ANTICANCER RESEARCH 29: 3065-3068 (2009)
Mutation of Ameloblastin Gene in Calcifying Epithelial Odontogenic Tumor
PAÔLLA FREITAS PERDIGÃO1,2, VINICIUS MAGALHÃES CARVALHO1, LUIZ DE MARCO2 and RICARDO SANTIAGO GOMEZ1
1Department of Oral Surgery and Pathology, School of Dentistry, and 2Department of Pharmacology, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
Abstract. Background: Ameloblastin (AMBN) gene expresses (2). AF occurs mainly in the first and second decades of life, an important protein that acts as a cell adhesion molecule. affecting mandible rather than the maxilla (3). COC is a This protein plays an important role in maintaining the cystic odontogenic tumor with an epithelial lining containing ameloblast secretory stage of differentiation by binding to ghost cells which may undergo calcification, closely them and inhibiting their proliferation. Due to the relationship resembling an ameloblastoma (4). Although the underlying of this protein in the differentiation and proliferation of genetic alterations are poorly understood in AF, mutations in odontogenic cells, here, we investigated this gene in different the β catenin gene were described in COC (5). types of odontogenic tumors. Materials and Methods: Some proteins of the enamel matrix have important roles Sequencing of the all encoding region of AMBN gene was in initiating cytodifferentiation of the dental tissue (6). carried out in four frozen cases of odontogenic tumors: one Ameloblastin (AMBN) is the most important protein case of calcifying epithelial odontogenic tumor (CEOT), two involved in these processes and is highly expressed during calcifying odontogenic cysts (COC) and one ameloblastic the differentiation of inner enamel epithelium (6, 7). The fibroma (AF). Results: DNA sequencing was modified in an AMBN protein is a cell adhesion molecule essential for important domain of the AMBN only in the CEOT. Conclusion: amelogenesis. It is localized near the cell surface and helps The present study suggests that AMBN gene alterations may maintaining the ameloblast secretory stage of differentiation be relevant to the pathogenesis of CEOT. by binding to them and inhibiting their proliferation (8). The human AMBN gene has been cloned and comprises a single- The presence of odontogenic epithelium and/or odontogenic copy gene containing 13 exons with an open reading frame ectomesenchyme is important in the classification of of 1,341 bp that encodes 447 amino acid 11 and maps to odontogenic tumors. The calcifying epithelial odontogenic chromosome 4q21 (9). tumor (CEOT) is a benign epithelial odontogenic tumor We have previously demonstrated that AMBN gene mutations characterized by a locally invasive behavior and affects are associated with the development of ameloblastoma, individuals ranging from 30 to 50 years of age (1). However, adenomatoid odontogenic tumor and squamous odontogenic the molecular mechanisms associated with its development tumor (10). As AMBN protein has an important role in the have not been established. differentiation of ameloblasts and epithelium-mesenchyme Ameloblastic fibroma (AF) and calcifying odontogenic signaling during odontogenesis, we prompted to investigate this cyst (COC, calcifying cystic odontogenic tumor according to gene in CEOT, COC and AF. WHO’s classification of 2005) are tumors that contain odontogenic epithelium and odontogenic ectomesenchyme Materials and Methods
In this report, we studied one CEOT, two COC and one AF. Fragments of these tumors were obtained during surgical removal Correspondence to: Ricardo Santiago Gomez, Faculdade de procedure, after an informed written consent was signed by all Odontologia, Universidade Federal de Minas Gerais, Av. Antonio patients. The University Ethics Committee approved this work. Carlos, 6627 Belo Horizonte, CEP 31270-901 MG, Brazil. Tel: +55 One fragment was fixed in 10% formalin buffer and paraffin- 3134092477, Fax: +55 3134092430, e-mail: [email protected] embedded tissue blocks were used for routine histological staining to confirm the clinical diagnosis. All four tumors studied showed Key Words: Ameloblastin, odontogenic tumors, calcifying epithelial typical histological features described in the literature (2, 4, 11). odontogenic tumor, calcifying odontogenic cyst, ameloblastic Specimen samples were immediately stored at –70˚C for fibroma, mutation. subsequent molecular analyses. In addition, contra-lateral oral
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Figure 2. Sequences of the human AMBN gene and its homologs and/or orthologs/paralogs around the c. 338A>T and c. 339G>A mutation site.
Figure 1. DNA sequence data showing a heterozygous mutation 338A>T and 339G>A (GenBank: NM 0165194).
mucosa swabs were taken from each patient for DNA extraction Discussion followed by DNA sequencing. DNA extraction was carried out as previously described and all coding regions of the AMBN gene studied using specific PCR reaction conditions as previously COC represents about 1% of jaw cysts and although it may described (10, 12). Briefly, PCR reactions were performed in a occur in soft tissue, it presents more commonly within the final volume of 50 μl containing 100 ng of template DNA, 200 bone (14). Both the intra- and extraosseous forms occur with μmol/l dNTPs, 10 pmol/l of each primer and 1.25 U of equal frequency in the maxilla and mandible, mainly in the proofreading taq DNA polymerase (Platinum® Taq DNA incisor and canine areas (15). To date, available data show that Polymerase High Fidelity; Invitrogen, USA). Thirty-five cycles of amplification were performed in a PTC-100-60 thermocycler (MJ β-catenin mutation is the main molecular alteration in COC Research, Watertown, MA, USA) with the appropriate parameters. (5). AF is a benign neoplasm of the odontogenic apparatus DNA sequencing reactions were carried out using the BigDye that occurs mainly in the first and second decades of life (3). terminator kit and DNA Sequencer ABI PRISM 310 Genetic Whilst the majority of AFs are true neoplasms with a potential Analyzer (Applied Biosystems, Foster City, CA, USA). to recur, some of them could represent the primitive stage of a Sequencing was performed in duplicate, both strands, from developing odontoma (16). In the present study, none of these different amplification products. Mutation nomenclature follows tumor types demonstrated molecular alterations of the AMBN published guidelines (13). Numbering of nucleotide and aminoacid refer to the complete cDNA sequence (GenBank: NP gene. Although COC and AF present some histological 057603 and NM 01651941). similarities to ameloblastoma, such as the presence of epithelial basal layer of palisade columnar cells and loosely Results arranged cells resembling stellate reticulum, AMBN gene mutations are restricted to the ameloblastoma (10). Sequencing analyses demonstrated that the AMBN gene was Considering that AMBN has an important role in ameloblast altered in only one of the four tumors investigated. The maturation to the secretory phase, we speculate that the CEOT presented a heterozygous mutation, with the exchange alteration of this protein in ameloblastoma, not found in COC of two base pairs in exon 6 of the AMBN (Figure 1). These and AF, could be related to the absence of an inductive effect alterations were somatic in origin because of their absence on the former. This inductive effect is sometimes reported in in the matching germline DNA obtained from the contra- COC and AF and is characterized by dentinoid formation and lateral oral mucosa. The mutation was characterized by two juxtaepithelial hyalinization, respectively (17). consecutive changes of nucleotide, a transversion (338A>T) Many case reports of CEOT have been described. This and a transition (339G>A) according to GenBank: NM tumor is characterized by the presence of islands and sheets 0165194. These mutations cause the exchange of the of polygonal cells with distinct cellular bridges (4). Although glutamine to leucine (Q113L) in an important and the CEOT shows eosinophilic amyloid-like material, no dentin conservative phosphorylation site PPLPSQPSL (Figure 2). formation is found. In the present study, we demonstrated a To predict whether the amino acid substitution will affect heterozygous mutation in exon 6 of the AMBN in the single protein function, we used the algorithm Sorting Intolerant CEOT studied. The mutation found led to the exchange of from Tolerant (SIFT) (http://blocks.fhcrc.org/sift/SIFT.html). CAG to CTA. The alteration of the DNA modifies the mRNA A SIFT score less than 0.05 is considered deleterious. In the and AMBN protein, leading to an exchange of glutamine, a present study, the amino acid substitutions Q113L showed a neutral and polar hydrophilic amino acid, to leucine, a SIFT score of 0.03. The SIFT scores showed that the amino nonpolar and therefore a hydrophobic amino acid (Q113L). acid substitutions in the AMB protein were deleterious, This mutation occurred in an important phosphorylation site affecting protein function. and in a proline-rich region PPLPSQPSL. However, functional
3066 Perdigão et al: Ameloblastin and Calcifying Epithelial Odontogenic Tumor studies of these mutations have not been performed, their 4 Slootweg PJ: Odontogenic tumours – An update. Curr Diag association with disease, their location in areas of the protein Pathol 12: 54-65, 2006. highly conserved between species (Figure 2) and, most 5 Sekine S, Sato S, Takata T, Fukuda Y, Ishida T, Kishino M, Shibata T, Kanai Y and Hirohashi S: β-Catenin mutations are important, by their absence in normal mucosa suggest that frequent in calcifying odontogenic cysts, but rare in they are pathogenic. ameloblastomas. Am J Pathol 163: 1707-1712, 2003. Areas of eosinophilic cells with prominent intercellular 6 Fincham AG, Moradian-Oldak J and Simmer JP: The structural bridges similar to those observed in CEOT have been described biology of the developing dental enamel matrix. J Struct Biol in a few cases of adenomatoid odontogenic tumors (4). In a 126: 270-299, 1999. previous study, we demonstrated the presence of AMBN gene 7 Begue-Kirn C, Krebsbach PH, Bartlett JD and Butler WT: mutations in adenomatoid odontogenic tumor suggesting that Dentin sialoprotein, dentin phosphoprotein, enamelysin and ameloblastin: tooth-specific molecules that are distinctively both lesions share at least some common genetic pathways expressed during murine dental differentiation. Eur J Oral Sci (10). Additional genetic or epigenetic damage in CEOT could 106: 963-970, 1998. explain its distinct local invasiveness growth. 8 Fukumoto S, Kiba T, Hall B, Iehara N, Nakamura T, Up-to-date molecular biology features have not been used in Longenecker G, Krebsbach PH, Nanci A, Kulkarni AB and the classification of odontogenic tumors. The development and Yamada Y: Ameloblastin is a cell adhesion molecule required for progression of odontogenic tumors are affected by alterations maintaining the differentiation state of ameloblasts. J Cell Biol of many genes and molecules that have been recently reviewed 167: 973-983, 2005. 9 Toyosawa S, Fujiwara T, Ooshima T, Shintani S, Sato A, Ogawa (18). Understanding the underlying molecular mechanisms will Y, Sobue S and Ijuhin N: Cloning and characterization of the help to predict the course of odontogenic tumors and the human ameloblastin gene. Gene 256: 1-11, 2000. development of new therapeutic targets for these lesions (18). It 10 Perdigão PF, Gomez RS, Pimenta FJGS and De Marco L: is important to note that until now, all odontogenic tumors that Ameloblastin gene (AMBN) mutations associated with epithelial had AMBN gene mutations, i.e. CEOT, ameloblastoma, odontogenic tumours. Oral Oncol 40: 841-846, 2004. adenomatoid odontogenic tumor and squamous odontogenic 11 Melrose RJ: Benign epithelial odontogenic tumours. Seminars tumor, belong to the group of odontogenic epithelium with a Diag Pathol 16: 271-287, 1999. 12 Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van mature, fibrous stroma, and without odontogenic Dillen PM and van der Noordaa J: Rapid and simple method for ectomesenchyme. On the other hand, tumors with odontogenic purification of nucleic acids. J Clin Microbiol 28: 495-503, epithelium and odontogenic ectomesenchyme (COC and AF) 1990. studied in the present investigation, albeit in reduced numbers, 13 den Dunnen JT and Antonarakis SE: Nomenclature for the did not show AMBN gene alterations. description of human sequence variations. Hum Genet 109: 121- In conclusion, the present study together with the 124, 2001. literature show that odontogenic tumors with a mature, 14 Shamaskin RG, Svirsky JA and Kaugards GE: Intraosseous and extraosseous calcifying odontogenic cyst (Gorlin Cyst). J Oral fibrous stroma, and without odontogenic ectomesenchyme Maxillofac Surg 47: 562-564, 1989. present AMBN gene alteration. Further studies are necessary 15 Buchner A: The central (intraosseous) calcifying odontogenic to demonstrate the impact of this alteration on the cyst: an analysis of 215 cases. J Oral Maxillofac Surg 49: 330- tumorigenesis of this group of lesions. 339, 1991. 16 Chen Y, Li TJ and Yu SF: Ameloblastic fibroma and related Acknowledgements lesions: a clinicopathologic study with reference to their nature and interrelationship. J Oral Pathol Med 34: 588-595, 2005. This study was supported in part by grants from Fundação de 17 Neville BD, Damm DD, Allen CM and Bouquot JE: Oral & Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Maxillofacial Pathology. Philadelphia: WB Saunders Company, from Milênio/Conselho Nacional de Desenvolvimento Científico e 2002. Tecnológico (CNPq), Brazil. Dr. L De Marco and RS Gomez are 18 Kumamoto H: Molecular pathology of odontogenic tumors. research fellows of CNPq. J Oral Pathol Med 35: 65-74, 2006.
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