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A Novel Missense Mutation (p.P52R) in Amelogenin Gene Causing X-linked Amelogenesis Imperfecta
M. Kida1,
Y. Sakiyama2,
A. Matsuda3,
S. Takabayashi4,
H. Ochi5,
H. Sekiguchi5,
S. Minamitake5 and
T. Ariga1,*
1 Departments of Pediatrics and
2 Human Gene Therapy, Hokkaido University Graduate School of Medicine, N-15, W-7, Kita-ku, Sapporo, 060-8638, Hokkaido, Japan;
3 Sapporo Railway Hospital, N-3, E-1, Chuo-ku, Sapporo, 060-0033, Hokkaido, Japan;
4 Pediatric Dentistry Poplar Clinic, 3-275 Shinn-matsudo, Matsudo, 270-0034, Chiba, Japan; and
5 Shimadzu Analytical & Measuring Center, Inc., 380-1, Horiyama-shita, Hadano, 259-1304, Kanagawa, Japan
Correspondence: * corresponding author, tada-ari{at}med.hokudai.ac.jp
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ABSTRACT
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Amelogenesis imperfecta (AI) is a hereditary disease with abnormal dental enamel formation. Here we report a Japanese family with X-linked AI transmitted over at least four generations. Mutation analysis revealed a novel mutation (p.P52R) in exon 5 of the amelogenin gene. The mutation was detected as heterozygous in affected females and as hemizygous in their affected father. The affected sisters exhibited vertical ridges on the enamel surfaces, whereas the affected father had thin, smooth, yellowish enamel with distinct widening of inter-dental spaces. To study the pathological cause underlying the disease in this family, we synthesized the mutant amelogenin p.P52R protein and evaluated it in vitro. Furthermore, we studied differences in the chemical composition between normal and affected teeth by x-ray diffraction analysis and x-ray fluorescence analysis. We believe that these results will greatly aid our understanding of the pathogenesis of X-linked AI.
Key Words: amelogenesis imperfecta • X-linked • AMELX • mutant • X-ray analysis
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INTRODUCTION
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Tooth enamel, which is a highly mineralized tissue of ectodermal origin, is the hardest material in the human body. Amelogenesis imperfecta (AI) is an inherited disease exhibiting tooth enamel defects. The prevalence of AI has been reported at 1/700 in northern Sweden (Bäckman and Holm, 1986) and 1/14,000 in the USA (Witkop and Sauk, 1976). There are various modes of reported inheritance, and thus far, 5 responsible genes for AI have been identified (Stephanopoulos et al., 2005): the amelogenin gene (AMELX) (Kim et al., 2004), the enamelin gene (ENAM) (Rajpar et al., 2001; Kida et al., 2002; Mardh et al., 2002; PS Hart et al., 2003; TC Hart et al., 2003; Kim et al., 2005a,b), the enamelysin gene (MMP-20) (Kim et al., 2005b), the kallikrein-4 (KLK-4) (Hart et al., 2004), and the distal-less homeobox3 gene (DLX3) (Dong et al., 2005). AI is currently classified into 14 distinct subtypes based on phenotype and mode of inheritance (Witkop, 1988); however, the gene corresponding to each subtype has not been well-defined.
Amelogenin is the major extracellular matrix protein in developing enamel and is thought to be critical for the formation of normal enamel crystallite morphology. In normal enamel development, amelogenin is first secreted by ameloblasts, and is subsequently removed by enamel-specific proteinases (Woessner, 1998). Mutations in the human amelogenin gene (AMELX), located at Xp22.1–p22.3, are now known to be associated with X-linked AI. To date, 14 allelic AMELX mutations have been reported (Kim et al., 2004). The phenotype of individuals with AMELX mutations can vary from thin hypoplastic enamel to normal enamel thickness, but with reduced mineral and increased protein content compared with normal. Affected heterozygous females often exhibit alternating vertical ridges in both the deciduous and permanent dentition. The ridges consist of bands of the normal and affected enamel, indicating X chromosome inactivation of either the normal or the mutated gene early in development (the Lyon hypothesis) (Witkop, 1967).
In this report, we present a large Japanese X-linked AI family harboring a novel missense AMELX mutation, p.P52R. To characterize the pathological basis of abnormal amelogenesis in this family, we synthesized the mutant amelogenin protein and evaluated it in vitro. Moreover, we compared the mineral composition of the patient’s teeth with that of normal individuals by x-ray analysis. These results, together with those from other basic AI studies, will contribute to our understanding of the pathogenesis of AI.
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MATERIALS & METHODS
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Family Studies
The pedigree of the family in this study is shown in Fig. 1A . Informed consent, provided according to the Declaration of Helsinki, was obtained from all participants prior to this study. Approval was obtained from the institutional review board.
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Figure 1. The Japanese X-linked AI family included in this study. (A) Pedigree of the family. The arrow indicates the proband. (B) The III-6 family member. His teeth showed generalized thin enamel, resulting in small tooth size, widened inter-dental spacing, brownish color, and generally hypoplastic appearance. (C) The IV-1 proband. Her teeth showed hypoplasia in the form of distinct vertical ridges. (D-F) Sequencing results of the amelogenin gene from the family members. Results of mutation detection in the members [III-7 (D), III-6 (E), and IV-1 (F)] are shown. The mutation detected is indicated by an asterisk (*).
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Mutation Analysis
Genomic DNA isolated from peripheral blood mononuclear cells from family members, as well as from control individuals, was analyzed. Coding exons of the AMELX, together with their flanking introns, were PCR-amplified with appropriate primers as described previously (Lagerström-Fermer et al., 1995; Sekiguchi et al., 2001). PCR products were purified, and were then directly sequenced by means of an ABI Gene analyzer 310 (PE Applied Biosystems, Foster City, CA, USA).
X-ray Diffraction (XRD) Analysis and X-ray Fluorescence (XFD) Analysis
Extracted teeth from the proband and control maxillary deciduous second molars were used as test samples. XRD patterns were measured with a Shimadzu x-ray diffractometer (XRD-6100) and a graphite mono-chrometer (Shimadzu, Hadano, Japan) (Zhang et al., 2000). The diffraction pattern of the sample was characteristic of its constituent material, and, on the basis of this, we were able to conduct qualitative analysis of the material. Therefore, the diffraction of the tooth enamel provided information about the basic mineral composition of the surface material. The scanning XRD conditions were: Cu radiation, 40-kV accelerating voltage, 40-mA current, 20°–60° scanning range, and 0.02° step scan.
We performed XRF using an automated Shimadzu x-ray fluorescence spectrometer (XRF-1800) (Shimadzu, Hadano, Japan). The wavelengths of the fluorescent x-rays corresponded to each element. We estimated the elemental composition of the material by analyzing the fluorescent x-rays emitted from the sample and determining the wavelengths of the spectral peaks. Also, we estimated the concentrations (%) of the elements in the material by measuring the x-ray intensity of the spectral lines. The scanning conditions for XRF were: Rh radiation, with an accelerating voltage of 40 kV, 95-mA current, 8°–148° scanning range, either 4° or 8°/min scan speed, and a 3-mm-diameter collimator set to He mode. Elemental analysis was performed at the Analytical & Measuring Center of the Shimadzu Corporation (Ochi and Okashita, 1988).
Synthesis of Wild and Mutant Amelogenin Proteins
Total RNA was prepared from normal human tooth germs, and a 2-µg quantity of total RNA was used for reverse transcription. Full-length amelogenin cDNA was amplified by RT-PCR with optimal primers, and mutant amelogenin cDNA was then created by overlap PCR methods with mutation-incorporative primers. An EcoRI restriction site prior to the translation initiation codon and an XhoI site following the termination codon were prepared. The wild and mutant amelogenin coding regions were excised with EcoRI and XhoI-digestion and subcloned into a pGEX-4T-1 expression vector (Amersham Biosciences Corp., Piscataway, NJ, USA), respectively. Recombinant protein was overexpressed in the E. coli DH5 strain: Bacteria harboring the plasmid were incubated in LB medium containing glucose (10 µmol) and ampicillin (100 µg/ml) at 37°C until 0.8 O.D. at 600 nm. A 100-nmol quantity of isopropyl-β-d-thio-galacto-pyranoside (IPTG) was then added and incubated for another 4 hrs. The resulting bacterial cells were pelleted, re-suspended in loading buffer, and boiled for 5 min before fractionation by sodium dodecyl sulfate (SDS)-12% polyacrylamide gel electrophoresis (PAGE). The amount of protein was then evaluated with Coomassie brilliant blue staining. The antigen specificity and reactivity of the GST-amelogenin and -mutant amelogenin fusion proteins to anti-amelogenin monoclonal antibody (Hokudo Co., Ltd, Hokkaido, Japan) were studied by Western blotting.
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RESULTS
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Clinical Features
Inquiries were conducted with proband family members to ascertain whether there was systemic or generalized disease associated with their AI. Information for four generations of the family members was available (Fig. 1A ). It was noted that the phenotype of AI in the family was quite different between females and males: Two sisters showed the vertical ridges in their teeth (Fig. 1C), whereas the father showed small yellow-brown-mottled teeth (Fig. 1B). Anterior open bite was not found in the affected family members. Collectively, the X-linked dominant inherited pattern was suspected for this AI family. The phenotype of this AI family appeared consistent with Witkop’s classification type IE: the hypoplastic, smooth X-linked dominant type (Witkop, 1988).
Mutation Analysis
Sequencing of the DNA fragment revealed a single base alteration (g.3458C>G) in exon 5 of the X-chromosomal amelogenin gene, resulting in a substitution of proline at position 52 by arginine (p.P52R) (Figs. 1E, 1F). This alteration was confirmed only in the affected members in the family, and was heterozygous for affected sisters (IV-1,2), and hemizygous for their father (III-6). This defect was not found in any unaffected family members (Fig. 1D) or in 100 healthy control individuals (data not shown).
XRD and XRF Analyses
We performed XRD analysis to investigate the bulk and surface crystallization effects in the various phases. Extracted normal control teeth and teeth from the patient in the family (IV-1) were analyzed. The results showed that the surface structures of tooth enamel were hydroxyapatite [Ca5(PO4)3OH] in both the normal individual and the patient (Figs. 2A, 2B). Other crystalline materials were not confirmed; no differences were observed between samples. Results of XRF analysis revealed the production of deposits that contained large amounts of CaO and P2O5; however, this was also similar between normal control and affected teeth (Table). Collectively, no significant differences between the affected and normal teeth were observed by x-ray analysis.
Characterization of the Mutant (p.P52R) Amelogenin Protein Synthesized in vitro
The supernatant from the DH5 lysate containing the pGEX-4T-1 plasmid with the mutant amelogenin cDNA was examined on 12% SDS-PAGE. The results revealed that the P52R mutant amelogenin was highly expressed as a 50-kDa-fusion protein (Fig. 3A), similar to that of the normal amelogenin protein. Western blot analysis revealed that both the mutant and normal amelogenins were detected by the affinity-purified antibody against amelogenin (Fig. 3B). The recombinant p.P52R mutant product, as a GST-fusion protein, exhibited in vitro size and stability similar to those of the wild-type product.
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Figure 3. Synthesis of GST-fusion mutant and wild-type amelogenins in vitro. (A) SDS-PAGE analysis of the recombinant GST-fusion products in the E. coli lysates. (B) Immunoblotting with anti-amelogenin antibody. Lane M: molecular markers. Other lanes indicate cell lysates of an E. coli strain containing the vacant expression vector (lanes 1,2), GST-tagged wild amelogenin (lanes 3,4), and GST-tagged mutant amelogenin (lanes 5,6). Blotting under conditions with (lanes 2,4,6) or without (lanes 1,3,5) isopropyl-β-d-thio-galactopyranoside (ITPG)-induction. The faster-migrating bands in lanes 4 and 6 are suspected to be the cleavage products of GST-fusion amelogenin (wild or mutant) protein.
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DISCUSSION
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In this study, we have identified a novel nucleotide alteration of the AMELX encoding for amelogenin protein in a Japanese family with X-linked AI. The alteration was g.3458C>G in exon 5, predicting p.P52R. We concluded that this is a novel mutation of the AMELX, because this g.3458C>G defect co-segregated with all the affected individuals, and was not detected in any unaffected family members or in 100 unrelated control individuals. Furthermore, amelogenin proline 52 is highly conserved among other mammalian species such as bovine, murine, rat, capra, and equidae. So far, 14 allelic AMELX mutations have been reported; among them, only 4 were missense mutations. These mutations include: W4S (Kim et al., 2004), T51I (Lench and Winter, 1995), P70T (Collier et al., 1997; Hart et al., 2000) and H77L (Hart et al., 2002b), all of which were located within highly conserved regions. Thus, this is the fifth AMELX missense mutation resulting in AI. Interestingly, this is the third mutation in exon 5 of the AMELX; the p.P52R mutation is located next to the T51I mutation, and is the same codon as p.P52fx53 (Aldred et al., 1992; Lench et al., 1994), indicating that an important functional domain might be located within this region, and that this region might be a mutation ‘hot spot’.
The phenotype of AI in the present family was quite different between the females and the male; two sisters showed vertical ridges on normal-sized teeth, whereas their father showed small, discolored teeth. These characteristic clinical features led us to consider the diagnosis of AI with X-linked inheritance. The pattern of the individuals’ pedigree was also consistent with X-linked inheritance. Indeed, it has often been reported (Hart et al., 2002a; Kim et al., 2004) that females with X-linked AI show vertical ridges of seemingly normal enamel size, just as in our females. This phenomenon was consistent with the Lyon hypothesis. Thus, although most phenotype-genotype relationships in AI and its causative gene are not yet well-understood, specific features observed in this family appear to be a promising diagnostic indicator for AMELX defects.
To characterize the mutant amelogenin p.P52R, we synthesized it as a recombinant GST fusion protein in vitro. The mutant p.P52R was successfully detected by an anti-amelogenin antibody with Western blotting, similar to the wild-type amelogenin, demonstrating that the p.P52R mutant was stable and of a molecular size similar to that of the wild-type amelogenin. Functional evaluation for the p.P52R amelogenin protein, although unavailable at present, is necessary for us to gain a better understanding of the molecular pathogenesis of AI in this family.
The crystal and chemical compositions of the proband’s teeth were analyzed by XRD and XRF, and compared with that of normal teeth. No differences between affected and unaffected teeth were detected by these analyses. The XRD patterns indicated the presence of hydroxyapatite crystals as the major constituent, and chemical components such as CaO and P2O5 showed no differences (Table), indicating that, despite the abnormal vertical zones in the affected persons’ teeth, the mutant p.P52R amelogenin failed to affect the basic chemical and mineral tooth composition qualitatively. However, it remains unclear if the same results would be obtained upon analysis of the affected father’s teeth, which showed a much more severe phenotype.
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ACKNOWLEDGMENTS
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This study was supported by grants 25060018-15 from the Ministry of Health, Labor and Welfare, 16390293 from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and 17008766 from the Japan Society for the Promotion of Science. We thank Dr. James R. McMillan for proofreading our manuscript.
Received for publication April 4, 2006.
Revision received August 4, 2006.
Accepted for publication October 5, 2006.
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Journal of Dental Research, Vol. 86, No. 1,
69-72 (2007)
DOI: 10.1177/154405910708600111
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ABSTRACT
INTRODUCTION
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