K., Matsuki, Y., Kozak, C.A., Yamada, K.M. and Yamada, Y.: Full-length sequence, localization, and chromosomal mapping of ameloblastin. A novel tooth-specific gene. J Biol Chem 271: 4431–4435, 1996. 2) Cerny, R., Slaby, I., Hammarström, L. and Wurtz, T.: A novel gene expressed in rat ameloblasts codes for
14)
15)
proteins with cell binding domains. J Bone Miner Res 11: 883–891, 1996. Hu, C.C., Fukae, M., Uchida, T., Qian, Q., Zhang, C.H., Ryu, O.H., Tanabe, T., Yamakoshi, Y., Murakami, C., Dohi, N., Shimizu, M. and Simmer, J.P: Sheathlin: cloning, cDNA/polypeptide sequences, and immunolocalization of porcine enamel sheath proteins. J Dent Res 76: 648–657, 1997. Uchida, T., Fukae, M., Tanabe, T., Yamakoshi, Y., Satoda, T., Murakami, C., Takahashi, O. and Shimizu, M.: Immunochemical and immunocytochemical study of a 15KDa non-amelogenin and related proteins in the porcine immature enamel: Proposal of a new group of enamel proteins ‘sheath proteins’. Biomed Res 16: 131–140, 1995. Fong, C.D., Slaby, I. and Hammarström, L.: Amelin: an enamel-related protein, transcribed in the cells of epithelial root sheath. J Bone Miner Res 11: 892– 898, 1996 Fong, C.D., Cerny, R., Hammarström, L. and Slaby, I.: Sequential expression of an amelin gene in mesenchymal and epithelial cells during odontogenesis in rats. Eur J Oral Sci 106(Suppl. 1): 324–330, 1998. Paine, M.L., Wang, H-J., Luo, W., Krebsbach, P.H. and Snead, M.L.: A transgenic animal model resembling amelogenesis imperfecta related to ameloblastin overexpression. J Boil Chem 278: 19447–19452, 2003. Toyosawa, S., Fujiwara, T., Ooshima, T., Shintani, S., Sato, A., Ogawa, Y., Sobue, S. and Ijuhin, N.: Cloning and characterization of the human ameloblastin gene. Gene 256: 1–11, 2000. Hu, J.C. and Yamakoshi, Y.: Enamelin and autosomaldominant amelogenesis imperfecta. Crit Rev Oral Biol Med 14: 387–398, 2003. Witkop, C.J. Jr.: Amelogenesis imperfecta, dentinogenesis imperfecta and dentin dysplasia revisited: problems in classification. J Oral Pathol 17: 547– 553, 1995. Kärrman, C., Bäckman, B., Holmgren, G. and Forsman, K.: Genetic heterogeneity of autosomal dominant amelogenesis imperfecta demonstrated by its exclusion from the AIH2 region on human chromosome 4Q. Arch Oral Biol 41: 893–900, 1996. Rajpar, M.H., Harley, K., Laing, C., Davies, R.M. and Dixon, M.J.: Mutation of the gene encoding the enamel-specific protein, enamelin, causes autosomaldominant amelogenesis imperfecta. Hum Mol Genet 10: 1673–1677, 2001. Kida, M., Ariga, T., Shirakawa, T., Oguchi, H. and Sakiyama, Y.: Autosomal-dominant hypoplastic form of amelogenesis imperfecta caused by an enamelin gene mutation at the exon-intron boundary. J Dent Res 81: 738–742, 2002. Mårdh Kärrman, C., Bäckman, B., Holmgren, G., Hu, J.C., Simmer, J.P. and Forsman-Semb, K.: A nonsense mutation in the enamelin gene causes local hypoplastic autosomal dominant amelogenesis imperfecta (AIH2). Hum Mol Genet 11: 1069–1074, 2002. Hart, T.C., Hart, P.S., Gorry, M.C., Michalec, M.D.,
AMELOBLASTIN POLYMORPHISMS IN JAPANESE
Ryu, O.H., Uygur, C., Ozdemir, D., Firatli, S., Aren, G. and Firatli, E.: Novel ENAM mutation responsible for autosomal recessive amelogenesis imperfecta and localised enamel defects. J Med Genet 40: 900–906, 2003. 16) Hart, P.S., Michalec, M.D., Seow, W.K., Hart, T.C. and Wright, J.T.: Identification of the enamelin (g.8344delG) mutation in a new kindred and presentation of a standardized ENAM nomenclature. Arch Oral Biol 48: 589–596, 2003. 17) Hart, P.S., Hart, T.C., Michalec, M.D., Ryu, O.H., Simmons, D., Hong, S. and Wright, J.T.: Mutation in kallikrein 4 causes autosomal recessive hypomaturation amelogenesis imperfecta. J Med Genet 41: 545–549, 2004. 18) Cooper, D.N.: Human gene mutation in pathology
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and evolution. J Inherit Metab Dis 25: 157–182, 2002. 19) Mårdh Kärrman, C., Bäckman, B., Simmons, D., Golovleva, I., Gu, T.T., Holmgren, G., MacDougall, M. and Forsman-Semb, K.: Human ameloblastin gene: genomic organization and mutation analysis in amelogenesis imperfecta patients. Eur J Oral Sci 109: 8–13, 2001. 20) Shintani, S., Kobata, M., Toyosawa, S., Fujiwara, T., Sato, A. and Ooshima, T.: Identification and characterization of ameloblastin gene in a reptile. Gene 283: 245–254, 2002. 21) Shintani, S., Kobata, M., Toyosawa, S. and Ooshima, T.: Identification and characterization of ameloblastin gene in an amphibian, Xenopus laevis. Gene 318: 125–136, 2003.
Ameloblastin gene polymorphisms in healthy Japanese Seikou Shintani*1, Mitsuhiko Kobata*1, Satoru Toyosawa*2, Yoshiko Tanaka*1, Chiaki Takeuchi*1 and Takashi Ooshima*1 *1 Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry 1-8 Yamada-oka, Suita, Osaka 565-0871, JAPAN *2 Department of Oral Pathology, Osaka University Graduate School of Dentistry 1-8 Yamada-oka, Suita, Osaka 565-0871, JAPAN
Abstract Ameloblastin is one of the extracellular matrix proteins in tooth enamel and may be responsible for autosomal amelogenesis imperfecta (AI), since it plays a significant role in enamel crystal growth. We investigated polymorphisms of the human ameloblastin gene by polymerase chain reaction, DNA sequencing and single-strand conformational polymorphism (SSCP) analysis using genomic DNA from 50 Japanese subjects with sound dentition. One single sequential trinucleotide deletion and 3 single-nucleotide polymorphisms (SNPs) were identified in the translated region. The nucleotide deletion results in the lack of an amino acid residue and 2 of the SNPs cause nonsynonymous substitutions of amino acid residues. These results provide important background information for the investigation of autosomal AI in Japanese patients.
Introduction Ameloblastin1), also known as amelin2) or sheathlin3), is one of the non-amelogenin proteins present in tooth enamel. In situ hybridization and immunohistological studies have shown that ameloblastin is present in the enamel prism sheath4) and principally expressed by enamel-forming ameloblast cells5,6). Hence, it is considered to play an important role in the control of enamel crystal growth and determination of the prismatic structure7). The human ameloblastin gene has been cloned from tooth germs8) and shown to be located on chromosome 4q13.29). Amelogenesis imperfecta (AI), a hereditary disorder that causes abnormalities in the quantity and/or quality of dental enamel, is generally classified into 14 distinct subtypes based on the mode of inheritance and clinical manifestation10). Further, it is generally classified into 2 forms, autosomal and X-linked, based on inheritance. The Received on September 7, 2004 Accepted on November 29, 2004
58
Key words Ameloblastin, Amelogenesis imperfecta, Gene polymorphisms, Japanese
autosomal forms of AI are the most prevalent, representing over 95% of all reported cases, and genetically heterogeneous11). Four distinct mutations of the human enamelin gene and one of kallikrein 4, which cause autosomal AI, have recently been reported12–17), while mutations in the X locus of the amelogenin gene is considered to cause the defects seen in X-linked AI patients. The ameloblastin gene, which is closely linked to the locus of enamelin, is also thought to be responsible for autosomal AI, though its linkage to the disease has not been proven9). Hereditary diseases are generally caused by mutations in functional genes, including substitutions or deletions, however, mutations do not always induce a pathogenic condition. Such non-pathogenic mutations are called polymorphisms and are either neutral or functionally significant18). Thus, nucleotide mutations found in ameloblastin genes may lead to not only pathogenic mutations causing autosomal AI, but also nonpathogenic polymorphisms. To evaluate whether the ameloblastin gene is responsible for AI, it is necessary to investigate polymorphisms of ameloblastin
AMELOBLASTIN POLYMORPHISMS IN JAPANESE
59
Fig. 1 Schematic representation of the ameloblastin gene showing relative positions of the oligonucleotide primers used for PCR and DNA sequencing. Boxes show exons and arrow heads indicate primer orientations.
Table 1 Primer sequences used to amplify the coding region of the ameloblastin gene Primers names
Exon numbers
AMB-26 AMB-53 AMB-47 AMB-54 AMB-55 AMB-56 AMB-57 AMB-58 AMB-71 AMB-48 AMB-60 AMB-36 AMB-61 AMB-62 AMB-63 AMB-66 AMB-67 AMB-68
1 1 2 2 3 3 4 4 5 5 6 6 7, 8, 9 7, 8, 9 10, 11, 12 10, 11, 12 13 13
Nucleotide sequences 5⬘-ATCTTGGTTGGCATCATCAGGC-3⬘ 5⬘-ATTCAGCTACTGGTGACAAATGAAGG-3⬘ 5⬘-CGGTGGTTTTTGTAAGAGCAGAGACT-3⬘ 5⬘-TTAAATCAAGTGAGTCTATGCGTGGAC-3⬘ 5⬘-GGGCATTGAAGGAAGTTTTGTACCA-3⬘ 5⬘-GCTAGGAAAACTGAGAAGCACACGATTA-3⬘ 5⬘-TTCCACCTTTCAGTGATGATTTGTGTC-3⬘ 5⬘-CACTACCACCACCATGAATACTTGCA-3⬘ 5⬘-AAGGAAAAGGAAAACCAAATATAACCAATG-3⬘ 5⬘-TGTTAATGCTAGGACTTGGCTGTTTCT-3⬘ 5⬘-TGGTGCTTGCTATGTAAACTCAACTTC-3⬘ 5⬘-GGTTAGCTGGTGATTCTGATCTG-3⬘ 5⬘-CACTTTGTCTATTTTGTTTATTTTTTGACTGA-3⬘ 5⬘-TTGCAAGACAGTGTCTCATTGAGA-3⬘ 5⬘-ACTGTTATGGGGATGTGCCTGTGAG-3⬘ 5⬘-AAGTGACTGTTCTTCCCTGGCCACT-3⬘ 5⬘-GGTATAGTTAATAGCATGTGATGATGGCA-3⬘ 5⬘-TTGAAAGCAAGAAGGGGACCTACACT-3⬘
from non-AI individuals and compare them with those from autosomal AI patients. In the present study, we investigated the presence of nucleotide polymorphisms of the ameloblastin gene in Japanese subjects who did not show signs of AI, as a method of screening for the mutations causing autosomal AI.
Orientation sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense
Product size (bp) 164 310 204 243 300 431 481 774 776
Materials and methods
DNA extraction kit (Qiagen, Hilden, Germany). All subjects were healthy and had sound dentition, and gave their written informed consent prior to the study. All procedures were carried out in full compliance with the Japanese Public Health Service and Osaka University Health Guidelines Involving Human Subjects, and were approved by our Institute Review Board.
Sample population and genomic DNA preparation Venous blood (10 ml) was collected using acidcitrate dextrose as an anticoagulant from 50 Japanese volunteers (28 males and 22 females, 25 to 55 years old) and genomic DNA was isolated using a Qiagen
PCR amplification Isolated genomic DNA (⬍1g) was amplified by a PCR procedure in 50l of PCR buffer using a TaKaRa LA PCR kit, (version 2.1, TAKARA, Otsu, Shiga, Japan), following adjustment as recommended by the manufacturer with a GeneAmp 2400 thermal cycler
60
Shintani, S., Kobata, M., Toyosawa, S. et al.
Table 2 Allelic frequencies of polymorphisms in the translated region of ameloblastin genes from 50 normal subjects Exon
Position
7 12 13 13
Codon 180 Codon 255 Codon 329 Codon 441
Polymorphism GGA (Gly) vs. ***(deletion) GCC (Ala) vs. GTC (Val) CCG (Pro) vs. CTG (Leu) GCA (Ala) vs. GCG (Ala)
Allelic frequencies
Mårdh Kärrman et al. (2001) & NCBI data
0.81 vs. 0.19 0.97 vs. 0.03 0.98 vs. 0.02 0.91 vs. 0.09
— 0.974 vs. 0.026 — 0.925 vs. 0.075
(Applied Biosystems, Mihama, Japan). The program consisted of 3 minutes of initial denaturation at 95°C followed by 35 amplification cycles. Each cycle consisted of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute, and the reaction was completed by a primer extension step for 10 minutes at 72°C. PCR products were evaluated under UV light following 1.5% agarose gel electrophoresis and staining with ethidium bromide. The primers were based on the 5⬘ and 3⬘ sequences flanking the exons (Fig. 1 and Table 1). Single-strand conformational polymorphism (SSCP) analysis by silver staining SSCP is the scanning method in common use and based on the differences in electrophoretic migration between mutant and wild-type DNAs. It can detect and map only a subset of mutations to an accuracy of Ⳳ200 bp. Amplified DNA segments from exons 1, 2, 3, 4, 5, 6, and 7 to 9 from 30 of the subjects were analyzed by SSCP. Approximately 10 ng of each PCR product was heat-denatured in the presence of 95% deionized formamide and electrophoresed at 18°C using a GeneGel Excel 12.5/24 Kit (Amersham Pharmacia Biotech), with a GenePhor Electrophoresis Unit (Amersham Pharmacia Biotech). DNA fragments were visualized using a DNA Silver Staining Kit (Amersham Pharmacia Biotech). DNA Sequencing The remaining PCR products were prepared using a Centricon 100 spin column (Millipore, Bedford, Mass, USA) for the DNA sequencing templates. The products were then analyzed using an automated DNA sequencer model 373 (Applied Biosystems, Buckinghamshire, UK) with a DYEnamic ET Terminator Cycle Sequencing kit (Amersham Pharmacia Biotech). Aberrant PCR products by SSCP were also sequenced.
(A)
(B)
Fig. 2 Molecular study of exon 7 to 9 of the ameloblastin gene in Japanese by PCR-SSCP. (A) control subject, (B) subject whose codon 180 is deleted.
Results One single sequential trinucleotide deletion and 3 single nucleotide polymorphisms (SNPs) were detected in the translated region of the ameloblastin gene in the Japanese subjects (Table 2). Further, a trinucleotide deletion at codon 180 in exon 7 resulted in the lack of an amino acid residue (Fig. 2). Among the 3 SNPs, 2 at codon 225 in exon 12 and codon 329 in exon 13 caused nonsynonymous substitutions of amino acid residues, while the other, at codon 441 in exon 13, was a synonymous substitution (Fig. 3). All polymorphisms were observed in a heterozygous state. The remaining nucleotide sequences of the translated regions were completely identical and all of the splice sites showed consensus splice sequences, with all donor sites containing ‘GT’ and all acceptor sites containing ‘AG’.
AMELOBLASTIN POLYMORPHISMS IN JAPANESE
Fig. 3 Nucleotide and translated amino acid sequences of the human ameloblastin gene. Amino acid residues are given in the IUPAC-IUB single letter code. The signal peptide is underlined with a dotted line and vertical lines indicate exon borders. Numbering shows codon positions. The polymorphic sites are indicated by the boxes. The allelic nucleotides and amino acids show beneath the consensuses.
61
62
Shintani, S., Kobata, M., Toyosawa, S. et al.
Discussion Nucleotide sequence polymorphisms in Swedish subjects were previously described19) and deposited in GenBank (accession no. AF209780, AY009121009124). The most common sequence in those was the portion coding for the N-terminal region of the protein and was completely identical with that in the present Japanese subjects, whereas the remaining part differed by several single nucleotide substitutions (2 nonsynonymous substitutions in exon 6, 1 nonsynonymous and 1 synonymous substitution in exon 7, and 6 nonsynonymous and 1 synonymous substitutions in exon 13). The trinucleotide deletion in exon 7 and 2 SNPs in exon 13 are reported here for the first time. Further, 2 nonsynonymous SNPs were previously reported in the Swedish subjects19), of which 1 was present in the Japanese subjects (codon 255 in exon 12), and the allele frequencies between the Japanese and Swedish subjects were very similar (3% vs. 2.6%). The other was not detected (codon 354 in exon 13) in the Japanese subjects. The trinucleotide deletion found in exon 7 caused a single glycine deletion, however, it is apparent that the deletion does not have a relationship to either autosomal dominant or recessive AI, because of the high allele frequency (19%). The human ameloblastin gene has 2 extra exons (exon 8 and 9), which are absent in other reported vertebrates20,21) that represent an internal sequence duplication of exon 78). The functional role of repetitive polypeptides has not been clarified, however, an advantage may be that exon 8 or 9 compensates for exon 7 when the part of the ameloblastin protein associated with exon 7 lacks its natural functions due to mutation. The ameloblastin gene is thought to be a good candidate for involvement in autosomal AI because of its role in amelogenesis. Our findings provide useful information for further investigation of the relationship between the ameloblastin gene and autosomal AI in Japanese.
3)
4)
5)
6)
7)
8)
9) 10)
11)
12)
13)
References 1) Krebsbach, P.H., Lee, S.K., Matsuki, Y., Kozak, C.A., Yamada, K.M. and Yamada, Y.: Full-length sequence, localization, and chromosomal mapping of ameloblastin. A novel tooth-specific gene. J Biol Chem 271: 4431–4435, 1996. 2) Cerny, R., Slaby, I., Hammarström, L. and Wurtz, T.: A novel gene expressed in rat ameloblasts codes for
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