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Aim: Biallelic loss-of-function FAM20A mutations cause amelogenesis imperfecta (AI) type IG, better known as enamel renal syndrome (ERS), characterized by severe enamel hypoplasia, delayed/failed tooth eruption, intrapulpal calcifications, gingival hyperplasia, and nephrocalcinosis. FAM20A binds to FAM20C, the Golgi casein kinase (GCK), and potentiates its function to phosphorylate secreted proteins critical for biomineralization. While many FAM20A pathogenic mutations have been reported, the pathogeneses of orodental anomalies in ERS remain to be elucidated. This study aimed to identify disease-causing mutations for patients with ERS phenotypes and to discern the molecular mechanism underlying ERS intrapupal calcifications. Methodology: Phenotypic characterization and whole exome analyses were conducted for 8 families and 2 sporadic cases with hypoplastic AI. A minigene assay was performed to investigate the molecular consequences of a FAM20A splice-site variant. RNA sequencing followed by transcription profiling and gene ontology (GO) analyses were carried out for dental pulp tissues of ERS and the control. Results: Biallelic FAM20A mutations were demonstrated for each affected individual, including 7 novel pathogenic variants: c.590-5T>A, c.625T>A (p.Cys209Ser), c.771del (p.Gln258Argfs*28), c.832_835delinsTGTCCGACGGTGTCCGACGGTGTCCA (p.Val278Cysfs*29), c.1232G>A (p.Arg411Gln), c.1297A>G (p.Arg433Gly), and c.1351del (p.Gln451Serfs*4). The c.590-5T>A splice-site mutation caused Exon 3 skipping, which resulted in an in-frame deletion of a unique region of the FAM20A protein, p.(Asp197_Ile214delinsVal). Analyses of differentially expressed genes in ERS pulp tissues demonstrated that genes involved in biomineralization, particularly dentinogenesis, were significantly upregulated, such as DSPP, MMP9, MMP20, and WNT10A. Enrichment analyses indicated over-representation of gene sets associated with BMP and SMAD signaling pathways. In contrast, GO terms related to inflammation and axon development were under-represented. Among BMP signaling genes, BMP agonists GDF7, GDF15, BMP3, BMP8A, BMP8B, BMP4, and BMP6 were upregulated, while BMP antagonists GREM1, BMPER, and VWC2 showed decreased expression in ERS dental pulp tissues. Conclusions: Upregulation of BMP signaling underlies intrapulpal calcifications in ERS. FAM20A plays an essential role in pulp tissue homeostasis and prevention of ectopic mineralization in soft tissues. This critical function probably depends upon MGP (matrix Gla protein), a potent mineralization inhibitor that must be properly phosphorylated by FAM20A-FAM20C kinase complex.

Dental enamel covers the crown of the vertebrate tooth and is considered to be the hardest tissue in the body. Enamel develops during secretion of an extracellular matrix by ameloblast cells in the tooth germ, prior to eruption of the tooth into the oral cavity. Secreted enamel proteins direct mineralization patterns during the maturation stage of amelogenesis as the tooth prepares to erupt. The amelogenins are the most abundant enamel proteins, and are required for normal enamel development. Phenotypic differences were observed between incisors from individual Amelx (Amelogenin) null mice that had a mixed 129xC57BL/6J genetic background, and between inbred wld-type (WT) mice with different genetic backgrounds (C57BL/6J, C3H/HEJ, FVB/NJ). We hypothesized this could be due to modifier genes, as human patients with a mutation in an enamel protein gene causing the enamel defect amelogenesis imperfecta (AI) also can have varied appearance of dentitions within a kindred. Enamel density measurements varied for all WT inbred strains midway during incisor development. Enamel thickness varied between some WT strains and, unexpectedly, dentin density varied extensively between incisors and molars of all WT and Amelx null strains studied. WT FVB/NJ incisors were more similar to Amelx null than to the other WT strains in incisor height/weight ratio and pattern of enamel mineralization. Strain-specific differences led to the conclusion that modifier genes may be implicated in determining both normal development and severity of enamel appearance in AI mouse models and may in future studies be related to phenotypic heterogeneity within human AI kindreds reported in the literature.

Caries and obesity are two common conditions affecting children in the United States and other developed countries. Caries in the teeth of susceptible children have often been associated with frequent ingestion of fermentable sugars such as sucrose, fructose, glucose, and maltose. Increased calorie intake associated with sugars and carbohydrates, especially when associated with physical inactivity, has been implicated in childhood obesity. Fortunately, nonnutritive artificial alternatives and non-/low-caloric natural sugars have been developed as alternatives to fermentable sugars and have shown promise in partially addressing these health issues. Diet counseling is an important adjunct to oral health instruction. Although there are only five artificial sweeteners that have been approved as food additives by the Food and Drug Administration (FDA), there are additional five non-/low caloric sweeteners that have FDA GRAS (Generally Recognized as Safe) designation. Given the health impact of sugars and other carbohydrates, dental professionals should be aware of the nonnutritive non-/low caloric sweeteners available on the market and both their benefits and potential risks. Dental health professionals should also be proactive in helping identify patients at risk for obesity and provide counseling and referral when appropriate.

This book is intended as a reference that will provide the practitioner with a framework for establishing a diagnosis and developing a suitable treatment plan in patients presenting with a range of developmental defects of the teeth. The conditions covered include failure of tooth eruption, hypodontia, premature tooth exfoliation, defects of enamel development, and defects of dentin development, with full consideration of both syndromic and non-syndromic defects. In each case the phenotype and genotype are first described, followed by diagnostic information, including the availability of genetic testing, and treatment options. Summarizing tables are used to highlight the key diagnostic features, and helpful illustrated case presentations are included. Cleft palate is also addressed, with details on etiology, phenotypes, treatment timing and approaches, and dental management. The closing chapter provides stimulating reflections on potential future directions in the diagnosis and treatment of these disorders, encompassing changes in management related to environmental–genetic interactions, tissue engineering, and materials.

Odontogenic tumors originate from the remains of migrating enamel epithelium after the completion of normal tooth genesis. These enamel epithelium remnants exhibit the ability to recapitulate the events that occur during tooth formation. Several lines of evidence suggest that aberrance in the signaling pathways similar to the ones that are used during tooth development, including the WNT pathway, might be the cause of odontogenic tumorigenesis and maintenance. In this study we demonstrated that WNT5A expression was intense in both the epithelial component of ameloblastomas, the most common epithelial odontogenic tumor, and in this tumor's likely precursor cell, the enamel epithelium located at the cervical loop of normal developing human tooth buds. Additionally, when WNT5A was overexpressed in enamel epithelium cells (LS-8), the clones expressing high levels of WNT5A (S) exhibited characteristics of tumorigenic cells, including growth factor independence, loss of anchorage dependence, loss of contact inhibition, and tumor formation in immunocompromised mice. Moreover, overexpression of WNT5A drastically increased LS-8 cell migration and actin reorganization when compared with controls. Suppression of endogenous WNT5A in LS-8 cells (AS) greatly impaired their migration and AS cells failed to form significant actin reorganization and membrane protrusion was rarely seen. Taken together, our data indicate that WNT5A signaling is important in modulating tumorigenic behaviors of enamel epithelium cells in ameloblastomas.

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.

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.

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.

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-β-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.

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.