Amelogenin-deficient mice display an amelogenesis imperfecta phenotype.

Dental enamel is the hardest tissue in the body and cannot be replaced or repaired, because the enamel secreting cells are lost at tooth eruption. X-linked amelogenesis imperfecta (MIM 301200), a phenotypically diverse hereditary disorder affecting enamel development, is caused by deletions or point mutations in the human X-chromosomal amelogenin gene. Although the precise functions of the amelogenin proteins in enamel formation are not well defined, these proteins constitute 90% of the enamel organic matrix. We have disrupted the amelogenin locus to generate amelogenin null mice, which display distinctly abnormal teeth as early as 2 weeks of age with chalky-white discoloration. Microradiography revealed broken tips of incisors and molars and scanning electron microscopy analysis indicated disorganized hypoplastic enamel. The amelogenin null phenotype reveals that the amelogenins are apparently not required for initiation of mineral crystal formation but rather for the organization of crystal pattern and regulation of enamel thickness. These null mice will be useful for understanding the functions of amelogenin proteins during enamel formation and for developing therapeutic approaches for treating this developmental defect that affects the enamel.     

Identification of a nonsense mutation in the amelogenin gene (AMELX) in a family with X-linked amelogenesis imperfecta (AIH1)

A family with X-linked amelogenesis imperfecta (XAI) is described in which the disease is associated with a nonsense mutation in exon 5 of the amelogenin gene. This mutation involves a single base deletion (CCCCCCC) in the exon in an affected male, his sister and his mother. The effect of this deletion is to alter the reading frame and to introduce an inappropriate TGA stop codon (an opal mutation) into the exonic sequence of the amelogenin gene immediately 3 of the mutation. The clinical features in the examined members of this family indicate that, in some individuals, the most noticeable defect is of enamel hypoplasia. In others, the hypoplastic changes are subtle and might have been overlooked on cursory examination; the most noticeable change is of enamel colour, indicating a degree of hypomineralisation. We propose that the amelogenin gene is implicated in both the formation of enamel of normal thickness and in the normal mineralisation process.     

Reduced hydrolysis of amelogenin may result in X-linked amelogenesis imperfecta

Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine to adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N terminal to the proteinase cleavage site in amelogenin for enamel matrix metalloproteinase-20 (MMP-20), also known as enamelysin. MMP-20 effects the release of tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate MMP-20 hydrolyzes the putative mutated amelogenin cleavage site. The proteolytic site was modeled as a substrate by two synthetic peptides, P1 (SYGYEPMGGWLHHQ) and M1 (SYGYETMGGWLHHQ), selected from residue 36-49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the oligopeptides and the truncated peptides were separated by reversed phase HPLC and identified by mass spectrometry. The results demonstrate that both peptides are cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. However, the apparent first order rate of digestion of the mutation containing peptide by rMMP-20 was approximately 25 times slower than that of the non-mutated peptide. This study suggests that the reduced rate of TRAP formation due to a single amino acid substitution may alter enamel formation and consequently result in amelogenesis imperfecta.     

Evidence for regulation of amelogenin gene expression by 1,25-dihydroxyvitamin D(3) in vivo

The unique hereditary enamel defect clearly related to the disturbance of one enamel matrix protein is X-linked amelogenesis imperfecta (AI), in which several mutations of amelogenin gene have been identified. The clinical phenotype of many of these subjects shows similarities with enamel defects related to rickets. Therefore, we hypothesized that rachitic dental dysplasia is related to disturbances in the amelogenin pathway. In order to test this hypothesis, combined qualitative and quantitative studies in experimental vitamin D-deficient (-D) rat model systems were performed. First, Western blot analysis of microdissected enamel matrix (secretion and maturation stages) showed no clear evidence of dysregulation of amelogenin protein processing in -D rats as compared with the controls. Second, the ultrastructural investigation permitted identification of the internal tissular defect of rachitic enamel, the irregular absence of intraprismatic enamel observed in -D animals, suggesting a possible link between prism morphogenesis and vitamin D. In addition, the steady-state levels of amelogenin mRNAs measured in microdissected dental cells was decreased in -D rats and up-regulated by an unique injection of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). The present study shows evidences that amelogenin expression is regulated by vitamin D. This is the first study of an hormonal regulation of tooth-specific genes.     

Enamelysin (matrix metalloproteinase 20)-deficient mice display an amelogenesis imperfecta phenotype

Enamelysin is a tooth-specific matrix metalloproteinase that is expressed during the early through middle stages of enamel development. The enamel matrix proteins amelogenin, ameloblastin, and enamelin are also expressed during this same approximate developmental time period, suggesting that enamelysin may play a role in their hydrolysis. In support of this interpretation, recombinant enamelysin was previously demonstrated to cleave recombinant amelogenin at virtually all of the precise sites known to occur in vivo. Thus, enamelysin is likely an important amelogenin-processing enzyme. To characterize the in vivo biological role of enamelysin during tooth development, we generated an enamelysin-deficient mouse by gene targeting. Although mice heterozygous for the mutation have no apparent phenotype, the enamelysin null mouse has a severe and profound tooth phenotype. Specifically, the null mouse does not process amelogenin properly, possesses an altered enamel matrix and rod pattern, has hypoplastic enamel that delaminates from the dentin, and has a deteriorating enamel organ morphology as development progresses. Our findings demonstrate that enamelysin activity is essential for proper enamel development.     

Amelogenin sequence and enamel biomineralization in Rana pipiens

The amelogenin gene contributes the majority of tooth enamel proteins and plays a significant role in enamel biomineralization. While several mammalian and reptilian amelogenins have been cloned and sequenced, basal vertebrate amelogenin evolution remains to be understood. In order to start elucidating the structure and function of amelogenins in the evolution of enamel, the leopard frog (Rana pipiens) was used as a model. Tissues from Rana pipiens teeth were analyzed for enamel structure and RNA extracts were processed for sequence analysis. Electron microscopy revealed that Rana pipiens enamel contains long and parallel crystals similar to mammalian enamel, while immunoreactions confirmed the site-specific localization of cross-reactive amelogenins in Rana pipiens enamel. Sequencing of amelogenin PCR products revealed a 782bp cDNA with a 546-nucleotide coding sequence encoding 181 amino acids. The homology of the newly discovered Rana pipiens amelogenin nucleotide and amino acid sequence with the published mouse amelogenin was 38.6% and 45%, respectively. These findings report the first complete amelogenin cDNA sequence in amphibians and indicate a close homology between mammalian enamel formation and Rana pipiens enamel biomineralization.     

Identification and characterization of a squamate reptilian amelogenin gene: Iguana iguana

As the principal components of the developing tooth enamel matrix, amelogenins play a significant role in tooth enamel formation and organization. In order to elucidate the structure and function of amelogenins in the evolution of enamel, we have selected the Iguana iguana as a squamate model organism. Here we report the first complete squamate amelogenin sequence available as of yet and document unique features of Iguana amelogenins and enamel. Transmission electron microscopy documented randomly oriented Iguana enamel crystals during the elongation phase compared with organized enamel crystal patterns at comparable stages in mammals. Sequencing of PCR amplified products revealed a full-length I. iguana amelogenin cDNA containing 877 nucleotides with a 564 nucleotide coding sequence encoding 187 amino acids. The homologies of the newly discovered I. iguana amelogenin amino acid sequence with the published mouse, caiman (Palaeosuchus), and snake (Elaphe) amelogenin were 41.3%, 53.5%, and 55.5%, respectively. On Western blots one major protein with a molecular weight of 24 kDa, and two minor proteins with molecular weights of 28 and 13.5 kDa, respectively, were detected based on the cross-reactivity of antisera against recombinant Rana pipiens amelogenin proteins. Sequence analysis revealed a moderate sequence homology between mammalian and reptilian amelogenin genes. A significant alteration was the deletion of the hydrophilic GSP sequence from exon 3 in the mouse sequence resulting in a conversion to a hydrophobic region in Iguana. Together, these findings identified a novel amelogenin cDNA sequence in the squamate reptilian I. iguana and functional implications for the evolution of amelogenins and enamel in squamates.