No abstract

Enamelin is critical for proper dental enamel formation, and defects in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. We used gene targeting to generate a knock-in mouse carrying a null allele of enamelin (Enam) that has a lacZ reporter gene replacing the Enam translation initiation site and gene sequences through exon 7. Correct targeting of the transgene was confirmed by Southern blotting and PCR analyses. No enamelin protein could be detected by Western blotting in the Enam-null mice. Histochemical 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-gal) staining demonstrated ameloblast-specific expression of enamelin. The enamel of the Enam ؉/؊ mice was nearly normal in the maxillary incisors, but the mandibular incisors were discolored and tended to wear rapidly where they contacted the maxillary incisors. The Enam ؊/؊ mice showed no true enamel. Radiography, microcomputed tomography, and light and scanning electron microscopy were used to document changes in the enamel of Enam ؊/؊ mice but did not discern any perturbations of bone, dentin, or any other tissue besides the enamel layer. Although a thick layer of enamel proteins covered normal-appearing dentin of unerupted teeth, von Kossa staining revealed almost a complete absence of mineral formation in this protein layer. However, a thin, highly irregular, mineralized crust covered the dentin on erupted teeth, apparently arising from the formation and fusion of small mineralization foci (calcospherites) in the deeper part of the accumulated enamel protein layer. These results demonstrate ameloblast-specific expression of enamelin and reveal that enamelin is essential for proper enamel matrix organization and mineralization.

Within nine dentin dysplasia (type II) and dentinogenesis imperfecta (type II and III) patient/families, seven have one of four net −1 deletions within the ~2kb coding repeat domain of the DSPP gene while the remaining two patients had splice-site mutations. All frameshift mutations are predicted to change the highly soluble DSPP protein into proteins with long hydrophobic amino acid repeats that could interfere with processing of normal DSPP and/or other secreted matrix proteins. We propose that all previously reported missense, nonsense, and splice-site DSPP mutations (all associated with exons 2 and 3) result in dominant phenotypes due to disruption of signal peptide-processing and/or related biochemical events that also result in interference with protein processing. This would bring the currently known dominant forms of the human disease phenotype in agreement with the normal phenotype of the heterozygous null Dspp (−/+) mice. A study of 188 normal human chromosomes revealed a hypervariable DSPP repeat domain with extraordinary rates of change including 20 slip-replication indel events and 37 predominantly C-to-T transition SNPs. The most frequent transition in the primordial 9-bp DNA repeat was a sense-strand CpG site while a CpNpG (CAG) transition was the second most frequent SNP. Bisulfite-sequencing of genomic DNA showed that DSPP repeat can be methylated at both motifs. This suggests that, like plants and some animals, human methylate some CpNpG sequences. Analysis of 37 haplotypes of the highly variable DSPP gene from geographically diverse people suggests it may be a useful autosomal marker in human migration studies.

Formation of tooth enamel is a very complex process in which a specific set of proteins secreted by ameloblasts play a primordial role. As part of a screening procedure to identify novel proteins secreted by EO (enamel organ) cells of rat incisors, we isolated a partial cDNA fragment (EO-017) that is the homologue of the recently described mouse Amtn (amelotin) gene [Iwasaki, Bajenova, Somogyi-Ganss, Miller, Nguyen, Nourkeyhani, Gao, Wendel and Ganss (2005) J. Dent. Res. 84, 1127-1132]. Presented herein is the cloning of rat and pig full-length cDNAs with their deduced protein sequences. Detailed expression profiling by Northern-blot analysis and RT (reverse transcriptase)-PCR on rat and mouse tissues revealed highest expression in the mandible, more specifically in the maturation stage of the EO. Among all tissues tested, low expression was detected only in periodontal ligament, lung, thymus and gingiva. In silico analyses revealed that the Amtn gene is highly conserved in seven other mammals, but is absent from fish, birds and amphibians. The Amtn protein is enriched in proline, leucine, glutamine and threonine (52% of total) and contains a perfectly conserved protein kinase CK2 phosphorylation site. Transient transfection experiments in HEK-293 cells (human embryonic kidney cells) showed that secreted Amtn is post-translationally modified possibly through O-linked oligosaccharides on threonine residues. In concordance with its predominant expression site, immunofluorescence localization within the rat and mouse mandibles revealed Amtn localized to the basal lamina of maturation stage ameloblasts of incisors and unerupted molars. Intense Amtn protein expression was also detected in the internal basal lamina of junctional epithelium in molars. The peculiar and unique cellular localization of Amtn suggests a role in cell adhesion.

Amelogenesis imperfecta (AI) represents hereditary conditions affecting the quality and quantity of enamel. Six genes are known to cause AI (AMELX, ENAM, MMP20, KLK4, FAM83H, and WDR72). Our aim was to determine the distribution of different gene mutations in a large AI population and evaluate phenotype-genotype relationships. Affected and unaffected family members were evaluated clinically and radiographically by one examiner. Genotyping was completed using genomic DNA obtained from blood or saliva. A total of 494 individuals were enrolled, with 430 (224 affected, 202 unaffected, and 4 not definitive) belonging to 71 families with conditions consistent with the diagnosis of AI. Diverse clinical phenotypes were observed (i.e. hypoplastic, hypocalcified, and hypomaturation). Genotyping revealed mutations in all 6 candidate genes. A molecular diagnosis was made in 132 affected individuals (59%) and in 26 of the families (37%). Mutations involved 12 families with FAM83H (46%), 6 families with AMELX (23%), 3 families with ENAM (11%), 2 families with KLK4 and MMP20 (8% for each gene), and 1 family with a WDR72 mutation (4%). Phenotypic variants were associated with allelic FAM83H and AMELX mutations. Two seemingly unrelated families had the same KLK4 mutation. Families affected with AI where candidate gene mutations were not identified could have mutations not identifiable by traditional gene sequencing (e.g. exon deletion) or they could have promoter sequence mutations not evaluated in this study. However, the results suggest that there remain new AI causative genes to be identified.

The amelogenesis imperfectas (AIs) are a clinically and genetically diverse group of conditions that are caused by mutations in a variety of genes that are critical for normal enamel formation. To date, mutations have been identified in four genes (AMELX, ENAM, KLK4, MMP20) known to be involved in enamel formation. Additional yet to be identified genes also are implicated in the etiology of AI based on linkage studies. The diverse and often unique phenotypes resulting from the different allelic and non-allelic mutations in these genes provide an opportunity to better understand the role of these genes and their related proteins in enamel formation. Understanding the AI phenotypes also provides an aid to clinicians in directing molecular studies aimed at delineating the genetic basis underlying these diverse clinical conditions. Our current knowledge of the known mutations and associated phenotypes of the different AI subtypes are reviewed.

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An international advisory group met at the National Institutes of Health in Bethesda, Maryland in 2017, to discuss a new classification system for the ectodermal dysplasias (EDs) that would integrate both clinical and molecular information. We propose the following, a working definition of the EDs building on previous classification systems and incorporating current approaches to diagnosis: EDs are genetic conditions affecting the development and/or homeostasis of two or more ectodermal derivatives, including hair, teeth, nails, and certain glands. Genetic variations in genes known to be associated with EDs that affect only one derivative of the ectoderm (attenuated phenotype) will be grouped as non-syndromic traits of the causative gene (e.g., nonsyndromic hypodontia or missing teeth associated with pathogenic variants of EDA "ectodysplasin"). Information for categorization and cataloging includes the phenotypic features, Online Dedication: We dedicate this article to the late Dr. Carlos Salinas, long-time leader who sought continuous improvement in our understanding and classification of the ectodermal dysplasias.