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Abstract Dentin sialophosphoprotein (DSPP) is primarily expressed by differentiated odontoblasts (dentin-forming cells), and transiently expressed by presecretory ameloblasts (enamel-forming cells). Disease-causing DSPP mutations predominantly fall into two categories: 5’ mutations affecting targeting and trafficking, and 3’ − 1 frameshift mutations converting the repetitive, hydrophilic, acidic C-terminal domain into a hydrophobic one. We characterized the dental phenotypes and investigated the pathological mechanisms of Dspp P19L and Dspp −1fs mice that replicate the two categories of human DSPP mutations. In Dspp P19L mice, dentin is less mineralized but contains dentinal tubules. Enamel mineral density is reduced. Intracellular accumulation and ER retention of DSPP is observed in odontoblasts and ameloblasts. In Dspp −1fs mice, a thin layer of reparative dentin lacking dentinal tubules is deposited. Odontoblasts show severe pathosis, including intracellular accumulation and ER retention of DSPP, strong ubiquitin and autophagy activity, ER-phagy, and sporadic apoptosis. Ultrastructurally, odontoblasts show extensive autophagic vacuoles, some of which contain fragmented ER. Enamel formation is comparable to wild type. These findings distinguish molecular mechanisms underlying the dental phenotypes of Dspp P19L and Dspp −1fs mice and support the recently revised Shields classification of dentinogenesis imperfecta caused by DSPP mutations in humans. The Dspp −1fs mice may be valuable for the study of autophagy and ER-phagy.

Abstract Human ACP4 (OMIM*606362) encodes a transmembrane protein that belongs to histidine acid phosphatase (ACP) family. Recessive mutations in ACP4 cause non-syndromic hypoplastic amelogenesis imperfecta (AI1J, OMIM#617297). While ACP activity has long been detected in developing teeth, its functions during tooth development and the pathogenesis of ACP4-associated AI remain largely unknown. Here, we characterized 2 AI1J families and identified a novel ACP4 disease-causing mutation: c.774_775del, p.Gly260Aspfs*29. To investigate the role of ACP4 during amelogenesis, we generated and characterized Acp4 R110C mice that carry the p.(Arg110Cys) loss-of-function mutation. Mouse Acp4 expression was the strongest at secretory stage ameloblasts, and the protein localized primarily at Tomes’ processes. While Acp4 heterozygous (Acp4 +/R110C) mice showed no phenotypes, incisors and molars of homozygous (Acp4 R110C/R110C) mice exhibited a thin layer of aplastic enamel with numerous ectopic mineralized nodules. Acp4 R110C/R110C ameloblasts appeared normal initially but underwent pathology at mid-way of secretory stage. Ultrastructurally, sporadic enamel ribbons grew on mineralized dentin but failed to elongate, and aberrant needle-like crystals formed instead. Globs of organic matrix accumulated by the distal membranes of defective Tomes’ processes. These results demonstrated a critical role for ACP4 in appositional growth of dental enamel probably by processing and regulating enamel matrix proteins around mineralization front apparatus.

Abstract Background Ameloblastin (AMBN) is a secreted matrix protein that is critical for the formation of dental enamel and is enamel‐specific with respect to its essential functions. Biallelic AMBN defects cause non‐syndromic autosomal recessive amelogenesis imperfecta. Homozygous Ambn mutant mice expressing an internally truncated AMBN protein deposit only a soft mineral crust on the surface of dentin. Methods We characterized a family with hypoplastic amelogenesis imperfecta caused by AMBN compound heterozygous mutations (c.1061T>C; p.Leu354Pro/ c.1340C>T; p.Pro447Leu). We generated and characterized Ambn knockout/NLS‐lacZ (AmbnlacZ/lacZ) knockin mice. Results No AMBN protein was detected using immunohistochemistry in null mice. ß‐galactosidase activity was specific for ameloblasts in incisors and molars, and islands of cells along developing molar roots. AmbnlacZ/lacZ 7‐week incisors and unerupted (D14) first molars showed extreme enamel surface roughness. No abnormalities were observed in dentin mineralization or in nondental tissues. Ameloblasts in the AmbnlacZ/lacZ mice were unable to initiate appositional growth and started to degenerate and deposit ectopic mineral. No layer of initial enamel ribbons formed in the AmbnlacZ/lacZ mice, but pockets of amelogenin accumulated on the dentin surface along the ameloblast distal membrane and within the enamel organ epithelia (EOE). NLS‐lacZ signal was positive in the epididymis and nasal epithelium, but negative in ovary, oviduct, uterus, prostate, seminal vesicles, testis, submandibular salivary gland, kidney, liver, bladder, and bone, even after 15 hr of incubation with X‐gal. Conclusions Ameloblastin is critical for the initiation of enamel ribbon formation, and its absence results in pathological mineralization within the enamel organ epithelia.

Enamel-renal syndrome (ERS) is an autosomal recessive disorder characterized by severe enamel hypoplasia, failed tooth eruption, intrapulpal calcifications, enlarged gingiva, and nephrocalcinosis. Recently, mutations in FAM20A were reported to cause amelogenesis imperfecta and gingival fibromatosis syndrome (AIGFS), which closely resembles ERS except for the renal calcifications. We characterized three families with AIGFS and identified, in each case, recessive FAM20A mutations: family 1 (c.992G>A; g.63853G>A; p.Gly331Asp), family 2 (c.720-2A>G; g.62232A>G; p.Gln241_Arg271del), and family 3 (c.406C>T; g.50213C>T; p.Arg136* and c.1432C>T; g.68284C>T; p.Arg478*). Significantly, a kidney ultrasound of the family 2 proband revealed nephrocalcinosis, revising the diagnosis from AIGFS to ERS. By characterizing teeth extracted from the family 3 proband, we demonstrated that FAM20A(-/-) molars lacked true enamel, showed extensive crown and root resorption, hypercementosis, and partial replacement of resorbed mineral with bone or coalesced mineral spheres. Supported by the observation of severe ectopic calcifications in the kidneys of Fam20a null mice, we conclude that FAM20A, which has a kinase homology domain and localizes to the Golgi, is a putative Golgi kinase that plays a significant role in the regulation of biomineralization processes, and that mutations in FAM20A cause both AIGFS and ERS.

Several nonsense mutations observed in the FAM83H gene are identified and studied in two families with autosomal-dominant hypocalcified amelogenesis imperfecta (AI). The analysis proves that the FAM83H gene is extremely significant for the proper calcification of the dental-enamel.

Inherited dentin defects are classified into three types of dentinogenesis imperfecta (DGI) and two types of dentin dysplasia (DD). The genetic etiology of DD-I is unknown. Defects in dentin sialophosphoprotein (DSPP) cause DD type II and DGI types II and III. DGI type I is the oral manifestation of osteogenesis imperfecta (OI), a systemic disease typically caused by defects in COL1A1 or COL1A2. Mutations in MSX1, PAX9, AXIN2, EDA and WNT10A can cause non-syndromic familial tooth agenesis. In this study a simplex pattern of clinical dentinogenesis imperfecta juxtaposed with a dominant pattern of hypodontia (mild tooth agenesis) was evaluated, and available family members were recruited. Mutational analyses of the candidate genes for DGI and hypodontia were performed and the results validated. A spontaneous novel mutation in COL1A2 (c.1171G>A; p.Gly391Ser) causing only dentin defects and a novel mutation in PAX9 (c.43T>A; p.Phe15Ile) causing hypodontia were identified and correlated with the phenotypic presentations in the family. Bone radiographs of the proband's dominant leg and foot were within normal limits. We conclude that when no DSPP mutation is identified in clinically determined isolated DGI cases, COL1A1 and COL1A2 should be considered as candidate genes. PAX9 mutation p.Phe15Ile within the N-terminal β-hairpin structure of the PAX9 paired domain causes tooth agenesis.

Enamel formation requires consecutive stages of development to achieve its characteristic extreme mineral hardness. Mineralization depends on the initial presence then removal of degraded enamel proteins from the matrix via endocytosis. The ameloblast membrane resides at the interface between matrix and cell. Enamel formation is controlled by ameloblasts that produce enamel in stages to build the enamel layer (secretory stage) and to reach final mineralization (maturation stage). Each stage has specific functional requirements for the ameloblasts. Ameloblasts adopt different cell morphologies during each stage. Protein trafficking including the secretion and endocytosis of enamel proteins is a fundamental task in ameloblasts. The sites of internalization of enamel proteins on the ameloblast membrane are specific for every stage. In this review, an overview of endocytosis and trafficking of vesicles in ameloblasts is presented. The pathways for internalization and routing of vesicles are described. Endocytosis is proposed as a mechanism to remove debris of degraded enamel protein and to obtain feedback from the matrix on the status of the maturing enamel. [ABSTRACT FROM AUTHOR]

AIM: Using a nationwide population-based dataset, this study investigated the relationship between retinal vein occlusion (RVO) and subsequent acute myocardial infarction (AMI). METHODS: This study is based on a nationwide database released by the Taiwan National Health Research Institute. The study cohort consisted of all ambulatory care patients who were diagnosed as having RVO during 2000∼2003 (n = 591), while the control cohort comprised 2955 randomly selected patients extracted from the same dataset; five patients for every RVO patient, matched by age and gender. Each patient was individually tracked for 3 years from their index ambulatory care visit. Cox proportional hazard regressions were performed to compute the adjusted 3-year AMI-free survival rates, comparing these two cohorts. RESULTS: RVO patients had a significantly higher rate of AMI (1.86% vs 0.78%) during the 3-year follow-up period than patients in the comparison group (p = 0.032). However, after adjusting for the patients’ gender, age, geographic region and comorbid medical disorders, there was no significant difference between the central retinal vein occlusion, branch retinal vein occlusion patients and the comparison group in terms of the hazard of AMI during the 3-year follow-up period. CONCLUSION: RVO did not independently increase the risk of AMI. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the clinical and biological activity of apremilast in patients with severe plaque-type psoriasis.Research design and methods: Apremilast, a phosphodiesterase-4 inhibitor, inhibits in vitro activity of multiple inflammatory factors implicated in the pathogenesis of psoriasis. Patients received 20 mg apremilast orally for 29 days. Immunohistological analysis was conducted on lesional-skin biopsies for psoriasis-associated inflammatory markers. Lipopolysaccharide-stimulated tumor necrosis factor-α levels were evaluated in blood. Psoriasis Area and Severity Index (PASI), static Physician's Global Assessment, and Body Surface Area were used to monitor disease severity.Results: There were 19 patients enrolled in this study, of whom 17 completed the study. Epidermal thickness was reduced by a mean of 20.5% from baseline to day 29. Among the responders, T cells were reduced by 28.8% and 42.6% in the dermis and epidermis, respectively. Similarly, CD11c cells were reduced by 18.5% and 40.2% in the dermis and epidermis, respectively. Fourteen of the 19 (73.7%) patients demonstrated an improvement in their PASI scores.Limitations: This was a small, single-arm, open-label pilot study; therefore there was neither a placebo nor a comparison group.Conclusion: Apremilast demonstrated biological activity and improved psoriasis clinical efficacy scores in patients with severe plaque-type psoriasis. The majority of adverse events were mild in nature. Two adverse events (fatigue and dizziness) were judged by the investigator to be moderate and related to apremilast. In addition, there were no clinically-relevant abnormal laboratory test results in subjects treated with apremilast for 29 days. [ABSTRACT FROM AUTHOR]

The initiation of enamel crystals at the dentino-enamel junction is associated with the expression of dentin sialophosphoprotein (DSPP, a gene normally linked with dentin formation), three ‘structural’ enamel proteins – amelogenin (AMELX), enamelin (ENAM), and ameloblastin (AMBN) – and a matrix metalloproteinase, enamelysin (MMP20). Enamel formation proceeds with the steady elongation of the enamel crystals at a mineralization front just beneath the ameloblast distal membrane, where these proteins are secreted. As the crystal ribbons lengthen, enamelysin processes the secreted proteins. Some of the cleavage products accumulate in the matrix, others are reabsorbed back into the ameloblast. Once crystal elongation is complete and the enamel layer reaches its final thickness, kallikrein 4 (KLK4) facilitates the breakdown and reabsorption of accumulated enamel matrix proteins. The importance of the extracellular matrix proteins to proper tooth development is best illustrated by the dramatic dental phenotypes observed in the targeted knockouts of enamel matrix genes in mice (Dspp,Amelx,Ambn,Mmp20) and in human kindreds with defined mutations in the genes (DSPP,AMELX,ENAM,MMP20,KLK4) encoding these matrix proteins. However, ablation studies alone cannot give specific mechanistic information on how enamel matrix proteins combine to catalyze the formation of enamel crystals. The best approach for determining the molecular mechanism of dental enamel formation is to reconstitute the matrix and synthesize enamel crystals in vitro. Here, we report refinements to the procedures used to isolate porcine enamel and dentin proteins, recent advances in the characterization of enamel matrix protein posttranslational modifications, and summarize the results of human genetic studies that associate specific mutations in the genes encoding matrix proteins with a range of dental phenotypes. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

To understand carbon cycling in marginal seas better, particulate organic carbon (POC) concentrations, POC fluxes and primary production (PP) were measured in the East China Sea (ECS) in summer 2007. Higher concentrations of POC were observed in the inner shelf, and lower POC values were found in the outer shelf. Similar to POC concentrations, elevated uncorrected POC fluxes (720–7300 mg C m−2 d−1) were found in the inner shelf, and lower POC fluxes (80–150 mg C m−2 d−1) were in the outer shelf, respectively. PP values (~ 340–3380 mg C m−2 d−1) had analogous distribution patterns to POC fluxes, while some of PP values were significantly lower than POC fluxes, suggesting that contributions of resuspended particles to POC fluxes need to be appropriately corrected. A vertical mixing model was used to correct effects of bottom sediment resuspension, and the lowest and highest corrected POC fluxes were in the outer shelf (58 ± 33 mg C m−2 d−1) and the inner shelf (785 ± 438 mg C m−2 d−1), respectively. The corrected POC fluxes (486 to 785 mg C m−2 d−1) in the inner shelf could be the minimum value because we could not exactly distinguish the effect of POC flux from Changjiang influence with turbid waters. The results suggest that 27–93% of the POC flux in the ECS might be from the contribution of resuspension of bottom sediments rather than from the actual biogenic carbon sinking flux. While the vertical mixing model is not a perfect model to solve sediment resuspension because it ignores biological degradation of sinking particles, Changjiang plume (or terrestrial) inputs and lateral transport, it makes significant progress in both correcting the resuspension problem and in assessing a reasonable quantitative estimate of POC flux in a marginal sea.

To assess the impact of rising atmospheric CO2 and eutrophication on the carbonate chemistry of the East China Sea shelf waters, saturation states (Ω) for two important biologically relevant carbonate minerals – calcite (Ωc) and aragonite (Ωa) – were calculated throughout the water column from dissolved inorganic carbon (DIC) and total alkalinity (TA) data collected in spring and summer of 2009. Results show that the highest Ωc (∼9.0) and Ωa (∼5.8) values were found in surface water of the Changjiang plume area in summer, whereas the lowest values (Ωc = ∼2.7 and Ωa = ∼1.7) were concurrently observed in the bottom water of the same area. This divergent behavior of saturation states in surface and bottom waters was driven by intensive biological production and strong stratification of the water column. The high rate of phytoplankton production, stimulated by the enormous nutrient discharge from the Changjiang, acts to decrease the ratio of DIC to TA, and thereby increases Ω values. In contrast, remineralization of organic matter in the bottom water acts to increase the DIC to TA ratio, and thus decreases Ω values. The projected result shows that continued increases of atmospheric CO2 under the IS92a emission scenario will decrease Ω values by 40–50% by the end of this century, but both the surface and bottom waters will remain supersaturated with respect to calcite and aragonite. Nevertheless, superimposed on such Ω decrease is the increasing eutrophication, which would mitigate or enhance the Ω decline caused by anthropogenic CO2 uptake in surface and bottom waters, respectively. Our simulation reveals that, under the combined impact of eutrophication and augmentation of atmospheric CO2, the bottom water of the Changjiang plume area will become undersaturated with respect to aragonite (Ωa = ∼0.8) by the end of this century, which would threaten the health of the benthic ecosystem.

A tremendous amount of organic carbon respired by plankton communities has been found in summer in the East China Sea (ECS), and this rate has been significantly correlated with fluvial discharge from the Changjiang River. However, respiration data has rarely been collected in other seasons. To evaluate and reveal the potential controlling mechanism of organic carbon consumption in spring in the ECS, two cruises covering almost the entire ECS shelf were conducted in the spring of 2009 and 2010. These results showed that although the fluvial discharge rates were comparable to the high riverine flow in summer, the plankton community respiration (CR) varied widely between the two springs. In 2009, the level of CR was double that of 2010, with mean (± SD) values of 111.7 (±76.3) and 50.7 (±62.9) mg C m−3 d−1, respectively. The CR was positively correlated with concentrations of particulate organic carbon and/or chlorophyll a (Chl a) in 2009 (all p < 0.01). These results suggest that the high CR rate in 2009 can be attributed to high planktonic biomasses. During this period, phytoplankton growth flourished due to allochthonous nutrients discharged from the Changjiang River. Furthermore, higher phytoplankton growth led to the absorption of an enormous amount of fugacity of CO2 (fCO2) in the surface waters, even with a significant amount of inorganic carbon regenerated via CR. In 2010, even more riverine runoff nutrients were measured in the ECS than in 2009. Surprisingly, the growth of phytoplankton in 2010 was not stimulated by enriched nutrients, and its growth was likely limited by low water temperature and/or low light intensity. Low temperature might also suppress planktonic metabolism, which could explain why the CR was lower in 2010. During this period, lower surface water fCO2 may have been driven mainly by physical process(es). To conclude, these results indicate that high organic carbon consumption (i.e. CR) in the spring of 2009 could be attributed to high planktonic biomasses, and the lower CR rate during the cold spring of 2010 might be likely limited by low temperature in the ECS. This further suggests that the high inter-annual variability of organic carbon consumption needs to be kept in mind when budgeting the annual carbon balance.

Severe tropical storms play an important role in triggering phytoplankton blooms, but the extent to which such storms influence biogenic carbon flux from the euphotic zone is unclear. In 2008, typhoon Fengwong provided a unique opportunity to study the in situ biological responses including phytoplankton blooms and particulate organic carbon fluxes associated with a severe storm in the southern East China Sea (SECS). After passage of the typhoon, the sea surface temperature (SST) in the SECS was markedly cooler (∼25 to 26 °C) than before typhoon passage (∼28 to 29 °C). The POC flux 5 days after passage of the typhoon was 265 ± 14 mg C m−2 d−1, which was ∼1.7-fold that (140–180 mg C m−2 d−1) recorded during a period (June–August, 2007) when no typhoons occurred. A somewhat smaller but nevertheless significant increase in POC flux (224–225 mg C m−2 d−1) was detected following typhoon Sinlaku which occurred approximately 1 month after typhoon Fengwong, indicating that typhoon events can increase biogenic carbon flux efficiency in the SECS. Remarkably, phytoplankton uptake accounted for only about 5% of the nitrate injected into the euphotic zone by typhoon Fengwong. It is likely that phytoplankton population growth was constrained by a combination of light limitation and grazing pressure. Modeled estimates of new/export production were remarkably consistent with the average of new and export production following typhoon Fengwong. The same model suggested that during non-typhoon conditions approximately half of the export of organic carbon occurs via convective mixing of dissolved organic carbon, a conclusion consistent with earlier work at comparable latitudes in the open ocean.

For the interpretation of the storm event-induced landslide distribution for an area, deterministic methods are frequently used, while a region's landslide susceptibility is commonly predicted via a statistical approach based upon multi-temporal landslide inventories and environmental factors. In this study we try to use an event-based landslide inventory, a set of environmental variables and a triggering factor to build a susceptibility model for a region which is solved using a multivariate statistical method. Data for shallow landslides triggered by the 2002 typhoon, Toraji, in central western Taiwan, are selected for training the susceptibility model. The maximum rainfall intensity of the storm event is found to be an effective triggering factor affecting the landslide distribution and this is used in the model. The model is built for the Kuohsing region and validated using data from the neighboring Tungshih area and a subsequent storm event – the 2004 typhoon, Mindulle, which affected both the Kuohsing and the Tungshih areas. The results show that we can accurately interpret the landslide distribution in the study area and predict the occurrence of landslides in the neighboring region in a subsequent typhoon event. The advantage of this statistical method is that neither hydrological data, strength data, failure depth, nor a long-period landslide inventory is needed as input.

We have developed a new strategy for differential screening of genes that are expressed in two or more tissues, and have used it to identify genes that are preferentially expressed in both testis and ovary. In this approach, testis-specific cDNAs were first generated using the suppression subtractive hybridization technique, cloned and arrayed in microplates. The inserts from the subtracted testis-specific library were amplified by PCR and spotted on filters. The dot blots were screened by hybridization using either testis- or ovary-specific subtracted cDNA mixtures as probes. By screening about 2000 putative clones from the subtracted testis-specific library, we identified 14 candidate genes that hybridized to both cDNA probes. Northern blot analysis showed that 12 clones were preferentially or exclusively expressed in testis and ovary. Nucleotide sequence analysis indicated that three of the identified clones represented cDNAs from novel genes.

Abstract Non-syndromic inherited defects of tooth dentin are caused by two classes of dominant negative/gain-of-function mutations in dentin sialophosphoprotein (DSPP): 5′ mutations affecting an N-terminal targeting sequence and 3′ mutations that shift translation into the − 1 reading frame. DSPP defects cause an overlapping spectrum of phenotypes classified as dentin dysplasia type II and dentinogenesis imperfecta types II and III. Using CRISPR/Cas9, we generated a Dspp −1fs mouse model by introducing a FLAG-tag followed by a single nucleotide deletion that translated 493 extraneous amino acids before termination. Developing incisors and/or molars from this mouse and a Dspp P19L mouse were characterized by morphological assessment, bSEM, nanohardness testing, histological analysis, in situ hybridization and immunohistochemistry. Dspp P19L dentin contained dentinal tubules but grew slowly and was softer and less mineralized than the wild-type. Dspp P19L incisor enamel was softer than normal, while molar enamel showed reduced rod/interrod definition. Dspp −1fs dentin formation was analogous to reparative dentin: it lacked dentinal tubules, contained cellular debris, and was significantly softer and thinner than Dspp +/+ and Dspp P19L dentin. The Dspp −1fs incisor enamel appeared normal and was comparable to the wild-type in hardness. We conclude that 5′ and 3′ Dspp mutations cause dental malformations through different pathological mechanisms and can be regarded as distinct disorders.

Abstract Oligodontia is the congenital absence of six or more teeth and comprises the more severe forms of tooth agenesis. Many genes have been implicated in the etiology of tooth agenesis, which is highly variable in its clinical presentation. The purpose of this study was to identify associations between genetic mutations and clinical features of oligodontia patients. An online systematic search of papers published from January 1992 to June 2021 identified 381 oligodontia cases meeting the eligibility criteria of causative gene mutation, phenotype description, and radiographic records. Additionally, ten families with oligodontia were recruited and their genetic etiologies were determined by whole-exome sequence analyses. We identified a novel mutation in WNT10A (c.99_105dup) and eight previously reported mutations in WNT10A (c.433 G > A; c.682 T > A; c.318 C > G; c.511.C > T; c.321 C > A), EDAR (c.581 C > T), and LRP6 (c.1003 C > T, c.2747 G > T). Collectively, 20 different causative genes were implicated among those 393 cases with oligodontia. For each causative gene, the mean number of missing teeth per case and the frequency of teeth missing at each position were calculated. Genotype–phenotype correlation analysis indicated that molars agenesis is more likely linked to PAX9 mutations, mandibular first premolar agenesis is least associated with PAX9 mutations. Mandibular incisors and maxillary lateral incisor agenesis are most closely linked to EDA mutations.

Abstract Dental enamel forms extracellularly as thin ribbons of amorphous calcium phosphate (ACP) that initiate on dentin mineral in close proximity to the ameloblast distal membrane. Secreted proteins are critical for this process. Enam −/− and Ambn −/− mice fail to form enamel. We characterize enamel ribbon formation in wild-type (WT), Amelx −/− and Mmp20 −/− mouse mandibular incisors using focused ion beam scanning electron microscopy (FIB-SEM) in inverted backscatter mode. In Amelx −/− mice, initial enamel mineral ribbons extending from dentin are similar in form to those of WT mice. As early enamel development progresses, the Amelx −/− mineral ribbons develop multiple branches, resembling the staves of a Japanese fan. These striking fan-shaped structures cease growing after attaining ~ 20 µm of enamel thickness (WT is ~ 120 µm). The initial enamel mineral ribbons in Mmp20 −/− mice, like those of the Amelx −/− and WT, extend from the dentin surface to the ameloblast membrane, but appear to be fewer in number and coated on their sides with organic material. Remarkably, Mmp20 −/− mineral ribbons also form fan-like structures that extend to ~ 20 µm from the dentin surface. However, these fans are subsequently capped with a hard, disorganized outer mineral layer. Amelogenin cleavage products are the only matrix components absent in both Amelx −/− and Mmp20 −/− mice. We conclude that MMP20 and amelogenin are not critical for enamel mineral ribbon initiation, orientation, or initial shape. The pathological fan-like plates in these mice may form from the lack of amelogenin cleavage products, which appear necessary to form ordered hydroxyapatite.

Abstract Mutations of Odontogenesis-Associated Phosphoprotein (ODAPH, OMIM *614829) cause autosomal recessive amelogenesis imperfecta, however, the function of ODAPH during amelogenesis is unknown. Here we characterized normal Odaph expression by in situ hybridization, generated Odaph truncation mice using CRISPR/Cas9 to replace the TGC codon encoding Cys41 into a TGA translation termination codon, and characterized and compared molar and incisor tooth formation in Odaph +/+, Odaph +/C41*, and Odaph C41*/C41* mice. We also searched genomes to determine when Odaph first appeared phylogenetically. We determined that tooth development in Odaph +/+ and Odaph +/C41* mice was indistinguishable in all respects, so the condition in mice is inherited in a recessive pattern, as it is in humans. Odaph is specifically expressed by ameloblasts starting with the onset of post-secretory transition and continues until mid-maturation. Based upon histological and ultrastructural analyses, we determined that the secretory stage of amelogenesis is not affected in Odaph C41*/C41* mice. The enamel layer achieves a normal shape and contour, normal thickness, and normal rod decussation. The fundamental problem in Odaph C41*/C41* mice starts during post-secretory transition, which fails to generate maturation stage ameloblasts. At the onset of what should be enamel maturation, a cyst forms that separates flattened ameloblasts from the enamel surface. The maturation stage fails completely.

Abstract Background Matrix metallopeptidase 20 (MMP20) is an evolutionarily conserved protease that is essential for processing enamel matrix proteins during dental enamel formation. MMP20 mutations cause human autosomal recessive pigmented hypomaturation‐type amelogenesis imperfecta (AI2A2; OMIM #612529). MMP20 is expressed in both odontoblasts and ameloblasts, but its function during dentinogenesis is unclear. Methods We characterized 10 AI kindreds with MMP20 defects, characterized human third molars and/or Mmp20−/− mice by histology, Backscattered Scanning Electron Microscopy (bSEM), µCT, and nanohardness testing. Results We identified six novel MMP20 disease‐causing mutations. Four pathogenic variants were associated with exons encoding the MMP20 hemopexin‐like (PEX) domain, suggesting a necessary regulatory function. Mutant human enamel hardness was softest (13% of normal) midway between the dentinoenamel junction (DEJ) and the enamel surface. bSEM and µCT analyses of the third molars revealed reduced mineral density in both enamel and dentin. Dentin close to the DEJ showed an average hardness number 62%–69% of control. Characterization of Mmp20−/− mouse dentin revealed a significant reduction in dentin thickness and mineral density and a transient increase in predentin thickness, indicating disturbances in dentin matrix secretion and mineralization. Conclusion These results expand the spectrum of MMP20 disease‐causing mutations and provide the first evidence for MMP20 function during dentin formation.

Abstract Background ENAM mutations cause autosomal dominant or recessive amelogenesis imperfecta (AI) and show a dose effect: enamel malformations are more severe or only penetrant when both ENAM alleles are defective. Methods Whole exome sequences of recruited AI probands were initially screened for mutations in known AI candidate genes. Sanger sequencing was used to confirm sequence variations and their segregation with the disease phenotype. The co‐occurrence of ENAM and LAMA3 mutations in one family raised the possibility of digenic inheritance. Enamel formed in Enam+/+Ambn+/+, Enam+/−, Ambn+/−, and Enam+/−Ambn+/− mice was characterized by dissection and backscattered scanning electron microscopy (bSEM). Results ENAM mutations segregating with AI in five families were identified. Two novel ENAM frameshift mutations were identified. A single‐nucleotide duplication (c.395dupA/p.Pro133Alafs*13) replaced amino acids 133‐1142 with a 12 amino acid (ATTKAAFEAAIT*) sequence, and a single‐nucleotide deletion (c.2763delT/p.Asp921Glufs*32) replaced amino acids 921‐1142 with 31 amino acids (ESSPQQASYQAKETAQRRGKAKTLLEMMCPR*). Three families were heterozygous for a previously reported single‐nucleotide ENAM deletion (c.588+1delG/p.Asn197Ilefs*81). One of these families also harbored a heterozygous LAMA3 mutation (c.1559G>A/p.Cys520Tyr) that cosegregated with both the AI phenotype and the ENAM mutation. In mice, Ambn+/− maxillary incisors were normal. Ambn+/− molars were also normal, except for minor surface roughness. Ambn+/− mandibular incisors were sometimes chalky and showed minor chipping. Enam+/− incisor enamel was thinner than normal with ectopic mineral deposited laterally. Enam+/− molars were sometimes chalky and rough surfaced. Enam+/−Ambn+/− enamel was thin and rough, in part due to ectopic mineralization, but also underwent accelerated attrition. Conclusion Novel ENAM mutations causing AI were identified, raising to 22 the number of ENAM variations known to cause AI. The severity of the enamel phenotype in Enam+/−Ambn+/− double heterozygous mice is caused by composite digenic effects. Digenic inheritance should be explored as a cause of AI in humans.

Abstract Background Truncation FAM83H mutations cause human autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI), an inherited disorder characterized by severe hardness defects in dental enamel. No enamel defects were observed in Fam83h null mice suggesting that Fam83h truncation mice would better replicate human mutations. Methods We generated and characterized a mouse model (Fam83hTr/Tr) expressing a truncated FAM83H protein (amino acids 1–296), which recapitulated the ADHCAI‐causing human FAM83H p.Tyr297* mutation. Results Day 14 and 7‐week Fam83hTr/Tr molars exhibited rough enamel surfaces and slender cusps resulting from hypoplastic enamel defects. The lateral third of the Fam83hTr/Tr incisor enamel layer was thinner, with surface roughness and altered enamel rod orientation, suggesting disturbed enamel matrix secretion. Regular electron density in mandibular incisor enamel indicated normal enamel maturation. Only mildly increased posteruption attrition of Fam83hTr/Tr molar enamel was observed at 7‐weeks. Histologically, the Fam83hTr/Tr enamel organ, including ameloblasts, and enamel matrices at sequential stages of amelogenesis exhibited comparable morphology without overt abnormalities, except irregular and less evident ameloblast Tomes' processes in specific areas. Conclusions Considering Fam83h−/− mice showed no enamel phenotype, while Fam83hTr/Tr (p.Tyr297*) mice displayed obvious enamel malformations, we conclude that FAM83H truncation mutations causing ADHCAI in humans disturb amelogenesis through a neomorphic mechanism, rather than haploinsufficiency.

Amelogenesis imperfecta (AI) is a collection of rare genetic conditions affecting tooth enamel. The affected enamel can be of insufficient quantity and/or altered quality, impacting structural content, surface integrity and coloration. Heterozygous mutations in ENAM result in hypoplastic AI without other syndromic phenotypes, with variable expressivity and reduced penetrance, unlike other AI-associated genes. In this study, we recruited a Caucasian family with hypoplastic AI. Mutational analysis (using whole exome sequencing) revealed a splicing donor site mutation (NM_031889.3: c. −61 + 1G > A). Mutational effects caused by this variant were investigated with a minigene splicing assay and in vitro expression analysis. The mutation resulted in a retention of intron 1 and exon 2 (a normally skipped exon), and this elongated 5′ UTR sequence attenuated the translation from the mutant mRNA. Structure and translation predictions raised the possibility that the long complex structures—especially a hairpin structure located right before the translation initiation codon of the mutant mRNA—caused reduced protein expression. However, there could be additional contributing factors, including additional uORFs. For the first time, we determined that a mutation altered the ENAM 5′ UTR, but maintained the normal coding amino acid sequence, causing hypoplastic AI.

Amelogenesis imperfecta is a group of rare inherited disorders that affect tooth enamel formation, quantitatively and/or qualitatively. The aim of this study was to identify the genetic etiologies of two families presenting with hypomaturation amelogenesis imperfecta. DNA was isolated from peripheral blood samples obtained from participating family members. Whole exome sequencing was performed using DNA samples from the two probands. Sequencing data was aligned to the NCBI human reference genome (NCBI build 37.2, hg19) and sequence variations were annotated with the dbSNP build 138. Mutations in MMP20 were identified in both probands. A homozygous missense mutation (c.678T>A; p.His226Gln) was identified in the consanguineous Family 1. Compound heterozygous MMP20 mutations (c.540T>A, p.Tyr180* and c.389C>T, p.Thr130Ile) were identified in the non-consanguineous Family 2. Affected persons in Family 1 showed hypomaturation AI with dark brown discoloration, which is similar to the clinical phenotype in a previous report with the same mutation. However, the dentition of the Family 2 proband exhibited slight yellowish discoloration with reduced transparency. Functional analysis showed that the p.Thr130Ile mutant protein had reduced activity of MMP20, while there was no functional MMP20 in the Family 1 proband. These results expand the mutational spectrum of the MMP20 and broaden our understanding of genotype-phenotype correlations in amelogenesis imperfecta.

The structure, magnetic properties and magnetostriction of high-pressure synthesized Pr1−xDyx(Fe0.8Co0.2)1.93 compounds were investigated. These compounds, which could not be readily synthesized under ambient pressure, exhibit single cubic Laves phase owing to the effects of high-pressure annealing. The Curie temperature increases with increasing x, indicating that 3d-4f coupling becomes stronger with the increase of Dy concentration. The saturation magnetization decreases with increasing x, which can be ascribed to the competition of sublattice magnetization. The easy magnetization direction of the compound lies along with x ≤ 0.05 while lies along when x ≥ 0.10. Meanwhile, the low-field magnetostriction λ∥ − λ⊥ of the compound system peaks at x = 0.05 and then decreases with increasing x, which reveals that the composition anisotropy compensation between Pr3+ and Dy3+ might be realized in Pr1−xDyx(Fe0.8Co0.2)1.93 system. Pr0.95Dy0.05(Fe0.8Co0.2)1.93 compound combines a large low-field magnetostriction (648 ppm at 3 kOe) and the merits of low-cost raw materials, which may make it a potential material for magnetostrictive application.

Mutations in the human enamelin gene cause autosomal dominant hypoplastic amelogenesis imperfecta in which the affected enamel is thin or absent. Study of enamelin knockout NLS-lacZ knockin mice revealed that mineralization along the distal membrane of ameloblast is deficient, resulting in no true enamel formation. To determine the function of enamelin during enamel formation, we characterized the developing teeth of the Enam-/- mice, generated amelogenin-driven enamelin transgenic mouse models, and then introduced enamelin transgenes into the Enam-/- mice to rescue enamel defects. Mice at specific stages of development were subjected to morphologic and structural analysis using β-galactosidase staining, immunohistochemistry, and transmission and scanning electron microscopy. Enamelin expression was ameloblast-specific. In the absence of enamelin, ameloblasts pathology became evident at the onset of the secretory stage. Although the aggregated ameloblasts generated matrix-containing amelogenin, they were not able to create a well-defined enamel space or produce normal enamel crystals. When enamelin is present at half of the normal quantity, enamel was thinner with enamel rods not as tightly arranged as in wild type suggesting that a specific quantity of enamelin is critical for normal enamel formation. Enamelin dosage effect was further demonstrated in transgenic mouse lines over expressing enamelin. Introducing enamelin transgene at various expression levels into the Enam-/- background did not fully recover enamel formation while a medium expresser in the Enam+/- background did. Too much or too little enamelin abolishes the production of enamel crystals and prism structure. Enamelin is essential for ameloblast integrity and enamel formation.

Amelogenesis imperfecta (AI) is a group of inherited conditions featuring isolated enamel malformations. About 5% of AI cases show an X-linked pattern of inheritance, which are caused by mutations in AMELX. In humans there are two, non-allelic amelogenin genes: AMELX (Xp22.3) and AMELY (Yp11.2). About 90% of amelogenin expression is from AMELX, which is nested within intron 1 of the gene encoding Rho GTPase activating protein 6 (ARHGAP6). We recruited two AI families and determined that their disease-causing mutations were partial deletions in ARHGAP6 that completely deleted AMELX. Affected males in both families had a distinctive enamel phenotype resembling "snow-capped" teeth. The 96,240 bp deletion in family 1 was confined to intron 1 of ARHGAP6 (g.302534_398773del96240), but removed alternative ARHGAP6 promoters 1c and 1d. Analyses of developing teeth in mice showed that ARHGAP6 is not expressed from these promoters in ameloblasts. The 52,654 bp deletion in family 2 (g.363924_416577del52654insA) removed ARHGAP6 promoter 1d and exon 2, precluding normal expression of ARHGAP6. The male proband of family 2 had slightly thinner enamel with greater surface roughness, but exhibited the same pattern of enamel malformations characteristic of males in family 1, which themselves showed minor variations in their enamel phenotypes. We conclude that the enamel defects in both families were caused by amelogenin insufficiency, that deletion of AMELX results in males with a characteristic snow-capped enamel phenotype, and failed ARHGAP6 expression did not appreciably alter the severity of enamel defects when AMELX was absent.