Enamelin Research Articles

Comparative expression of the four enamel matrix protein genes, amelogenin, ameloblastin, enamelin and amelotin during amelogenesis in the lizard Anolis carolinensis.

BackgroundIn a recent study, we have demonstrated that amelotin (AMTN) gene structure and its expression during amelogenesis have changed during tetrapod evolution. Indeed, this gene is expressed throughout enamel matrix deposition and maturation in non-mammalian tetrapods, while in mammals its expression is restricted to the transition and maturation stages of amelogenesis. Previous studies of amelogenin (AMEL) gene expression in a lizard and a salamander have shown similar expression pattern to that in mammals, but to our knowledge there are no data regarding ameloblastin (AMBN) and enamelin (ENAM) expression in non-mammalian tetrapods. The present study aims to look at, and compare, the structure and expression of four enamel matrix protein genes, AMEL, AMBN, ENAM and AMTN during amelogenesis in the lizard Anolis carolinensis.ResultsWe provide the full-length cDNA sequence of A. carolinensisAMEL and AMBN, and show for the first time the expression of ENAM and AMBN in a non-mammalian species. During amelogenesis in A. carolinensis, AMEL, AMBN and ENAM expression in ameloblasts is similar to that described in mammals. It is noteworthy that AMEL and AMBN expression is also found in odontoblasts.ConclusionsOur findings indicate that AMTN is the only enamel matrix protein gene that is differentially expressed in ameloblasts between mammals and sauropsids. Changes in AMTN structure and expression could be the key to explain the structural differences between mammalian and reptilian enamel, i.e. prismatic versus non-prismatic.

New genomic and fossil data illuminate the origin of enamel.

Enamel, the hardest vertebrate tissue, covers the teeth of almost all sarcopterygians (lobe-finned bony fishes and tetrapods) as well as the scales and dermal bones of many fossil lobe-fins. Enamel deposition requires an organic matrix containing the unique enamel matrix proteins (EMPs) amelogenin (AMEL), enamelin (ENAM) and ameloblastin (AMBN). Chondrichthyans (cartilaginous fishes) lack both enamel and EMP genes. Many fossil and a few living non-teleost actinopterygians (ray-finned bony fishes) such as the gar, Lepisosteus, have scales and dermal bones covered with a proposed enamel homologue called ganoine. However, no gene or transcript data for EMPs have been described from actinopterygians. Here we show that Psarolepis romeri, a bony fish from the the Early Devonian period, combines enamel-covered dermal odontodes on scales and skull bones with teeth of naked dentine, and that Lepisosteus oculatus (the spotted gar) has enam and ambn genes that are expressed in the skin, probably associated with ganoine formation. The genetic evidence strengthens the hypothesis that ganoine is homologous with enamel. The fossil evidence, further supported by the Silurian bony fish Andreolepis, which has enamel-covered scales but teeth and odontodes on its dermal bones made of naked dentine, indicates that this tissue originated on the dermal skeleton, probably on the scales. It subsequently underwent heterotopic expansion across two highly conserved patterning boundaries (scales/head-shoulder and dermal/oral) within the odontode skeleton.

Interordinal gene capture, the phylogenetic position of Steller’s sea cow based on molecular and morphological data, and the macroevolutionary history of Sirenia

The recently extinct (ca. 1768) Steller’s sea cow (Hydrodamalis gigas) was a large, edentulous North Pacific sirenian. The phylogenetic affinities of this taxon to other members of this clade, living and extinct, are uncertain based on previous morphological and molecular studies. We employed hybridization capture methods and second generation sequencing technology to obtain >30kb of exon sequences from 26 nuclear genes for both H. gigas and Dugong dugon. We also obtained complete coding sequences for the tooth-related enamelin (ENAM) gene. Hybridization probes designed using dugong and manatee sequences were both highly effective in retrieving sequences from H. gigas (mean=98.8% coverage), as were more divergent probes for regions of ENAM (99.0% coverage) that were designed exclusively from a proboscidean (African elephant) and a hyracoid (Cape hyrax). New sequences were combined with available sequences for representatives of all other afrotherian orders. We also expanded a previously published morphological matrix for living and fossil Sirenia by adding both new taxa and nine new postcranial characters. Maximum likelihood and parsimony analyses of the molecular data provide robust support for an association of H. gigas and D. dugon to the exclusion of living trichechids (manatees). Parsimony analyses of the morphological data also support the inclusion of H. gigas in Dugongidae with D. dugon and fossil dugongids. Timetree analyses based on calibration density approaches with hard- and soft-bounded constraints suggest that H. gigas and D. dugon diverged in the Oligocene and that crown sirenians last shared a common ancestor in the Eocene. The coding sequence for the ENAM gene in H. gigas does not contain frameshift mutations or stop codons, but there is a transversion mutation (AG to CG) in the acceptor splice site of intron 2. This disruption in the edentulous Steller’s sea cow is consistent with previous studies that have documented inactivating mutations in tooth-specific loci of a variety of edentulous and enamelless vertebrates including birds, turtles, aardvarks, pangolins, xenarthrans, and baleen whales. Further, branch-site dN/dS analyses provide evidence for positive selection in ENAM on the stem dugongid branch where extensive tooth reduction occurred, followed by neutral evolution on the Hydrodamalis branch. Finally, we present a synthetic evolutionary tree for living and fossil sirenians showing several key innovations in the history of this clade including character state changes that parallel those that occurred in the evolutionary history of cetaceans.

ENAM Mutations with Incomplete Penetrance

Amelogenesis imperfecta (AI) is a genetic disease affecting tooth enamel formation. AI can be an isolated entity or a phenotype of syndromes. To date, more than 10 genes have been associated with various forms of AI. We have identified 2 unrelated Turkish families with hypoplastic AI and performed mutational analysis. Whole-exome sequencing identified 2 novel heterozygous nonsense mutations in the ENAM gene (c.454G>T p.Glu152* in family 1, c.358C>T p.Gln120* in family 2) in the probands. Affected individuals were heterozygous for the mutation in each family. Segregation analysis within each family revealed individuals with incomplete penetrance or extremely mild enamel phenotype, in spite of having the same mutation with the other affected individuals. We believe that these findings will broaden our understanding of the clinical phenotype of AI caused by ENAM mutations.

Molecular basis of human enamel defects

During eruption of teeth in the oral cavity, the effect of gene variations and environmental factors can result in morphological and structural changes in teeth. Amelogenesis imperfecta is a failure which is detected on the enamel of the teeth and clinical picture varies by the severity and type of the disease. Classification of the types of amelogenesis imperfecta is determined by histological, genetic, clinical and radiographic criteria. Specifically, there are 4 types of amelogenesis imperfecta (according to Witkop): hypoplastic form, hypo-maturation form, hypo-calcified form, and hypo-maturation/hypoplasia form with taurodontism and 14 subcategories. The diagnosis and classification of amelogenesis imperfecta has traditionally been based on clinical presentation or phenotype and the inheritance pattern. Several genes can be mutated and cause the disease. Millions of genes, possibly more than 10,000 genes produce proteins that regulate synthesis of enamel. Some of the genes and gene products that are likely associated with amelogenesis imperfecta are: amelogenin (AMELX, AMELY genes), ameloblastin (AMBN gene), enamelin (ENAM gene), enamelysin (MMP20 gene), kalikryn 4 (KLK 4 gene), tuftelins (Tuftelin gene), FAM83H (FAM83H gene) and WDR72 (WDR72 gene). Particular attention should be given by the dentist in recognition and correlation of phenotypes with genotypes, in order to diagnose quickly and accurately such a possible disease and to prevent or treat it easily and quickly. Modern dentistry should restore these lesions in order to guarantee aesthetics and functionality, usually in collaboration with a group of dentists.

Induction of dental epithelial cell differentiation marker gene expression in non-odontogenic human keratinocytes by transfection with thymosin beta 4

Previous studies have shown that the recombination of cells liberated from developing tooth germs develop into teeth. However, it is difficult to use human developing tooth germ as a source of cells because of ethical issues. Previous studies have reported that thymosin beta 4 (Tmsb4x) is closely related to the initiation and development of the tooth germ. We herein attempted to establish odontogenic epithelial cells from non-odontogenic HaCaT cells by transfection with TMSB4X. TMSB4X-transfected cells formed nodules that were positive for Alizarin-red S (ALZ) and von Kossa staining (calcium phosphate deposits) when cultured in calcification-inducing medium. Three selected clones showing larger amounts of calcium deposits than the other clones, expressed PITX2, Cytokeratin 14, and Sonic Hedgehog. The upregulation of odontogenesis-related genes, such as runt-related transcription factor 2 (RUNX2), Amelogenin (AMELX), Ameloblastin (AMBN) and Enamelin (ENAM) was also detected. These proteins were immunohistochemically observed in nodules positive for the ALZ and von Kossa staining. RUNX2-positive selected TMSB4X-transfected cells implanted into the dorsal subcutaneous tissue of nude mice formed matrix deposits. Immunohistochemically, AMELX, AMBN and ENAM were observed in the matrix deposits. This study demonstrated the possibility of induction of dental epithelial cell differentiation marker gene expression in non-odontogenic HaCaT cells by TMSB4X.

Genes expressed in dental enamel development are associated with molar-incisor hypomineralization

Genetic disturbances during dental development influence variation of number and shape of the dentition. In this study, we tested if genetic variation in enamel formation genes is associated with molar-incisor hypomineralization (MIH), also taking into consideration caries experience. DNA samples from 163 cases with MIH and 82 unaffected controls from Turkey, and 71 cases with MIH and 89 unaffected controls from Brazil were studied. Eleven markers in five genes [ameloblastin (AMBN), amelogenin (AMELX), enamelin (ENAM), tuftelin (TUFT1), and tuftelin-interacting protein 11 (TFIP11)] were genotyped by the TaqMan method. Chi-square was used to compare allele and genotype frequencies between cases with MIH and controls. In the Brazilian data, distinct caries experience within the MIH group was also tested for association with genetic variation in enamel formation genes. The ENAM rs3796704 marker was associated with MIH in both populations (Brazil: p=0.03; OR=0.28; 95% C.I.=0.06–1.0; Turkey: p=1.22e−012; OR=17.36; 95% C.I.=5.98–56.78). Associations between TFIP11 (p=0.02), ENAM (p=0.00001), and AMELX (p=0.01) could be seen with caries independent of having MIH or genomic DNA copies of Streptococcus mutans detected by real time PCR in the Brazilian sample. Several genes involved in enamel formation appear to contribute to MIH.

Simple recessive mutation in ENAM is associated with amelogenesis imperfecta in Italian Greyhounds

We report a familial enamel hypoplasia in Italian Greyhounds resembling non-syndromic autosomal recessive amelogenesis imperfecta (AI) of humans. The condition uniformly affects deciduous and permanent teeth and is manifested by enamel roughening/thinning and brownish mottling. Affected teeth are often small and pointed with increased gaps. However, basic tooth structure is usually maintained throughout life, and fractures and dental cavities are not a serious problem as in humans. No tissues or organs other than teeth were affected by this mutation, and there was no relationship between enamel hypoplasia and either autoimmunity or periodontal disease, which also are prevalent in the breed. The enamel hypoplasia was associated with a 5-bp deletion in exon 10 of the enamelin (ENAM) gene. The prevalence of the enamel defect in Italian Greyhounds was 14%, and 30% of dogs with normal teeth were carriers. Genome analyses suggest that the trait is under inadvertent positive selection. Based on the deletion detected in the ENAM gene, a genetic test was developed for identifying mutation carriers, which would enable breeders to manage the trait.

Molecular decay of enamel matrix protein genes in turtles and other edentulous amniotes

BackgroundSecondary edentulism (toothlessness) has evolved on multiple occasions in amniotes including several mammalian lineages (pangolins, anteaters, baleen whales), birds, and turtles. All edentulous amniote clades have evolved from ancestors with enamel-capped teeth. Previous studies have documented the molecular decay of tooth-specific genes in edentulous mammals, all of which lost their teeth in the Cenozoic, and birds, which lost their teeth in the Cretaceous. By contrast with mammals and birds, tooth loss in turtles occurred in the Jurassic (201.6-145.5 Ma), providing an extended time window for tooth gene degradation in this clade. The release of the painted turtle and Chinese softshell turtle genomes provides an opportunity to recover the decayed remains of tooth-specific genes in Testudines.ResultsWe queried available genomes of Testudines (Chrysemys picta [painted turtle], Pelodiscus sinensis [Chinese softshell turtle]), Aves (Anas platyrhynchos [duck], Gallus gallus [chicken], Meleagris gallopavo [turkey], Melopsittacus undulatus [budgerigar], Taeniopygia guttata [zebra finch]), and enamelless mammals (Orycteropus afer [aardvark], Choloepus hoffmanni [Hoffmann’s two-toed sloth], Dasypus novemcinctus [nine-banded armadillo]) for remnants of three enamel matrix protein (EMP) genes with putative enamel-specific functions. Remnants of the AMBN and ENAM genes were recovered in Chrysemys and retain their original synteny. Remnants of AMEL were recovered in both testudines, although there are no shared frameshifts. We also show that there are inactivated copies of AMBN, AMEL and ENAM in representatives of divergent avian lineages including Galloanserae, Passeriformes, and Psittaciformes, and that there are shared frameshift mutations in all three genes that predate the basal split in Neognathae. Among enamelless mammals, all three EMP genes exhibit inactivating mutations in Orycteropus and Choloepus.ConclusionsOur results highlight the power of combining fossil and genomic evidence to decipher macroevolutionary transitions and characterize the functional range of different loci involved in tooth development. The fossil record and phylogenetics combine to predict the occurrence of molecular fossils of tooth-specific genes in the genomes of edentulous amniotes, and in every case these molecular fossils have been discovered. The widespread occurrence of EMP pseudogenes in turtles, birds, and edentulous/enamelless mammals also provides compelling evidence that in amniotes, the only unique, non-redundant function of these genes is in enamel formation.

Immunohistochemical expression of thymosin β4 in ameloblastomas and odontomas.

Ameloblastoma is regarded to be a benign odontogenic tumor, but it is destructive, locally invasive and presents a high rate of recurrence. Thymosin β4 (Tβ4) is closely associated with tooth germ development. Tβ4 also plays a role in malignant progression and invasion. However, little is known about the function of Tβ4 in odontogenic tumors. Thus, we investigated Tβ4 expression in ameloblastomas and compared it with odontomas. We immunohistochemically evaluated the expression of Tβ4, ameloblastin (AMBN), amelogenin (AMEL) and enamelin (ENAM) in 57 samples of ameloblastomas from 40 patients, and also assessed the expression of these molecules in 11 cases of odontomas, two of ameloblastic fibro-odontomas and one of tooth germ-like structures without the formation of enamel and dentin. Tβ4 signals were observed in almost all of the ameloblastomas. The signals were observed in both peripheral columnar cells and central polyhedral/angular cells. Similar findings were observed in tooth germ-like structures, and in the ameloblastomatous nests in the ameloblastic fibro-odontomas. These samples had negative results for AMBN, AMEL and ENAM. Meanwhile, Tβ4 signals were not seen in the odontomas, although immunolabeling for AMBN, AMEL and ENAM was observed in the enamel matrix and in some ameloblasts. Ectomesenhymal regions in the odontomas were negative for staining with the antibodies for AMBN, AMEL and ENAM. These results suggest that Tβ4 could be associated with morphogenesis and tumor invasion in the ameloblastoma, and that Tβ4 may play a role in the behavior of ameloblastoma.

Candidate-gene exclusion in a family with inherited non-syndromic dental disorders

ObjectivesAmelogenesis imperfecta, dentinogenesis imperfecta, and dentin dysplasia are the most common non-syndromic dental disorders. In this study, we analysed and localised the gene(s) responsible for inherited non-syndromic dental disorders in a Chinese family. MethodsThis study identified and researched non-syndromic dental disorders in a four-generation Chinese family, including four affected individuals whose clinical phenotype was atypical. Linkage analysis with seven polymorphic markers that localise to six different autochromosomes showed that the family was linked through chromosome 4q. All exons and exon–intron boundaries of dentin sialophosphoprotein (DSPP), enamelin (ENAM), and ameloblastin (AMBN), which are located on chromosome 4q, were sequenced in nine of the family members. ResultsDirect DNA sequence analysis revealed the existence of a G to A transversion in exon 4 (g.13081786G>A, c.727G>A, p.Asp243Asn, based on reference sequences NM_014208.3) of the DSPP gene, and this sequence variation correlated exactly with the presence of the disease. ConclusionThese results indicate that mutation p.Asp243Asn is a highly probable cause of non-syndromic dental disorder in this Chinese family. The presence of symptom heterogeneity is possible, as the clinical classification system is hampered by the lack of close correlation between the subtype and the molecular defect.

Targeting the Sonic Hedgehog Pathway in Keratocystic Odontogenic Tumor

Keratocystic odontogenic tumors (KCOT) may occur sporadically or associated with the nevoid basal cell carcinoma syndrome. It is a benign aggressive tumor of odontogenic epithelial origin with a high rate of recurrence. A primary human keratocystic odontogenic tumor cell population, KCOT-1, has been established from a tumor explant culture. The KCOT-1 cells were characterized by growth rate, gene expression profiles of major tooth enamel matrix proteins (EMPs), amelogenin (AMELX), enamelin (ENAM), ameloblastin (AMBN), amelotin (AMTN), tumor-related proteins enamelysin (MMP-20), kallikrein-4 (KLK-4), and odontogenic ameloblast-associated protein (ODAM) using quantitative real-time reverse transcription-polymerase chain reaction. Cytokeratin 14 (CK14) was examined by immunohistochemistry. In addition, expression of the members of the sonic hedgehog (SHH) pathway, SHH, patched (PTCH-1), smoothened (SMO), GLI-1, and GLI-2 and of the NOTCH signaling pathway, NOTCH-1, NOTCH-2, NOTCH-3, JAG-2 (Jagged-2), and Delta-like-1 (DLL-1) were evaluated. KCOT-1 cells were treated with SMO antagonist cyclopamine. We found that cyclopamine significantly arrested the growth of KCOT-1 cells in a dose-dependent manner and that the effects of cyclopamine were abolished by adding SHH protein. The protein expression of the SHH pathway was down-regulated by cyclopamine, further confirming that cyclopamine inhibits the SHH signaling pathway; SHH down-regulation correlated with the down-regulation of the NOTCH signaling pathway as well. In conclusion, using an established KCOT-1 cell population, we characterized the gene expression profiles related to the EMPs, SHH, and NOTCH signaling pathway and confirmed that cyclopamine significantly arrested the growth of KCOT-1 cells and may be a viable agent as a novel therapeutic.

Molecular Characterization of Amelogenesis Imperfecta in Chinese Patients

Background: Mutations in 6 genes have been identified as being part of the etiology of amelogenesis imperfecta (AI) with various phenotypes in an isolated condition. Among them the FAM83H gene is the major contributor to the etiology of AI with unknown function. Objective: This study aims to determine the phenotypic and molecular characterization of Chinese AI patients and to analyze the structure and function of the FAM83H protein. Methods: We enrolled 6 hypocalcified AI and 3 hypoplastic AI families from the Chinese population. Mutation analysis was performed by amplifying and sequencing all exons including intron-exon borders for FAM83H and ENAM genes. Structural modeling and function analysis on the FAM83H protein were carried out by bioinformatic processing. Results: No obvious anterior open bite was observed in all the investigated individuals. Five mutations (c.906T>G, c.924dupT, c.973C>T, c.1354C>T and c.2029C>T) in the C-terminal of the FAM83H gene were revealed, respectively, in 5 out of 6 hypocalcified AI families, and a splicing mutation c.534 + 1G>A in the ENAM gene was identified in 1 out of 3 hypoplastic AI families. Structural models of the N- and C-terminal regions of FAM83H were generated by homology modeling. The predicted structure of the FAM83H N-terminal shows resemblance to that of glycosyltransferases with GT-A folds, and the predicted structure of the FAM83H C-terminal possesses similarity to type I collagen protein. Conclusions: To our knowledge, this is the first report of AI with specific molecular variations in families of Chinese descent. Our study provides new insights into the structure and function of the FAM83H protein.

Clinical and molecular analysis of the enamelin gene ENAM in Colombian families with autosomal dominant amelogenesis imperfecta

In this study, we analyzed the phenotype, clinical characteristics and presence of mutations in the enamelin gene ENAM in five Colombian families with autosomal dominant amelogenesis imperfecta (ADAI). 22 individuals (15 affected and seven unaffected) belonging to five Colombian families with ADAI and eight individuals (three affected and five unaffected) belonging to three Colombian families with autosomal recessive amelogenesis imperfecta (ARAI) that served as controls for molecular alterations and inheritance patterns were studied. Clinical, radiographic and genetic evaluations were done in all individuals. Eight exons and three intron-exon boundaries were sequenced for mutation analysis. Two of the five families with ADAI had the hypoplasic phenotype, two had the hypocalcified phenotype and one had the hypomaturative phenotype. Anterior open bite and mandibular retrognathism were the most frequent skeletal abnormalities in the families with ADAI. No mutations were found. These findings suggest that ADAI in these Colombian families was unrelated to previously described mutations in the ENAM gene. These results also indicate that other regions not included in this investigation, such as the promoter region, introns and other genes should be considered as potential ADAI candidates.

Target gene analyses of 39 amelogenesis imperfecta kindreds.

Previously, mutational analyses identified six disease-causing mutations in 24 amelogenesis imperfecta (AI) kindreds. We have since expanded the number of AI kindreds to 39, and performed mutation analyses covering the coding exons and adjoining intron sequences for the six proven AI candidate genes [amelogenin (AMELX), enamelin (ENAM), family with sequence similarity 83, member H (FAM83H), WD repeat containing domain 72 (WDR72), enamelysin (MMP20), and kallikrein-related peptidase 4 (KLK4)] and for ameloblastin (AMBN) (a suspected candidate gene). All four of the X-linked AI families (100%) had disease-causing mutations in AMELX, suggesting that AMELX is the only gene involved in the aetiology of X-linked AI. Eighteen families showed an autosomal-dominant pattern of inheritance. Disease-causing mutations were identified in 12 (67%): eight in FAM83H, and four in ENAM. No FAM83H coding-region or splice-junction mutations were identified in three probands with autosomal-dominant hypocalcification AI (ADHCAI), suggesting that a second gene may contribute to the aetiology of ADHCAI. Six families showed an autosomal-recessive pattern of inheritance, and disease-causing mutations were identified in three (50%): two in MMP20, and one in WDR72. No disease-causing mutations were found in 11 families with only one affected member. We conclude that mutation analyses of the current candidate genes for AI have about a 50% chance of identifying the disease-causing mutation in a given kindred.

Mutation Screening of ENAM , KLK4, MMP20 and FAM83H Genes among the Members of Five Iranian Families Affected with Autosomal Recessive Hypoplastic Amelogenesis Imperfecta

Amelogenesis Imperfectas (AIs) are clinically and genetically heterogeneous conditions characterized by a wide range of clinical features. These abnormalities of enamel formation are categorized into three main groups, hypoplastic, hypomaturation and hypocalcified with different modes of inheritance such as autosomal recessive (AR), autosomal dominant (AD) and X-lined recessive (XLR). In spite of the fact that frequent studies have explained the histological features of AIs, our knowledge regarding the molecular etiology of the affected enamel is not adequate. Up to now, different loci have been suggested to associate with the causation of AIs. Several genetic mutations including enamelin ( ENAM ), amelogenin ( AMELX ), ameloblastin ( AMBN ), tuftelin (TUFT1), kallikrein 4 ( KLK4), matrix metalloproteinase 20 ( MMP20 ) and family with sequence similarity 83, member H (FAM83H) have been suggested to play critical roles in the pathogenesis of these disorders. Therefore, the aim of this investigation was to study of mutation screening in ENAM, KLK4, MMP20 and FAM83H genes, responsible for AIs development in five Iranian families in which the probands were diagnosed with autosomal recessive hypoplastic amelogenesis imperfecta (ARHAI). Genomic DNA was extracted from probands and exon/intron boundaries of afore-mentioned genes were amplified by PCR and subjected to direct sequencing. We could not detect any mutation in the studied genes however; two different novel polymorphisms including T18515C and G18504A were identified in the intron 4 of MMP20 in the probands of two families. Our findings support the notion that different genes may be involved in the development of amelogenesis imperfectas.

Identification of Amelotin‐ and ODAM‐interacting enamel matrix proteins using the yeast two‐hybrid system

The formation of dental enamel is a prototype of functional tissue development through biomineralization. Amelotin (AMTN) is a recently discovered secreted enamel protein predominantly expressed during the maturation stage of enamel formation. It accumulates in a basal lamina-like structure at the interface between ameloblasts and enamel mineral and it co-localizes with another recently described enamel protein, odontogenic ameloblast-associated protein (ODAM). The purpose of this study was to determine whether AMTN and ODAM bind to each other and/or to other well-established enamel matrix proteins. The coding sequences of all enamel proteins were cloned into appropriate vectors of the GAL4-based Matchmaker Gold Yeast Two-Hybrid System. The growth of yeast cells on selective media and color induction were used as indicators for reporter gene expression through protein-protein interactions in combinations of prey and bait constructs. We found that AMTN interacts with itself and with ODAM, but not with amelogenin (AMEL), ameloblastin (AMBN), or enamelin (ENAM). Using ODAM as bait, the interaction with AMTN was confirmed. Furthermore, ODAM was found to bind to itself and to AMBN, as well as weakly to AMEL but not to ENAM. We propose a model where the distinct expression of AMTN and ODAM and their interaction are involved in defining the enamel microstructure at the enamel surface.

A multidisciplinary approach for the diagnosis of hypocalcified amelogenesis imperfecta in two Chilean families

Objective. The purpose of this study was to conduct a multidisciplinary analysis of a specific type of tooth enamel disturbance (amelogenesis imperfecta) affecting two Chilean families to obtain a precise diagnosis and to investigate possible underlying mutations. Materials and methods. Two non-related families affected with amelogenesis imperfecta were evaluated with clinical, radiographic and histopathological methods. Furthermore, pedigrees of both families were constructed and the presence of eight mutations in the enamelin gene (ENAM) and three mutations in the enamelysin gene (MMP-20) were investigated by PCR and direct sequencing. Results. In the two affected patients, the dental malformation presented as soft and easily disintegrated enamel and exposed dark dentin. Neither of the affected individuals presented with a dental and skeletal open bite. Histologically, a high level of an organic matrix with prismatic organization was found. Genetic analysis indicated that the condition is autosomal recessive in one family and either autosomal recessive or due to a new mutation in the other family. Molecular mutational analysis revealed that none of the eight mutations previously described in the ENAM gene or the three mutations in the MMP-20 gene were present in the probands. Conclusion. A multidisciplinary analysis allowed for a diagnosis of hypocalcified amelogenesis imperfecta, Witkop type III, which was unrelated to previously described mutations in the ENAM or MMP-20 genes.

Craniofacial characteristics and genotypes of amelogenesis imperfecta patients

The aims of the study were to identify craniofacial characteristics in patients with the rough hypoplastic amelogenesis imperfecta (AI) phenotype and to evaluate whether craniofacial variables are related to a mutation in either of the two genes associated with AI, enamelin (ENAM) and amelogenin (AMGX). Eight children (five males and three females) with rough hypoplastic AI phenotype, aged 6.5-15 years, from three families and their parents (three males and three females) were examined clinically, radiographically, and genetically. Seventeen variables were measured on lateral cephalometric radiographs in AI affected (n = 11) and AI unaffected (n = 3) members. Craniofacial measurements were statistically analysed using a Student's t-test. In all 14 individuals, mutation analysis of the ENAM and AMGX genes was performed by direct sequencing of the coding region. All AI affected patients had hypoplastic enamel with a rough surface and malocclusions. In the vertical plane, all AI children presented an anterior and/or posterior open bite (OB). Craniofacial analysis confirmed increased vertical relationships, with increased vertical jaw relationships and higher values for gonial angle. In two AI affected families, A and B, the same heterozygous ENAM g.8344delG mutation was confirmed, while in the third family, neither ENAM nor AMGX mutation was found. All patients with rough hypoplastic AI had a moderate to severe malocclusion with increased vertical dimensions regardless of the presence or absence of the ENAM g.8344delG mutation. As an OB requires appropriate timing of therapy, it is important to diagnose these patients as early as possible.

Pseudogenization of the tooth gene enamelysin (MMP20) in the common ancestor of extant baleen whales.

Whales in the suborder Mysticeti are filter feeders that use baleen to sift zooplankton and small fish from ocean waters. Adult mysticetes lack teeth, although tooth buds are present in foetal stages. Cladistic analyses suggest that functional teeth were lost in the common ancestor of crown-group Mysticeti. DNA sequences for the tooth-specific genes, ameloblastin (AMBN), enamelin (ENAM) and amelogenin (AMEL), have frameshift mutations and/or stop codons in this taxon, but none of these molecular cavities are shared by all extant mysticetes. Here, we provide the first evidence for pseudogenization of a tooth gene, enamelysin (MMP20), in the common ancestor of living baleen whales. Specifically, pseudogenization resulted from the insertion of a CHR-2 SINE retroposon in exon 2 of MMP20. Genomic and palaeontological data now provide congruent support for the loss of enamel-capped teeth on the common ancestral branch of crown-group mysticetes. The new data for MMP20 also document a polymorphic stop codon in exon 2 of the pygmy sperm whale (Kogia breviceps), which has enamel-less teeth. These results, in conjunction with the evidence for pseudogenization of MMP20 in Hoffmann's two-toed sloth (Choloepus hoffmanni), another enamel-less species, support the hypothesis that the only unique, non-overlapping function of the MMP20 gene is in enamel formation.