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Autosomal-dominant Hypoplastic Form of Amelogenesis Imperfecta Caused by an Enamelin Gene Mutation at the Exon-Intron Boundary
1 Research Group of Human Gene Therapy, Hokkaido University Graduate School of Medicine, N-15, W-7, Kita-ku, Sapporo, 060-8638, Hokkaido, Japan; and Correspondence: * corresponding author, tada-ari{at}med.hokudai.ac.jp
Amelogenesis imperfecta (AI) is currently classified into 14 distinct subtypes based on various phenotypic criteria; however, the gene responsible for each phenotype has not been defined. We performed molecular genetic studies on a Japanese family with a possible autosomal-dominant form of AI. Previous studies have mapped an autosomal-dominant human AI locus to chromosome 4q11-q21, where two candidate genes, ameloblastin and enamelin, are located. We studied AI patients in this family, focusing on these genes, and found a mutation in the enamelin gene. The mutation detected was a heterozygous, single-G deletion within a series of 7 G residues at the exon 9-intron 9 boundary of the enamelin gene. The mutation was detected only in AI patients in the family and was not detected in other unaffected family members or control individuals. The male proband and his brother showed hypoplastic enamel in both their deciduous and permanent teeth, and their father showed local hypoplastic defects in the enamel of his permanent teeth. The clinical phenotype of these patients is similar to that of the first report of AI caused by an enamelin gene mutation. Thus, heterogeneous mutations in the enamelin gene are responsible for an autosomal-dominant hypoplastic form of AI.
Key Words: amelogenesis imperfecta • autosomal-dominant • enamelin • hypoplastic enamel
Amelogenesis imperfecta (AI) is a heterogeneous group of genetic disorders characterized by defects in tooth enamel formation in the absence of any generalized or systemic diseases. The prevalence of AI is reported at 1/700 in northern Sweden (Bäckman and Holm, 1986) and 1/14,000 in the US (Witkop and Sauk, 1976). There has been no comprehensive study of the prevalence of AI in Asian countries, although clinical phenotypes of hypoplastic and hypocalcified AI have been reported in Japan (Ooya et al., 1988; Sekiguchi et al., 1998). AI is currently classified into 14 distinct subtypes based on the clinical phenotype and mode of inheritance (Witkop, 1988); however, the gene responsible for every subtype has not yet been defined. Major enamel matrix proteins (amelogenin, enamelin, and ameloblastin) are suggested to contribute to the enamel formation of teeth (Uchida et al., 1991). During the secretory stage of enamel formation, these proteins are secreted by ameloblasts and play key roles in the growth of enamel crystal (Robinson et al., 1998). Several reports have shown that mutations in the amelogenin gene located at Xp22.1-p22.3 cause X-linked AI (Lagerström et al., 1991; Lagerstrom-Fermer et al., 1995; Hart et al., 2000). However, the X-linked AI presents in less than 5% of all reported cases. The most common type of AI is the autosomal-dominant form (Kärrman et al., 1996). The locus responsible for one autosomal-dominant form of local hypoplastic AI (AIH2) has been mapped to chromosome 4q11-q21 (Kärrman et al., 1997). A gene encoding a tooth-specific protein ameloblastin has been mapped to 4q21 (MacDougall et al., 1997), suggesting that ameloblastin might be the candidate gene responsible for the autosomal-dominant form of AI. However, to date, no case of AI has identified a mutation in the ameloblastin gene (Kärrman-Mårdh et al., 2001). The gene encoding another tooth-specific protein enamelin has also been mapped to chromosome 4q21 (Dong et al., 2000; Hu et al., 2000). Subsequently, a mutation in the enamelin gene was first identified in a family with the autosomal-dominant hypoplastic form of AI (Rajpar et al., 2001). In this paper, we report a Japanese family with an autosomal-dominant hypoplastic form of AI. We performed mutation analysis focusing on the ameloblastin and enamelin genes, and found a heterozygous single-base deletion at the exon 9-intron 9 border of the enamelin gene. Since the clinical phenotype of this family is similar to that of the first report caused by the enamelin gene mutation (Rajpar et al., 2001), it is possible that heterogeneous mutations within the enamelin gene might also be responsible for the autosomal-dominant hypoplastic forms of AI.
Familial Studies The pedigree of the AI family in this study and their clinical features are shown in Fig. 1  . The family individuals were clinically examined and the diagnosis of AI was made by several dentists according to Witkop’s classification (1988). Informed consent was obtained from all the participants prior to their entry into this study. Approval was obtained from the institutional review board for this study, and informed consent from all familial members in this article was provided according to the Declaration of Helsinki.
DNA Purification and PCR Analysis Peripheral-blood mononuclear cells (PBMC) of affected and unaffected individuals in the family were isolated by the Ficoll-Hypaque (Pharmacia Biotech, Piscataway, NJ, USA) gradient method. Genomic DNA was purified from PBMC by Sepa-Gene (Sanko-Junyaku, Tokyo, Japan). We performed the mutation analysis of the ameloblastin and enamelin genes. Primers used for ameloblastin were reported elsewhere (Kärrman-Mårdh et al., 2001). Primers originally designed by Rajpar et al. (2001) were used to amplify the enamelin gene (Table ). Since Hu et al. (2001) have proposed the presence of an additional exon of the human enamelin gene as exon 2, we have constructed an additional set of primers for it. Accordingly, the numbering of enamelin exons and introns was changed from that in Rajpar’s publication. PCR was performed in a 20-µL volume with the use of an AmpliTaq Gold PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 10 pM of forward and reverse primers, and 100 ng of sample DNA. Amplification was performed in a GeneAmp 9700 thermal cycler (Applied Biosystems). After the initial incubation for 9 min at 94°C, samples were subjected to 40 cycles of 30 sec at 94°C, 30 sec at 60°C, and 30 sec at 72°C. Then the final extension step was performed at 72°C for 7 min.
SSCP Analysis For the mutation screening in the AI patients, PCR products were analyzed by the single-strand conformation polymorphism (SSCP) method according to the previous method reported (Orita et al., 1989). The PCR samples, mixed with formamide dye (95% formamide with bromophenol blue and xylenecyanol), were heated to 100°C for 5 min, quenched on ice, and then loaded onto a Gene Gel Exel (12.5%/24-well Kit; Amersham Pharmacia Biotech, Piscataway, NJ, USA). Electrophoresis was performed with the conditions of 600 V, 25 mA, and 15 W at 15°C for 90 min in a GenePhor (Amersham Pharmacia Biotech). After electrophoresis, the gel was silver-stained by means of a DNA Silver Staining Kit (Amersham Pharmacia Biotech).
Sequence Analysis
Clinical Features We asked the family members whether there was any systemic or generalized disease associated with the family. The affected members of the family exhibited enamel hypoplasia without any general complications. The male proband boy and his younger brother showed hypoplastic enamel in both their deciduous and permanent teeth (Fig. 1B III-1, III-2  ) that resulted in hypersensitivity to cold stimuli. Anterior open bite was found in both of them in the primary dentition (Fig. 1B III-1). Their father showed local hypoplastic enamel defects demonstrating a horizontal lesion involving primarily the middle third of the permanent teeth (Fig. 1B II-1). In the molars, the lesion was further extended occlusally. In contrast to his sons, he showed normal sensitivity to cold stimuli in the affected teeth. We could not confirm the phenotype of their deciduous teeth, but as far as their permanent teeth were concerned, the affected brothers showed a more yellowish enamel than that of their father, and the enamel hypoplasia was more pronounced. Therefore, we concluded that the phenotype of AI in this family was the hypoplastic type, although the severity varied among the individual patients. Judging from this AI family’s pedigree, we considered that the inheritance pattern is autosomal-dominant. It should be noted that the probands’ paternal grandparents had normal enamel surfaces.
Mutation Analysis
Enamelin gene
Sequencing of the DNA fragment revealed a deletion of a single-G from a series of 7 G residues (AGGGGGGGTAAGT  AGGGGGGTAAGT) at the exon 9-intron 9 boundary of the enamelin gene (Fig. 2B, GenBank accession number AF530444). This mutation was confirmed in all affected individuals but was not found in unaffected family members or in the controls. The normal boundary of exon 9-intron 9 consists of AGGGGGG (3' site of exon 9)/GTAAGT (5' site of intron 9), where either a G of exon 9 or a G at a splice donor site of intron 9 has been deleted.
In this study, we found a mutation in the enamelin gene from a Japanese family with an autosomal-dominant AI. Mutational analysis revealed a single-G deletion at the exon 9-intron 9 boundary of the enamelin gene. The sequence of the mutation site in the enamelin gene is a series of 7 G residues at the 3' end of exon 9 and the 5' (donor) site of intron 9. Therefore, we cannot determine which "G" (in the exon or the intron) is involved in this deletion mutation. Although studies of the mutant enamelin mRNA of patients would provide useful information, this was not available because the patients’ tooth germ cells were the only samples obtained for the studies.
Two different consequences are proposed for this mutation. The new splice donor site created by a single G deletion (GT
Although there is an obvious difference in the phenotype between the affected brothers and their father, they all have the same mutation in the enamelin gene. What are the differences among them? Since neither of the probands’ paternal grandparents have the mutation in the enamelin gene, we speculated that a de novo mutation has occurred in the enamelin gene of the father, although their parent-child relationship was not fully verified. In that case, the mutation had originated in either gamete of the paternal grandparents or during subsequent cell division after fertilization. The latter case might result in a mosaicism of tooth germ cells in the father. Thus, this scenario might account for the reason why the father exhibited a less severe AI phenotype compared with his sons. However, an alternative scenario might be that the severity in this form of AI is affected by the other factors relating to the enamel formation in conjunction with this enamelin mutation. The phenotype of patients with X-linked AI who harbor mutations in the amelogenin gene is also likely to be varied. Further studies are needed to clarify the relation between the phenotype of AI and mutations in the genes concerned with the enamel formation.
We thank Prof. Michael Dixon, University of Manchester, UK, for kindly providing us with the PCR primer information to amplify the enamelin gene. This work is supported by the H12-genome-003 grant from the Ministry of Health, Labor and Welfare, Japan, and by grants 13670776 and 12470455 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Received for publication November 20, 2001. Revision received July 29, 2002. Accepted for publication August 7, 2002.
Journal of Dental Research, Vol. 81, No. 11,
738-742 (2002) This article has been cited by other articles:
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