Amelogenesis Imperfecta (AI) is a clinical diagnosis that encompasses a group of genetic mutations, each affecting processes involved in tooth enamel formation and thus, result in various enamel defects. The hypomaturation enamel phenotype has been described for mutations involved in the later stage of enamel formation, including Klk4, Mmp20, C4orf26, and Wdr72. Using a candidate gene approach we discovered a novel Wdr72 human mutation, resulting in a hypomaturation AI phenotype, to be a 5-base pair deletion (c.806_810delGGCAG; p.G255VfsX294). To gain insight into the function of WDR72, we used computer modeling of the full-length human WDR72 protein structure and found that the predicted N-terminal sequence forms two beta-propeller folds with an alpha-solenoid tail at the C-terminus. This domain iteration is characteristic of vesicle coat proteins, such as beta′-COP, suggesting a role for WDR72 in the formation of membrane deformation complexes to regulate intracellular trafficking. Our Wdr72 knockout mouse model (Wdr72−/−), containing a LacZ reporter knock-in, exhibited hypomineralized enamel similar to the AI phenotype observed in humans with Wdr72 mutations. MicroCT scans of Wdr72−/− mandibles affirmed the hypomineralized enamel phenotype occurring at the onset of the maturation stage. H&E staining revealed that Wdr72−/− ameloblasts were shorter, and the enamel matrix was retained during maturation stage. H+/Cl− exchange transporter 5 (CLC5), an early endosome acidifier, was co-localized with WDR72 in maturation-stage ameloblasts and decreased in Wdr72−/− maturation-stage ameloblasts. Other markers along the endocytic degradative pathway were disrupted in Wdr72−/− mice, including clathrin, Dynamin II, and ANXA8. There were no obvious differences in RAB4A and LAMP1 immunostaining of Wdr72−/− mice as compared to wildtype and heterozygous controls. Moreover, Wdr72−/− ameloblasts had reduced amelogenin immunoreactivity, suggesting defects in amelogenin fragment resorption from the matrix. In vivo and in vitro tracer studies with HRP and amelogenin showed delayed processing and loss of transport to lysosomes through the degradative pathway in our newly developed Wdr72−/− ameloblast-like cell line using CRISPR/Cas9. These cells enabled live cell mechanistic studies targeting WDR72 function in vesicle acidification and microtubule recruitment, revealing a complex relationship with a major vesicle acidifying protein, vacuolar H+-ATPase (v-ATPase), and a link to microtubule recruitment. Ultrastructure of Wdr72−/− ameloblasts also showed faulty vesicle formation and ruffled border formation that coincided with pH and calcium matrix defects. These data demonstrate that WDR72 has a major role in enamel mineralization, most notably during the maturation stage, and suggest a function involving endocytic vesicle trafficking, possibly in the removal of amelogenin proteins by regulating microtubule assembly and membrane turnover in the degradative pathway.

Purpose: The purpose was to evaluate the safety and efficacy of a moderate sedation drug regimen consisting of P.O. morphine (0.66 mg/kg, up to a maximum of 30 mg), hydroxyzine (25 mg flat dose), and ibuprofen (100 mg flat dose), as utilized with nitrous oxide and oxygen sedation.

Methods: A convenience sample of 595 sedation records were retrieved from one group of dentists all utilizing the same protocols. The sedation records were chronologically screened and those meeting inclusion and exclusion criteria were subsequently analyzed for descriptive data, oxygen saturation, cardiovascular stability, behavioral data, and adverse events.

Results: Of the original sample of 595 sedation records, 360 (60.5%) met inclusion and exclusion criteria for further analysis. Of the 360 records analyzed, the distribution of males and females was roughly equal (49.4% male, 50.6% female), with a median age of 5.0 years (range: 2.0 years to 11.8 years) and a median weight of 18.6 kg (range 9.5 kg to 43.18 kg). The median wait time was 63 minutes, and the median working time was 70 minutes. Asymptomatic hypotension occurred in 18 of 340 records (5.3%); asymptomatic tachycardia occurred in 20 of 327 records (6.1%), and asymptomatic bradycardia occurred in 14 of 349 records (4.3%). Sedated patients were quiet 72.1% of the time, crying 13.4% of the time, struggling 6.0% of the time, sleeping 4.0% of the time, and engaging in other behaviors 4.6% of the time. Significant adverse events occurred in 5 of 360 records (1.4%), with a true desaturation event occurring in one sedation record (SpO2=87), prolonged or excess sedation occurring in three sedation records, and intraoperative nausea/vomiting occurring in one sedation record.

Conclusions: The results suggest morphine may serve as a viable opiate in P.O. moderate sedation regimens in pediatric dentistry.

During amelogenesis (dental enamel formation), ameloblast cells undergo several morphological and functional changes as they form dental enamel. Interference in the timing of these changes results in enamel of poor quality, affecting the psychosocial development of children at a crucial age of their development. However, the mechanisms that facilitate these transitions in ameloblasts are not well understood. Using rodent models as a basis for study, I used microRNA, fluoride, and iron as tools to carry out histological, immunochemical, and quantitative PCR experiments that investigate the mechanisms that regulate and define ameloblast differentiation and function.

As ameloblasts differentiate from one phase to another during amelogenesis, many genes are simultaneously up- and down- regulated. MicroRNAs serve as one mechanism for rapid control of post-transcriptional expression of many genes. Amelogenin, the predominant protein found in the enamel matrix, is alternatively spiced and may serve as a source for microRNAs. I found that exon 4 of the amelogenin gene produces a microRNA that regulates expression of Runx2, a key transcription factor for when ameloblasts are secreting the enamel matrix.

Ameloblasts express and secrete KLK4, a crucial enzyme that facilitates the final phase of amelogenesis. However, it is unclear how ameloblasts begin increasing Klk4 expression at the right time. I found that androgen receptor (AR) serves as an intracellular regulator of expression for Klk4 and that exposure to fluoride inhibits AR translocation to result in decreased Klk4 expression and synthesis.

Normal rodent ameloblasts deposit iron onto the enamel surface, resulting in yellow-brown colored surface enamel. This pigment is lost in rodents sensitive to high levels of fluoride. Iron in the surface enamel is thought to increase wear resistance in the enamel, though it is unclear how ameloblasts acquire, manage, and transport iron for deposition into enamel. I found that fluoride exposure resulted in a significantly increased expression of ferroportin (iron-exporting protein), and significantly decreased expression of the heavy chain subunit of ferritin, which facilitates the safe storage of iron in cells. Furthermore, it appears that changes in these iron-related proteins by fluoride may be changing free intracellular iron levels to affect expression and synthesis of oxidative stress-related genes.

These studies have shown that multiple mechanisms are responsible for ameloblast differentiation. The effects of fluoride on ameloblast differentiation appear to be largely due to cellular effects of fluoride rather than fluoride related changes in the enamel matrix.

Identification of an Enamelin Defect Resulting in Amelogenesis Imperfecta

Jessica Claire Massie

ABSTRACT

Purpose: The purpose of this study was to identify the genetic defect in a child, which resulted in Amelogenesis Imperfecta (AI) using saliva collected from a routine patient visit.

Methods: A 10-year old female presented to the UCSF Pediatric Dental Clinic with sensitivity and esthetic concerns. She and her 59-year old father, both indicated a history significant for AI. Saliva was collected from the proband, her affected father, and her unaffected mother using the Oragene DNA collection kit from which their DNA was manually purified. A family history was taken to identify the mode of inheritance and enamelin (ENAM) was chosen as a candidate gene. Primer sets were generated to amplify the entire ENAM gene, and amplified products were sequenced.

Results: The family pedigree revealed an autosomal dominant inheritance pattern for AI. A clinical exam revealed a mixed dentition with generalized rough, pitted, yellow-brown enamel consistent with the hypoplastic phenotype reported for AI resulting from a defect in the enamel matrix protein, enamelin. PCR amplification of genomic DNA revealed a novel mutation in exon 7, at g.10602C>G that replaces Pro with Arg, which would alter the protein structure.

Conclusion: When patients present with inherited tooth defects, the dentist's role is to describe the phenotype and obtain a family pedigree of the inheritance pattern. Additionally, collecting a patient's saliva is a simple, painless procedure, which can be used for DNA purification, mutational analysis, and diagnosis of inherited tooth defects such as AI.

Background

Conclusion

Maintaining normal Ca2+ homeostasis is essential for all biological functions. Parathyroid glands (PTGs) were developed in land vertebrates to defend against hypocalcemic challenges by tightly regulated secretion of parathyroid hormone (PTH), which activates the PTH1R in target tissues to increase Ca2+ recycling in kidneys, Ca2+ absorption from the small intestine via indirect renal production of 1,25-dihydroxyvitamin D3 (1,25-D), and Ca2+ release from bone matrices. These PTH-mediated calciotropic activities are subsided by elevated concentrations of serum Ca2+ through actions of its putative receptor, the extracellular Ca2+-sensing receptor (CaSR), in PTGs to suppress PTH secretion, in kidneys to enhance Ca secretion, and in bone to remineralize matrices and suppress bone resorbing activity, together with PTH/ PTH1R, constituting a “yin/yang” feedback mechanism to maintain Ca2+ homeostasis at a steady state. Interestingly, intermittent PTH (iPTH), administered by once-daily injections, can produce skeletal anabolism in the presence of Ca2+ sufficiency. However, more effective dosages could not be achieved for clinical use, mainly due to its intolerable hypercalcemic side-effects. This dissertation explores interplays between the actions of PTH1R and CaSR and exploits these interactions to harness hypercalcemic effects of PTH while enhancing its skeletal anabolism to prevent bone loss and enhance bone fracture repair. Our strategy leverages the ability of co-injecting an allosteric agonist of CaSR (or calcimimetic) to normalize PTH induced hypercalcemia and synergize anabolic actions of CaSR and PTH1R in bone cells. The study employed state-of-the-art technologies, including high-resolution microCT imaging, automated comprehensive serological assays, Nanostring nCounter gene expression profiling, steady and dynamic histomorphometry, biomechanics testing, and novel genetically manipulated murine models. Our findings reveal novel synergistic actions of PTH and CaSR in bone and their underlying mechanisms, which hold powerful clinical implications for future strategies needed to treat osteoporotic disease and skeletal fractures.