Enamelin Research Articles

Human cementoblasts express enamel-associated molecules in vitro and in vivo

Cementum is a mineralized tissue that facilitates the attachment of periodontal ligament to the root and surrounding alveolar bone and plays a key role in the regeneration of periodontal tissues. The molecular mechanisms that regulate the proliferation and differentiation of cementoblasts, however, have not been elucidated to date. Enamel molecules are believed to regulate cementoblast differentiation and to initiate the formation of acellular extrinsic fiber cementum. The purpose of this study was therefore to isolate and culture human root-derived cells (HRDC) in order to determine whether they are able to express both cementum and specific enamel proteins and subsequently to confirm these findings in vivo. Human root-derived cells were isolated and expanded in vitro. Cells were characterized using RT-PCR, immunostaining, western blotting and by examination of total mRNA to determine the expression of cementum and enamel markers. Human periodontal tissues were also examined for the expression of enamel-related proteins by immunostaining. We showed that HRDC express mRNA corresponding to ameloblastin (AMBN), amelogenin (AMEL), enamelin (ENAM), tuftelin (TUFT) and cementum-associated molecules such as cementum protein 1 (CEMP1) and cementum attachment protein (CAP). Western blotting revealed that HRDC express both AMEL and AMBN gene products, as well as the cementum markers CEMP1 and CAP. In vivo, we have showed that AMBN and AMEL are expressed by cementoblasts lining cementum, paravascular cells and periodontal ligament cells. These results suggest that enamel-associated and cementum-associated proteins could act synergistically in regulating cementoblast differentiation and cementum deposition and offer new approaches on how the cementogenesis process is regulated.

The Enamelin Genes in Lizard, Crocodile, and Frog and the Pseudogene in the Chicken Provide New Insights on Enamelin Evolution in Tetrapods

Enamelin (ENAM) has been shown to be a crucial protein for enamel formation and mineralization. Previous molecular analyses have indicated a probable origin early in vertebrate evolution, which is supported by the presence of enamel/enameloid tissues in early vertebrates. In contrast to these hypotheses, ENAM was only characterized in mammals. Our aims were to 1) look for ENAM in representatives of nonmammalian tetrapods, 2) search for a pseudogene in the chicken genome, and 3) see whether the new sequences could bring new information on ENAM evolution. Using in silico approach and polymerase chain reaction, we obtained and characterized the messenger RNA sequences of ENAM in a frog, a lizard, and a crocodile; the genomic DNA sequences of ENAM in a frog and a lizard; and the putative sequence of chicken ENAM pseudogene. The comparison with mammalian ENAM sequences has revealed 1) the presence of an additional coding exon, named exon 8b, in sauropsids and marsupials, 2) a simpler 5'-untranslated region in nonmammalian ENAMs, 3) many sequence variations in the large exons while there are a few conserved regions in small exons, and 4) 25 amino acids that have been conserved during 350 million years of tetrapod evolution and hence of crucial biological importance. The chicken pseudogene was identified in a region that was not expected when considering the gene synteny in mammals. Together with the location of lizard ENAM in a homologous region, this result indicates that enamel genes were probably translocated in an ancestor of the sauropsid lineage. This study supports the origin of ENAM earlier in vertebrate evolution, confirms that tooth loss in modern birds led to the invalidation of enamel genes, and adds information on the important role played by, for example, the phosphorylated serines and the glycosylated asparagines for correct ENAM functions.

Gene expression and dental enamel structure in developing mouse incisor

At the mouse incisor tip the initially differentiated ameloblasts produce a thin, prism-free enamel, while further apically, in the immediate adjacent segment, the enamel thickness increases and the four-layered enamel of mouse incisor is formed. Comparative gene-expression profiling was carried out on RNA isolated from these two segments of incisor tooth germs at embryonic day (E)17.5 and at postnatal days (P)0, 1, 2, and 10 using microarrays to measure messenger RNA (mRNA) and microRNA (miRNA) species present in the segments. Validation of expression data was achieved using real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Bioinformatic data suggested enhanced cellular apoptosis in the incisal tip segment, which, together with diminished expression of the Amelx and Enam genes, may contribute to the production of the thin enamel seen in this tooth segment. For genes exhibiting higher levels of expression in the adjacent segment where complex enamel is being formed, bioinformatic analysis suggested significant associations with cellular functions involving the actin cytoskeleton, cellular development, morphology, and movement. This is suggested to reflect that ameloblasts with Tomes' process are being organized in transverse rows, facilitating the transverse movement that results in prism decussation in the inner enamel of the adjacent segment. Bioinformatic analysis of miRNA expression data lends support to these suggestions.

A Phylogeny and Timescale for the Evolution of Pseudocheiridae (Marsupialia: Diprotodontia) in Australia and New Guinea.

Pseudocheiridae (Marsupialia: Diprotodontia) is a family of endemic Australasian arboreal folivores, more commonly known as ringtail possums. Seventeen extant species are grouped into six genera (Pseudocheirus, Pseudochirulus, Hemibelideus, Petauroides, Pseudochirops, Petropseudes). Pseudochirops and Pseudochirulus are the only genera with representatives on New Guinea and surrounding western islands. Here, we examine phylogenetic relationships among 13 of the 17 extant pseudocheirid species based on protein-coding portions of the ApoB, BRCA1, ENAM, IRBP, Rag1, and vWF genes. Maximum parsimony, maximum likelihood, and Bayesian methods were used to estimate phylogenetic relationships. Two different relaxed molecular clock methods were used to estimate divergence times. Bayesian and maximum parsimony methods were used to reconstruct ancestral character states for geographic provenance and maximum elevation occupied. We find robust support for the monophyly of Pseudocheirinae (Pseudochirulus + Pseudocheirus), Hemibelidinae (Hemibelideus + Petauroides), and Pseudochiropsinae (Pseudochirops + Petropseudes), respectively, and for an association of Pseudocheirinae and Hemibelidinae to the exclusion of Pseudochiropsinae. Within Pseudochiropsinae, Petropseudes grouped more closely with the New Guinean Pseudochirops spp. than with the Australian Pseudochirops archeri, rendering Pseudochirops paraphyletic. New Guinean species belonging to Pseudochirops are monophyletic, as are New Guinean species belonging to Pseudochirulus. Molecular dates and ancestral reconstructions of geographic provenance combine to suggest that the ancestors of extant New Guinean Pseudochirops spp. and Pseudochirulus spp. dispersed from Australia to New Guinea ∼12.1–6.5 Ma (Pseudochirops) and ∼6.0–2.4 Ma (Pseudochirulus). Ancestral state reconstructions support the hypothesis that occupation of high elevations (>3000 m) is a derived feature that evolved on the terminal branch leading to Pseudochirops cupreus, and either evolved in the ancestor of Pseudochirulus forbesi, Pseudochirulus mayeri, and Pseudochirulus caroli, with subsequent loss in P. caroli, or evolved independently in P. mayeri and P. forbesi. Divergence times within the New Guinean Pseudochirops clade are generally coincident with the uplift of the central cordillera and other highlands. Diversification within New Guinean Pseudochirulus occurred in the Plio-Pleistocene after the establishment of the Central Range and other highlands.Electronic supplementary materialThe online version of this article (doi:10.1007/s10914-010-9129-7) contains supplementary material, which is available to authorized users.

Effect of fluoride therapy on remineralization and whiting of bleached enamel

Objective To evaluate the effect of fluorin dentifrice on the microhardness and whiting of dental enamel bleached with different concentration bleachers.Methods 6%carbamide peroxide(CP)and 30%CP were selected as bleachers.Twenty undefected molars were subjected to 3 groups.Groups A and B.which composed of 8 teeth each,were tested and group C was the control one.Group A(buccal surface)and group B(buccal surface)were treated with 6%CP.Group A (lingual surface)and group B(lingual surface)were treated with 30%CP.They were bleached 30 min every day for two weeks.The teeth in group B were brushed with fluorin dentifrice for 15 min after bleached every time and then all tested samples were kept in the artificial saliva at 37℃.Vickers microhardness of all teeth and color measurement of groups A and B were made before and at the end of bleaching procedure.Results The difference of microhardness values between the bleached and control samples were not statistically significant(P＞0.05).Brushed with fluorin agent significantly increased the hardness of enamelin group B(P＜0.05).The color change was not significant between bleached samples.Conclusion Using fluoride in the interphase of bleaching makes this therapy safer,and does not affect the whitening effect. Key words: Tooth bleaching; Fluoride; Microhardness; Color

Evolutionary Analysis of Mammalian Enamelin, The Largest Enamel Protein, Supports a Crucial Role for the 32-kDa Peptide and Reveals Selective Adaptation in Rodents and Primates

Enamelin (ENAM) plays an important role in the mineralization of the forming enamel matrix. We have performed an evolutionary analysis of mammalian ENAM to identify highly conserved residues or regions that could have important function (selective pressure), to predict mutations that could be associated with amelogenesis imperfecta in humans, and to identify possible adaptive evolution of ENAM during 200 million years ago of mammalian evolution. In order to fulfil these objectives, we obtained 36-ENAM sequences that are representative of the mammalian lineages. Our results show a remarkably high conservation pattern in the region of the 32-kDa fragment of ENAM, especially its phosphorylation, glycosylation, and proteolytic sites. In primates and rodents we also identified several sites under positive selection, which could indicate recent evolutionary changes in ENAM function. Furthermore, the analysis of the unusual signal peptide provided new insights on the possible regulation of ENAM secretion, a hypothesis that should be tested in the near future. Taken together, these findings improve our understanding of ENAM evolution and provide new information that would be useful for further investigation of ENAM function as well as for the validation of mutations leading to amelogenesis imperfecta.

The Structure and Composition of Deciduous Enamel Affected by Local Hypoplastic Autosomal Dominant Amelogenesis Imperfecta Resulting from an ENAM Mutation

In a group of families in northern Sweden, a mutation in the ENAM gene (predicted to produce a highly truncated protein) results in the local hypoplastic form of autosomal dominant amelogenesis imperfecta. In this study, sections of deciduous teeth from members of 3 of these families were examined by scanning electron microscopy (SEM) and the enamel mineral was analysed by energy dispersive X-ray spectroscopy (EDX). The sections were also probed with antibodies raised to a conserved sequence of the enamelin protein. Selected intact teeth were first analysed by digital imaging and ascribed with an ‘Enamel Defects Index’ (EDI) score. SEM of tooth sections revealed disrupted prism morphology and the prisms had a glass-like appearance in some areas. These areas of dysplasia were sometimes irregular but formed regular arrays in others. Comparison of EDI scores with SEM indicated that in one tooth the surface had no measurable defects but significant defects were present in the underlying enamel microstructure. SEM immunohistochemistry with the antibody raised to a fragment of the enamelin protein produced positive, but light, labelling throughout normal enamel. In dysplastic areas, however, the labelling intensity appeared to be reduced. The results indicate that the presence of functional enamelin in the correct amounts is necessary for correct prism morphogenesis. In addition, a combination of EDI and structural analysis indicate that defects in enamel microstructure are not necessarily visible as defects on the surface of the tooth, suggesting the possibility, at least, that some instances of under-diagnosis may occur.

Molecular Tooth Decay

Charles Darwin was intrigued with vestigial organs and, in fact, made them a part of his “long argument” for the existence of evolution in the natural world in Chapter 13 of On the Origin of Species. Now on the 150th anniversary of the publication of that seminal work, a study linking vestigial organs with molecular biology with paleontology has shown even more clearly how vestigial organs are of critical explanatory power in evolutionary theory. In the current issue of PLoS Genetics, Meredith et al. [1] present a comprehensive study of the Enamelin (ENAM) gene in placental mammals that sheds light on the evolutionary process. What is different about this study from others in the literature using gene sequences in the study of evolution is that ENAM sequences and functionality directly mirror the evolution of teeth in living and extinct placental mammals.

Molecular decay of the tooth gene Enamelin (ENAM) mirrors the loss of enamel in the fossil record of placental mammals.

Vestigial structures occur at both the anatomical and molecular levels, but studies documenting the co-occurrence of morphological degeneration in the fossil record and molecular decay in the genome are rare. Here, we use morphology, the fossil record, and phylogenetics to predict the occurrence of “molecular fossils” of the enamelin (ENAM) gene in four different orders of placental mammals (Tubulidentata, Pholidota, Cetacea, Xenarthra) with toothless and/or enamelless taxa. Our results support the “molecular fossil” hypothesis and demonstrate the occurrence of frameshift mutations and/or stop codons in all toothless and enamelless taxa. We then use a novel method based on selection intensity estimates for codons (ω) to calculate the timing of iterated enamel loss in the fossil record of aardvarks and pangolins, and further show that the molecular evolutionary history of ENAM predicts the occurrence of enamel in basal representatives of Xenarthra (sloths, anteaters, armadillos) even though frameshift mutations are ubiquitous in ENAM sequences of living xenarthrans. The molecular decay of ENAM parallels the morphological degeneration of enamel in the fossil record of placental mammals and provides manifest evidence for the predictive power of Darwin's theory.

Human and Mouse Enamel Phenotypes Resulting from Mutation or Altered Expression of AMEL, ENAM, MMP20 and KLK4

Amelogenesis imperfecta (AI) is caused by AMEL, ENAM, MMP20 and KLK4 gene mutations. Mice lacking expression of the AmelX, Enam and Mmp20 genes have been generated. These mouse models provide tools for understanding enamel formation and AI pathogenesis. This study describes the AI phenotypes and relates them to their mouse model counterparts. Human AI phenotypes were determined in a clinical population of AI families and published cases. Human and murine teeth were evaluated using light and electron microscopy. A total of 463 individuals from 54 families were evaluated and mutations in the AMEL, ENAM and KLK4 genes were identified. The majority of human mutations for genes coding enamel nonproteinase proteins (AMEL and ENAM) resulted in variable hypoplasia ranging from local pitting to a marked, generalized enamel thinning. Specific AMEL mutations were associated with abnormal mineralization and maturation defects. Amel and Enam null murine models displayed marked enamel hypoplasia and a complete loss of prism structure. Human mutations in genes coding for the enamel proteinases (MMP20 and KLK4) cause variable degrees of hypomineralization. The murine Mmp20 null mouse exhibits both hypoplastic and hypomineralized defects. The currently available Amel and Enam mouse models for AI exhibit enamel phenotypes (hypoplastic) that are generally similar to those seen in humans. Mmp20 null mice have a greater degree of hypoplasia than humans with MMP20 mutations. Mice lacking expression of the currently known genes associated with the human AI conditions provide useful models for understanding the pathogenesis of these conditions.

Identifying polymorphism in enamelin gene in amelogenesis imperfecta (AI)

Amelogenesis imperfecta (AI) is a developmental defect of dental enamel formation. This enamel defect can be caused by mutation in ENAM gene. Hence this study investigated the molecular defect in the enamelin gene in a patient with the clinical features of AI. The genomic DNA was extracted from patient's whole blood samples and the DNA was subjected to the polymerase chain reaction (PCR) in the presence of 16 pairs of oligonucleotide primers specifically designed to amplify all the 10 exons, g2382, g6395 and g8344 of the enamelin (ENAM) gene in the long arm of the chromosome 4. The PCR products were gel purified and sequenced to identify any mutation. The ENAM gene sequences from the patient were aligned with the reference sequence (GenBank accession no. AY167999) using VectorNTI software. We identified a single base difference at location g359 A → G on exon 1 between the reference sequence and patient's sequence. We successfully ruled out any possible mutation on exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, g2382, g6395 and g8344.

Morphological and Molecular Evidence for a Stepwise Evolutionary Transition from Teeth to Baleen in Mysticete Whales

The origin of baleen in mysticete whales represents a major transition in the phylogenetic history of Cetacea. This key specialization, a keratinous sieve that enables filter-feeding, permitted exploitation of a new ecological niche and heralded the evolution of modern baleen-bearing whales, the largest animals on Earth. To date, all formally described mysticete fossils conform to two types: toothed species from Oligocene-age rocks ( approximately 24 to 34 million years old) and toothless species that presumably utilized baleen to feed (Recent to approximately 30 million years old). Here, we show that several Oligocene toothed mysticetes have nutrient foramina and associated sulci on the lateral portions of their palates, homologous structures in extant mysticetes house vessels that nourish baleen. The simultaneous occurrence of teeth and nutrient foramina implies that both teeth and baleen were present in these early mysticetes. Phylogenetic analyses of a supermatrix that includes extinct taxa and new data for 11 nuclear genes consistently resolve relationships at the base of Mysticeti. The combined data set of 27,340 characters supports a stepwise transition from a toothed ancestor, to a mosaic intermediate with both teeth and baleen, to modern baleen whales that lack an adult dentition but retain developmental and genetic evidence of their ancestral toothed heritage. Comparative sequence data for ENAM (enamelin) and AMBN (ameloblastin) indicate that enamel-specific loci are present in Mysticeti but have degraded to pseudogenes in this group. The dramatic transformation in mysticete feeding anatomy documents an apparently rare, stepwise mode of evolution in which a composite phenotype bridged the gap between primitive and derived morphologies; a combination of fossil and molecular evidence provides a multifaceted record of this macroevolutionary pattern.

A Mutation in the Enamelin Gene in a Mouse Model

Amelogenesis imperfecta is an inherited disorder affecting tooth enamel formation. We previously isolated a mouse strain with an amelogenesis imperfecta phenotype (ATE1 mice) from a dominant ethylnitrosourea screen and mapped the disease-causing defect to a 9-cM region of mouse chromosome 5. In the current study, we tested the hypothesis that there is a mutation in enamelin (ENAM) or ameloblastin (AMBN), both of which are located within the linkage region, by sequencing these two candidate genes. Analysis of our data shows that the amelogenesis imperfecta phenotype is linked to a C > T transition in exon 8 of the enamelin gene. The mutation predicts a C826T transition, which is present in the enamelin transcript and changes the glutamine (Gln) codon at position 176 into a premature stop codon (Gln176X). Conversely, no mutation could be detected in the ameloblastin gene. These results define the ATE1 mice as a model for local hypoplastic autosomal-dominant amelogenesis imperfecta (AIH2), which is caused by enamelin truncation mutations in humans.

The Origin and Evolution of Enamel Mineralization Genes

Background/Aims: Enamel and enameloid were identified in early jawless vertebrates, about 500 million years ago (MYA). This suggests that enamel matrix proteins (EMPs) have at least the same age. We review the current data on the origin, evolution and relationships of enamel mineralization genes. Methods and Results: Three EMPs are secreted by ameloblasts during enamel formation: amelogenin (AMEL), ameloblastin (AMBN) and enamelin (ENAM). Recently, two new genes, amelotin (AMTN) and odontogenic ameloblast associated (ODAM), were found to be expressed by ameloblasts during maturation, increasing the group of ameloblast-secreted proteins to five members. The evolutionary analysis of these five genes indicates that they are related: AMEL is derived from AMBN, AMTN and ODAM are sister genes, and all are derived from ENAM. Using molecular dating, we showed that AMBN/AMEL duplication occurred >600 MYA. The large sequence dataset available for mammals and reptiles was used to study AMEL evolution. In the N- and C-terminal regions, numerous residues were unchanged during >200 million years, suggesting that they are important for the proper function of the protein. Conclusion: The evolutionary analysis of AMEL led to propose a dataset that will be useful to validate AMEL mutations leading to X- linked AI.

Identification of a novel mutation in the enamalin gene in a family with autosomal-dominant amelogenesis imperfecta

Amelogenesis imperfecta (AI) is a heterogeneous genetic disorder that affects the formation of the dental enamel matrix. Mutations in the enamelin ( ENAM) gene have been found in patients with this disorder. The objective of this research was to identify the mutations reported in exons 4, 7 and 9 of the ENAM gene in a single Colombian family with autosomal-dominant AI and to establish the phenotype. The fragments of exons 4, 7 and 9 of the ENAM gene were amplified by polymerase chain reaction and direct sequencing was performed. A mutation was found in exon 9 where guanine was substituted by thymine in one of the alleles in position 817, generating a change of arginine to methionine in codon 179 of the protein. The mutation was only found in affected members of this family who presented with the severe, generalised hypoplastic phenotype in all teeth. The genotype/phenotype correlation for different AI subtypes has not been established. These results support a possible correlation between hypoplastic AI and mutations in the ENAM gene; however, identification of additional mutations could be helpful in establishing phenotype/genotype relationships.

Phenotype and enamel ultrastructure characteristics in patients with ENAM gene mutations g.13185–13186insAG and 8344delG

Objective The main clinical manifestations of amelogenesis imperfecta (AI) include alteration in the quality and quantity of enamel. AI is associated with different mutations in four genes: enamelin ( ENAM), amelogenin ( AMGX), kallikrein ( KLK4) and enamelysin ( MMP-20). Seven different mutations have been identified in the enamelin gene ( ENAM). Design In this paper, we describe the phenotype and ultrastructure of enamel observed using scanning electron microscopy (SEM) in patients with two autosomal dominant (AD) mutations in the ENAM gene: g.13185–13186insAG and g.8344delG, each in one of two unrelated families. Mutations were confirmed by sequence analysis of PCR amplified products of all 10 exons and exon/intron boundaries of the ENAM gene. Results Phenotypic diversity was observed in patients with ENAM gene mutations g.13185–13186insAG with consecutive protein alteration designated as p.P422fsX488 within family 1. In the proband, the enamel of his entire dentition was chalky white with only mild local hypoplastic alteration, while the phenotypic appearance of his father's dentition was that of local hypoplastic AI. In patients with the ENAM gene mutation g.8344delG from family 2 with consecutive protein alteration designated as p.N197fsX277, generalised hypoplastic AI was observed. Conclusions Ultrastructural enamel changes in the patient with the autosomal dominant ENAM g.13185–13186insAG mutation, described for the first time in this study, were less pronounced compared to ultrastructural changes in patients with the autosomal dominant ENAM mutation 8344delG. Ultrastructural characteristics of the g.13185–13186insAG mutation revealed deformed prisms, an oval shape on the cross-section and wider interprism spaces, while enamel with the ENAM mutation 8344delG was laminated, but prismless.

The amelogenin story: origin and evolution

Genome sequencing and gene mapping have permitted the identification of HEVIN (SPARC-Like1) as the probable ancestor of the enamel matrix proteins (EMPs), amelogenin (AMEL), ameloblastin (AMBN) and enamelin (ENAM). We have undertaken a phylogenetic analysis to elucidate their relationships. AMEL genes available in databases, and new sequences obtained in blast searching genomes or expressed sequence tags, were compiled (22 full-length sequences), aligned, and the ancestral sequence calculated and used to search for similarities using psi-blast. Hits were obtained with the N-terminal region of AMBN, ENAM, and HEVIN. We retrieved all available AMBN (n=8), ENAM (n=3), and HEVIN (n=4) sequences. The sequences of the four proteins were aligned and analyzed phylogenetically. AMEL and AMBN are sister genes, which diverged after duplication of a common ancestor issued from ENAM. The latter derived from a copy of HEVIN. Comparisons of gene organization, amino acid sequences and location of ENAM and AMBN, adjacent on the same chromosome, suggest that AMBN is closer to ENAM than AMEL. This supports AMEL as being derived from AMBN duplication. This duplication occurred long before tetrapod differentiation, probably in an ancestral osteichthyan. The story of AMEL origin is completed as follows: SPARC-->HEVIN-->ENAM-->AMBN-->AMEL.

Phenotype of ENAM Mutations is Dosage-dependent

Five mutations in the ENAM gene have been found to cause hypoplastic amelogenesis imperfecta (AI), with phenotypes ranging from localized enamel pitting in carriers to severe hypoplastic AI. To determine the generality of ENAM mutations in hypoplastic AI, we sequenced the ENAM gene in ten Turkish families segregating autosomal hypoplastic AI. In two families, ENAM mutations were found. A novel nonsense mutation (g.12663C>A; p.S246X) was identified in one family segregating local hypoplastic AI as a dominant trait. Affected individuals in a second family segregating autosomal-recessive AI were compound heterozygotes for a novel insertion mutation (g.12946_12947insAGTCAGTACCAGTACTGTGTC) and a previously described insertion (g.13185_13186insAG) mutation. Heterozygous carriers of either insertion had a localized enamel-pitting phenotype. These findings substantiate that enamel phenotypes of ENAM mutations may be dose-dependent, with generalized hypoplastic AI segregating as a recessive trait and localized enamel pitting segregating as a dominant trait.

Variation in dental and skeletal open bite malocclusion in humans with amelogenesis imperfecta

The amelogenesis imperfectas (AI) are a diverse group of genetic disorders primarily affecting the quality and or quantity of enamel, however, affected individuals often have an open bite malocclusion. Three main AI types are recognized based on the perceived developmental mechanisms involved and the enamel phenotype. The purpose of this investigation was to evaluate the association of the AI enamel defect with craniofacial features characteristic of an open bite malocclusion. The sample consisted of 54 AI affected and 34 unaffected family members from 18 different kindreds. Lateral cephalograms were digitized and measurements evaluated for vertical plane alterations using Z-scores. Forty two percent of AI affected individuals and 12% of unaffected family members had dental or skeletal open bite malocclusions. Skeletal open bite malocclusion was variably expressed in AI affected individuals. The enamel phenotype severity did not necessarily correspond with the presence or severity of open bite malocclussion. Open bite malocclusion occurred in individuals with AI caused by mutations in the AMELX and ENAM genes even though these genes are considered to be predominantly or exclusively expressed in teeth. Affected AI individuals with cephalometric values meeting our criteria of skeletal open bite malocclusion were observed in all three major AI types. The pathophysiological relationship between AI associated enamel defects and open bite malocclusion remains unknown.

Enamelin ( Enam ) is essential for amelogenesis: ENU-induced mouse mutants as models for different clinical subtypes of human amelogenesis imperfecta (AI)

Amelogenesis imperfecta (AI) is a group of commonly inherited defects of dental enamel formation, which exhibits marked genetic and clinical heterogeneity. The genetic basis of this heterogeneity is still poorly understood. Enamelin, the affected gene product in one form of AI (AIH2), is an extracellular matrix protein that is one of the components of enamel. We isolated three ENU-induced dominant mouse mutations, M100395, M100514 and M100521, which caused AI-like phenotypes in the incisors and molars of the affected individuals. Linkage analyses mapped each of the three mutations to a region of chromosome 5 that contained the genes encoding enamelin (Enam) and ameloblastin (Ambn). Sequence analysis revealed that each mutation was a single-base substitution in Enam. M100395 (Enam(Rgsc395)) and M100514 (Enam(Rgsc514)) were putative missense mutations that caused S to I and E to G substitutions at positions 55 and 57 of the translated protein, respectively. Enam(Rgsc395) and Enam(Rgsc514) heterozygotes showed severe breakage of the enamel surface, a phenotype that resembled local hypoplastic AI. The M100521 mutation (Enam(Rgsc521)) was a T to A substitution at the splicing donor site in intron 4. This mutation resulted in a frameshift that gave rise to a premature stop codon. The transcript of the Enam(Rgsc521) mutant allele was degraded, indicating that Enam(Rgsc521) is a loss-of-function mutation. Enam(Rgsc521) heterozygotes showed a hypomaturation-type AI phenotype in the incisors, possibly due to haploinsufficiency of Enam. Enam(Rgsc521) homozygotes showed complete loss of enamel on the incisors and the molars. Thus, we report here that the Enam gene is essential for amelogenesis, and that mice with different point mutations at Enam may provide good animal models to study the different clinical subtypes of AI.