FAM20A mutations and transcriptome analyses of dental pulp tissues of enamel renal syndrome.

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 intrapulpal 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_835delinsTGTCCGACGGTGTCCGACGGTGTC CA (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 overrepresentation of gene sets associated with BMP and SMAD signalling pathways. In contrast, GO terms related to inflammation and axon development were underrepresented. Among BMP signalling 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 signalling 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.

Digital restorative workflows for developmental dental defects in young patients: A case series.

BACKGROUND: Digital technology is rapidly changing the provision of oral health care, although its adoption for the oral health care of young patients has lagged. The authors describe digitally supported treatment approaches for managing treatment of developmental dental defects in the early permanent dentition. CASE DESCRIPTION: Four adolescent patients with amelogenesis imperfecta received transitional anterior restorations for esthetic and functional rehabilitation using a variety of digital workflows. Combinations of restoration type, materials, and fabrication methods were selected to meet the needs of each patient on the basis of their specific amelogenesis imperfecta phenotype and chief symptoms. These cases highlight the application of digital technology in pediatric and adolescent dentistry for managing the treatment of developmental dental defects. PRACTICAL IMPLICATIONS: Digitally supported restorative approaches, as described in this report, offer broad applicability of materials and techniques directed at treating the complex restorative needs of young patients in the transitional and early permanent dentition.

Diagnosing Developmental Defects of Enamel: Pilot Study of Online Training and Accuracy.

PURPOSE: The purpose of this study was to assess dentists' ability to correctly identify and classify development defects of enamel (DDE). METHODS: The modified DDE (MDDE) index was used to classify enamel defects into two types: (1) enamel hypoplasia-pitted, grooved, or missing enamel; or (2) enamel opacity-translucency of enamel not caused by dental caries or fluorosis (can be either demarcated or diffuse). A panel of six experts selected and scored 36 images using the MDDE, and the consensus score was used as the gold standard score in the evaluation of survey respondents. A short training table was developed to match training images to descriptors for the MDDE. A survey, including the training table, was then distributed electronically to 2,036 U.S. dentists and expanded function dental assistants from the Indian Health Service and 6,174 members of American Academy of Pediatric Dentistry. The percent of correct responses was evaluated for each image. RESULTS: Survey respondents (348 total) showed great variability in correct responses for each image, ranging from 41 to 97 percent, for each category of the MDDE. CONCLUSIONS: Enhanced training and calibration on the ability of dental providers is needed to identify the different types of development defects of enamel.

Appropriate Fluoride Toothpaste Application: Improving Caregiver Compliance.

Purpose: Fluoridated toothpaste ingestion is associated with increased dental fluorosis prevalence. Current guidelines recommend a smear of fluoridated toothpaste for children younger than three years old and a pea-sized amount for children age three years or older. The purpose of this study was to evaluate two educational approaches to improve caregiver toothpaste dosing. Methods: Eighty-five caregivers of one- to six-year-olds were surveyed and applied toothpaste to a toothbrush as they normally would for their child. All caregivers then received visual aid (VA) counseling on toothpaste dosing (smear equals 0.09 g, with 0.10 mg fluoride and pea equals 0.22 g with 0.25 mg fluoride). Forty-three caregivers were randomly assigned for teach-back (TB) counseling. Each toothpaste application was weighed. Results: Caregivers (N) equals 56) not familiar with toothpaste guidelines applied more toothpaste at baseline than caregivers familiar with the guidelines (P=0.02). The TB group dispensed toothpaste amounts closer to the ideal compared with the VA group. For all caregivers, VA improved dispensing ability (P=0.008). While TB improved dispensing ability, the difference in average deviation from ideal was not statistically significant (P=0.40) between the TB and VA groups. Conclusions: Teach-back and visual aid counseling methods help caregivers dispense more appropriate toothpaste quantities. Dentists should counsel caregivers of children six years old or younger on fluoride toothpaste dosing and use VA and TB to verify dispensing skills.

Meeting report: a hard look at the state of enamel research.

The Encouraging Novel Amelogenesis Models and Ex vivo cell Lines (ENAMEL) Development workshop was held on 23 June 2017 at the Bethesda headquarters of the National Institute of Dental and Craniofacial Research (NIDCR). Discussion topics included model organisms, stem cells/cell lines, and tissues/3D cell culture/organoids. Scientists from a number of disciplines, representing institutions from across the United States, gathered to discuss advances in our understanding of enamel, as well as future directions for the field.

A systematic review of the association between consumption of sugar-containing beverages and excess weight gain among children under age 12.

OBJECTIVE: A systematic review was conducted to address this clinical question: Does consumption of (non-dairy) sugar-containing beverages (SCBs) among children under age 12 result in excess weight gain? METHODS: The authors searched four databases for controlled trials (randomized and non-randomized) and cohort studies published in English through March 29, 2016: PubMed, EMBASE, Cochrane Database of Systematic Reviews, CINAHL. Initial and full-text screening, data abstraction, and risk of bias assessment were performed independently and in duplicate. RESULTS: Thirty-eight studies met inclusion criteria for this systematic review. One was a randomized controlled trial, and 37 were cohort studies. Though the results of these studies were mixed, the majority demonstrated a statistically significant positive association between SCB consumption in children under age 12 and total adiposity and central adiposity. In contrast, most studies that assessed 100 percent fruit juice consumption only with either total adiposity or central adiposity did not support an association. Among only children under age 5 at baseline, no studies examined central adiposity, but nearly all studies examining SCBs and total adiposity, and a majority examining only fruit juice consumption, demonstrated a statistically significant positive association. CONCLUSION: Our results support a statistically significant positive association between SCBs and total and central adiposity among children under age 12. This association is most consistent for total adiposity among children <5. Our results for 100 percent fruit juice only suggest differences by age, as most studies among those < 12 were negative but most among those <5 were positive.

DENTAL ENAMEL FORMATION AND IMPLICATIONS FOR ORAL HEALTH AND DISEASE.

Dental enamel is the hardest and most mineralized tissue in extinct and extant vertebrate species and provides maximum durability that allows teeth to function as weapons and/or tools as well as for food processing. Enamel development and mineralization is an intricate process tightly regulated by cells of the enamel organ called ameloblasts. These heavily polarized cells form a monolayer around the developing enamel tissue and move as a single forming front in specified directions as they lay down a proteinaceous matrix that serves as a template for crystal growth. Ameloblasts maintain intercellular connections creating a semi-permeable barrier that at one end (basal/proximal) receives nutrients and ions from blood vessels, and at the opposite end (secretory/apical/distal) forms extracellular crystals within specified pH conditions. In this unique environment, ameloblasts orchestrate crystal growth via multiple cellular activities including modulating the transport of minerals and ions, pH regulation, proteolysis, and endocytosis. In many vertebrates, the bulk of the enamel tissue volume is first formed and subsequently mineralized by these same cells as they retransform their morphology and function. Cell death by apoptosis and regression are the fates of many ameloblasts following enamel maturation, and what cells remain of the enamel organ are shed during tooth eruption, or are incorporated into the tooth's epithelial attachment to the oral gingiva. In this review, we examine key aspects of dental enamel formation, from its developmental genesis to the ever-increasing wealth of data on the mechanisms mediating ionic transport, as well as the clinical outcomes resulting from abnormal ameloblast function.

Shear bond strength of dentin and deproteinized enamel of amelogenesis imperfecta mouse incisors.

PURPOSE: The purposes of this study were to: (1) investigate adhesion through shear bond strength (SBS) testing of a resin composite bonded with a self-etching bonding system (SEB) to amelogenesis imperfecta (AI)-affected deproteinized mouse enamel or dentin; and (2) compare wild-type (WT), amelogenin null (AmelxKO), and matrix metalloproteinase-20 null (Mmp20KO) enamel and dentin phenotypes using micro-CT and nanoindentation. METHODS: Enamel incisor surfaces of WT, AmelxKO, and Mmp20KO mice were treated with SEB with and without sodium hypochlorite and tested for SBS. Incisor dentin was also treated with SEB and tested for SBS. These surfaces were further examined by scanning electron miscroscopy. Micro-CT and nanoindentation analyses were performed on mouse dentin and enamel. Data were analyzed for significance by analysis of variance. RESULTS: Deproteinization did not improve SBS of SEB to these AI-affected enamel surfaces. SBS of AmelxKO teeth was similar in dentin and enamel; however, it was higher in Mmp20KO dentin. The nanohardness of knockout enamel was significantly lower than WT, while knockout dentin nanohardness was not different from WT. CONCLUSIONS: Using animal amelogenesis imperfecta models, enamel sodium hypochlorite deproteinization of hypoplastic and hypoplastic-hypomaturation enamel did not increase shear bond strength, while removal of the defective enamel allowed optimal dentin bonding.

Fluoride toothpaste efficacy and safety in children younger than 6 years: a systematic review.

BACKGROUND: The authors conducted a systematic review to assess the efficacy and safety of fluoride toothpaste use in children younger than 6 years. METHODS: The authors defined research questions to formulate a search strategy. They screened studies, extracted data and assessed risk of bias systematically. They conducted meta-analyses to determine the effects of brushing with fluoride toothpaste. RESULTS: Use of fluoride toothpaste brushing had a statistically significant effect on mean decayed, missing and filled primary tooth surfaces and decayed, missing and filled primary teeth for populations at high risk of developing caries (standard mean difference [95 percent confidence interval {CI}], -0.25 [-0.36 to -0.14] and -0.19 [-0.32 to -0.06], respectively). The effects of using different fluoride concentration toothpastes on caries varied. Study findings showed either a decrease in the odds of having fluorosis (odds ratio [OR] [95 percent CI] = 0.66 [0.48-0.90]) when the use of fluoride toothpaste was initiated after 24 months or no statistically significant difference (OR [95 percent CI] = 0.92 [0.71-1.18]). Beginning use after 12 or 14 months of age decreased the risk of fluorosis (OR = 0.70 [0.57-0.88]). CONCLUSIONS: Limited scientific evidence demonstrates that for children younger than 6 years, fluoride toothpaste use is effective in caries control. Ingesting pea-sized amounts or more can lead to mild fluorosis. Practical Implications. To minimize the risk of fluorosis in children while maximizing the caries-prevention benefit for all age groups, the appropriate amount of fluoride toothpaste should be used by all children regardless of age. Dentists should counsel caregivers by using oral description, visual aids and actual demonstration to help ensure that the appropriate amount of toothpaste is used.

Topical fluoride for caries prevention: executive summary of the updated clinical recommendations and supporting systematic review.

BACKGROUND: A panel of experts convened by the American Dental Association (ADA) Council on Scientific Affairs presents evidence-based clinical recommendations regarding professionally applied and prescription-strength, home-use topical fluoride agents for caries prevention. These recommendations are an update of the 2006 ADA recommendations regarding professionally applied topical fluoride and were developed by using a new process that includes conducting a systematic review of primary studies. TYPES OF STUDIES REVIEWED: The authors conducted a search of MEDLINE and the Cochrane Library for clinical trials of professionally applied and prescription-strength topical fluoride agents--including mouthrinses, varnishes, gels, foams and pastes--with caries increment outcomes published in English through October 2012. RESULTS: The panel included 71 trials from 82 articles in its review and assessed the efficacy of various topical fluoride caries-preventive agents. The panel makes recommendations for further research. PRACTICAL IMPLICATIONS: The panel recommends the following for people at risk of developing dental caries: 2.26 percent fluoride varnish or 1.23 percent fluoride (acidulated phosphate fluoride) gel, or a prescription-strength, home-use 0.05 percent fluoride gel or paste or 0.09 percent fluoride mouthrinse for patients 6 years or older. Only 2.26 percent fluoride varnish is recommended for children younger than 6 years. The strengths of the recommendations for the recommended products varied from "in favor" to "expert opinion for." As part of the evidence-based approach to care, these clinical recommendations should be integrated with the practitioner's professional judgment and the patient's needs and preferences.

A systematic review of oral health outcomes produced by dental teams incorporating midlevel providers.

BACKGROUND: The authors conducted a systematic review on this research question: "In populations where nondentists conduct diagnostic, treatment planning, and/or irreversible/ surgical dental procedures, is there a change in disease increment, untreated dental disease, and/or cost-effectiveness of dental care?" METHODS: The authors searched 12 electronic databases for articles published through February 2012 and hand searched relevant articles. They assessed the risk of bias of included studies and extracted data. RESULTS: The authors screened 7,701 citations, resulting in 18 observational studies that met the inclusion criteria. They judged 13 of the studies to be at high risk of bias, five at moderate risk and one at low risk. The authors found no data regarding cost effectiveness, irreversible diagnostic procedures or diseases other than caries. CONCLUSIONS: The authors concluded that the quality of the evidence was poor. They found that in select groups in which participants received irreversible dental treatment from teams that included midlevel providers, caries increment, caries severity or both decreased across time; however, there was no difference in caries increment, caries severity or both compared with those in populations in which dentists provided all irreversible treatment. In select groups in which participants had received irreversible dental treatment from teams that included midlevel providers, there was a decrease in untreated caries across time and a decrease in untreated caries compared with that in populations in which dentists provided all treatment. CLINICAL IMPLICATIONS: Generalizability of results to populations other than those studied is limited owing to the age of some of the studies, as well as to clinical and methodological heterogeneity; consequently, the conclusions should be viewed with caution.

Mouse genetic background influences the dental phenotype.

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 wild-type (WT) mice with different genetic backgrounds (C57BL/6J, C3H/HeJ, FVB/NJ). We hypothesized that 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) can also 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. WTFVB/NJ incisors were more similar to those of Amelx null mice than to those of the other WT strains in terms of incisor height/width 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.

Nonnutritive, low caloric substitutes for food sugars: clinical implications for addressing the incidence of dental caries and overweight/obesity.

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.

Amelogenesis imperfecta: genotype-phenotype studies in 71 families.

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.

Primary failure of eruption and PTH1R: the importance of a genetic diagnosis for orthodontic treatment planning.

INTRODUCTION: Primary failure of eruption (PFE) is characterized by nonsyndromic eruption failure of permanent teeth in the absence of mechanical obstruction. Recent studies support that this dental phenotype is inherited and that mutations in PTH1R genes explain several familial cases of PFE. The objective of our study was to investigate how genetic analysis can be used with clinical diagnostic information for improved orthodontic management of PFE. METHODS: We evaluated a family (n = 12) that segregated an autosomal dominant form of PFE with 5 affected and 7 unaffected persons. Nine available family members (5 male, 4 female) were enrolled and subsequently characterized clinically and genetically. RESULTS: In this family, PFE segregated with a novel mutation in the PTH1R gene. A heterozygous c.1353-1 G>A sequence alteration caused a putative splice-site mutation and skipping of exon 15 that segregated with the PFE phenotype in all affected family members. CONCLUSIONS: A PTH1R mutation is strongly associated with failure of orthodontically assisted eruption or tooth movement and should therefore alert clinicians to treat PFE and ankylosed teeth with similar caution-ie, avoid orthodontic treatment with a continuous archwire.

Oral manifestations in the epidermolysis bullosa spectrum.

The craniofacial and oral manifestations of the different epidermolysis bullosa (EB) types vary markedly in character and severity depending largely on the EB type. The tissues affected and the phenotypes displayed are closely related to the specific abnormal or absent proteins resulting from the causative genetic mutations for these disorders. In this article, the major oral manifestations are reviewed for different EB subtypes and are related to the causative genetic mutations and gene expression.

Regulation of pH During Amelogenesis.

During amelogenesis, extracellular matrix proteins interact with growing hydroxyapatite crystals to create one of the most architecturally complex biological tissues. The process of enamel formation is a unique biomineralizing system characterized first by an increase in crystallite length during the secretory phase of amelogenesis, followed by a vast increase in crystallite width and thickness in the later maturation phase when organic complexes are enzymatically removed. Crystal growth is modulated by changes in the pH of the enamel microenvironment that is critical for proper enamel biomineralization. Whereas the genetic bases for most abnormal enamel phenotypes (amelogenesis imperfecta) are generally associated with mutations to enamel matrix specific genes, mutations to genes involved in pH regulation may result in severely affected enamel structure, highlighting the importance of pH regulation for normal enamel development. This review summarizes the intra- and extracellular mechanisms employed by the enamel-forming cells, ameloblasts, to maintain pH homeostasis and, also, discusses the enamel phenotypes associated with disruptions to genes involved in pH regulation.