A novel WNT10A variant impairs the homeostasis of alveolar bone mesenchymal stem cells.

OBJECTIVES: To explore the influence of a novel WNT10A variant on bone mineral density, proliferation, and osteogenic differentiation capacities of alveolar bone mesenchymal stem cells in humans. SUBJECTS AND METHODS: Whole-exome sequencing and Sanger sequencing were utilized to detect gene variants in a family with non-syndromic tooth agenesis (NSTA). The panoramic mandibular index was calculated on the proband with WNT10A variant and normal controls to evaluate bone mineral density. Alveolar bone mesenchymal stem cells from the proband with a novel WNT10A variant and normal controls were isolated and cultured, then proliferation and osteogenic differentiation capacities were evaluated and compared. RESULTS: We identified a novel WNT10A pathogenic missense variant (c.353A > G/p. Tyr118Cys) in a family with NSTA. The panoramic mandibular index of the proband implied a reduction in bone mineral density. Moreover, the proliferation and osteogenic differentiation capacities of alveolar bone mesenchymal stem cells from the proband with WNT10A Tyr118Cys variant were significantly decreased. CONCLUSIONS: Our findings broaden the spectrum of WNT10A variants in patients with non-syndromic oligodontia, suggest an association between WNT10A and the proliferation and osteogenic differentiation of alveolar bone mesenchymal stem cells, and demonstrate that WNT10A is involved in maintaining jaw bone homeostasis.

BMPR2 Variants Underlie Nonsyndromic Oligodontia.

Oligodontia manifests as a congenital reduction in the number of permanent teeth. Despite the major efforts that have been made, the genetic etiology of oligodontia remains largely unknown. Bone morphogenetic protein receptor type 2 (BMPR2) variants have been associated with pulmonary arterial hypertension (PAH). However, the genetic significance of BMPR2 in oligodontia has not been previously reported. In the present study, we identified a novel heterozygous variant (c.814C > T; p.Arg272Cys) of BMPR2 in a family with nonsyndromic oligodontia by performing whole-exome sequencing. In addition, we identified two additional heterozygous variants (c.1042G > A; p.Val348Ile and c.1429A > G; p.Lys477Glu) among a cohort of 130 unrelated individuals with nonsyndromic oligodontia by performing Sanger sequencing. Functional analysis demonstrated that the activities of phospho-SMAD1/5/8 were significantly inhibited in BMPR2-knockout 293T cells transfected with variant-expressing plasmids, and were significantly lower in BMPR2 heterozygosity simulation groups than in the wild-type group, indicating that haploinsufficiency may represent the genetic mechanism. RNAscope in situ hybridization revealed that BMPR2 transcripts were highly expressed in the dental papilla and adjacent inner enamel epithelium in mice tooth germs, suggesting that BMPR2 may play important roles in tooth development. Our findings broaden the genetic spectrum of oligodontia and provide clinical and genetic evidence supporting the importance of BMPR2 in nonsyndromic oligodontia.

Four Novel PAX9 Variants and the PAX9-Related Non-Syndromic Tooth Agenesis Patterns.

The purpose of this research was to investigate and identify PAX9 gene variants in four Chinese families with non-syndromic tooth agenesis. We identified pathogenic gene variants by whole-exome sequencing (WES) and Sanger sequencing and then studied the effects of these variants on function by bioinformatics analysis and in vitro experiments. Four novel PAX9 heterozygous variants were identified: two missense variants (c.191G > T (p.G64V) and c.350T > G (p.V117G)) and two frameshift variants (c.352delC (p.S119Pfs*2) and c.648_649insC(p.Y217Lfs*100)). The bioinformatics analysis showed that these variants might be pathogenic. The tertiary structure analysis showed that these four variants could cause structural damage to PAX9 proteins. In vitro functional studies demonstrated that (1) the p.Y217Lfs*100 variant greatly affects mRNA stability, thereby affecting endogenous expression; (2) the p. S119Pfs* 2 variant impairs the subcellular localization of the nuclear expression of the wild-type PAX9 protein; and (3) the four variants (p.G64V, p.V117G, p.S119Pfs*2, and p.Y217Lfs*100) all significantly affect the downstream transcriptional activity of the BMP4 gene. In addition, we summarized and analyzed tooth missing positions caused by PAX9 variants and found that the maxillary second molar (84.11%) and mandibular second molar (84.11%) were the most affected tooth positions by summarizing and analyzing the PAX9-related non-syndromic tooth agenesis positions. Our results broaden the variant spectrum of the PAX9 gene related to non-syndromic tooth agenesis and provide useful information for future genetic counseling.

KDF1 Novel Variant Causes Unique Dental and Oral Epithelial Defects.

Keratinocyte differentiation factor 1 (KDF1) is a recently identified and rare candidate gene for human tooth agenesis; however, KDF1-related morphological characteristics and pathological changes in dental tissue and the oral epithelium remain largely unknown. Here, we employed whole-exome sequencing (WES) and Sanger sequencing to screen for the suspected variants in a cohort of 151 tooth agenesis patients, and we segregated a novel KDF1 heterozygous missense variation, c.920G>C (p.R307P), in a non-syndromic tooth agenesis family. Essential bioinformatics analyses and tertiary structural predictions were performed to analyze the structural changes and functional impacts of the novel KDF1 variant. The subsequent functional assessment using a TOP-flash/FOP-flash luciferase reporter system demonstrated that KDF1 variants suppressed the activation of canonical Wnt signaling in 293T cells. To comprehensively investigate the KDF1-related oral morphological anomalies, we performed scanning electron microscopy and ground section of the lower right lateral deciduous incisor extracted from #285 proband, and histopathological assessment of the gingiva. The phenotypic analyses revealed a series of tooth morphological anomalies related to the KDF1 variant R307P, including a shovel-shaped lingual surface of incisors and cornicione-shaped marginal ridges with anomalous morphological occlusal grooves of premolars and molars. Notably, keratinized gingival epithelium abnormalities were revealed in the proband and characterized by epithelial dyskeratosis with residual nuclei, indistinct stratum granulosum, epithelial hyperproliferation, and impaired epithelial differentiation. Our findings revealed new developmental anomalies in the tooth and gingival epithelium of a non-syndromic tooth agenesis individual with a novel pathogenic KDF1 variant, broadening the phenotypic spectrum of KDF1-related disorders and providing new evidence for the crucial role of KDF1 in regulating human dental and oral epithelial development.

Bufalin Inhibits Tumorigenesis, Stemness, and Epithelial-Mesenchymal Transition in Colorectal Cancer through a C-Kit/Slug Signaling Axis.

Colorectal cancer (CRC) is a major source of morbidity and mortality, characterized by intratumoral heterogeneity and the presence of cancer stem cells (CSCs). Bufalin has potent activity against many tumors, but studies of its effect on CRC stemness are limited. We explored bufalin's function and mechanism using CRC patient-derived organoids (PDOs) and cell lines. In CRC cells, bufalin prevented nuclear translocation of ß-catenin and down-regulated CSC markers (CD44, CD133, LGR5), pluripotency factors, and epithelial-mesenchymal transition (EMT) markers (N-Cadherin, Slug, ZEB1). Functionally, bufalin inhibited CRC spheroid formation, aldehyde dehydrogenase activity, migration, and invasion. Network analysis identified a C-Kit/Slug signaling axis accounting for bufalin's anti-stemness activity. Bufalin treatment significantly downregulated C-Kit, as predicted. Furthermore, overexpression of C-Kit induced Slug expression, spheroid formation, and bufalin resistance. Similarly, overexpression of Slug resulted in increased expression of C-Kit and identical functional effects, demonstrating a pro-stemness feedback loop. For further study, we established PDOs from diagnostic colonoscopy. Bufalin differentially inhibited PDO growth and proliferation, induced apoptosis, restored E-cadherin, and downregulated CSC markers CD133 and C-Myc, dependent on C-Kit/Slug. These findings suggest that the C-Kit/Slug axis plays a pivotal role in regulating CRC stemness, and reveal that targeting this axis can inhibit CRC growth and progression.

Reversal of the intrusion of a natural tooth located between two implant-supported crowns: A 7-year follow-up report.

In patients with implant-supported restorations, intrusion rarely occurs in nonconnected natural teeth. This clinical report describes the intrusion of a natural tooth located between 2 implant-supported crowns after 4 months of normal function. The second premolar was intruded by 3 mm. The intrusion was completely reversed after interproximal contact adjustments, and the tooth position was stable at the 7-year follow-up.

Accuracy of a milled digital implant surgical guide: An in vitro study.

STATEMENT OF PROBLEM: An accurate surgical template for guided implant surgery is essential for the success of an implant restoration. However, reports on the accuracy of digitally designed and computer numeric controlled (CNC) machine-milled surgical templates are sparse. PURPOSE: The purpose of this in vitro study was to investigate the accuracy of an implant surgical guide digitally designed by using data from cone beam computed tomography (CBCT) scans and milled with a 5-axis CNC machine. MATERIAL AND METHODS: Six representative radiographic templates were prepared from radiopaque resin plates. For each guide, a CBCT scan was made, and the extracted Digital Imaging and Communications in Medicine (DICOM) data were imported into a planning software program (ORGANICAL Dental Implant). Nine implants were virtually designed for each guide. The design data were imported into a 5-axis CNC machine, and the radiographic guides were fixed onto the CNC machine (Organical Multi S). Bore holes for surgical guide sleeves were milled directly in the radiographic template, which was converted into a surgical template. After the milling process, the surgical guides were scanned by using a laboratory cast scanner. The deviation between the position of the sleeve bore hole in the milled template and that in the virtual implant planning was digitally calculated. RESULTS: The mean global deviation of the surgical guide was 0.16 ±0.06 mm in the circle center of the sleeve top, and the mean angular deviation was 0.61 ±0.40 degrees. The sleeve-implant distance and the sleeve axis angle showed no significant influence on the in vitro accuracy of the implant surgical guide. CONCLUSIONS: The mean deviation of the surgical guide prepared by using the virtual planning software program and 5-axis CNC milling procedure in this study was 0.16 ±0.06 mm in the center of the sleeve top. Thus, the guide had acceptable precision.

Functional study of novel PAX9 variants: The paired domain and non-syndromic oligodontia.

OBJECTIVES: To investigate pathogenic variants of the paired box 9 (PAX9) gene in patients with non-syndromic oligodontia, and the functional impact of these variants. SUBJECTS AND METHODS: Whole exome sequencing and Sanger sequencing were utilized to detect gene variants in a cohort of 80 patients diagnosed with non-syndromic oligodontia. Bioinformatic and conformational analyses, fluorescence microscopy and luciferase reporter assay were employed to explore the functional impact. RESULTS: We identified three novel variants in the PAX9, including two frameshift variants (c.211_212insA; p.I71Nfs*246 and c.236_237insAC; p.T80Lfs*6), and one missense variant (c.229C > G; p.R77G). Familial co-segregation verified an autosomal-dominant inheritance pattern. Conformational analyses revealed that the variants resided in the paired domain, and could cause corresponding structural impairment of the PAX9 protein. Fluorescence microscopy showed abnormal subcellular localizations of frameshift variants, and luciferase assay showed impaired downstream transactivation activities of the bone morphogenetic protein 4 (BMP4) gene in all variants. CONCLUSIONS: Our findings broaden the spectrum of PAX9 variants in patients with non-syndromic oligodontia and support that paired domain structural impairment and the dominant-negative effect are likely the underlying mechanisms of PAX9-related non-syndromic oligodontia. Our findings will facilitate genetic diagnosis and counselling, and help lay the foundation for precise oral health therapies.

Comparative analysis of rare EDAR mutations and tooth agenesis pattern in EDAR- and EDA-associated nonsyndromic oligodontia.

Nonsyndromic oligodontia is a rare congenital anomaly. Mutations in the ectodysplasin A receptor (EDAR) gene are the primary cause of hypohidrotic ectodermal dysplasia but are rarely reported in nonsyndromic oligodontia. This study investigated EDAR mutations in multiplex nonsyndromic oligodontia and comparatively analyzed the EDAR- and EDA-related tooth agenesis patterns. Mutation screening was carried out using whole-exome sequencing and familial segregation. Evolutionary conservation and conformational analyses were used to evaluate the potential pathogenic influence of EDAR mutants. EDAR mutations were found to occur in 10.7% of nonsyndromic oligodontia cases. We reported seven heterozygous mutations of EDAR, including five novel mutations (c.404G>A, c.871G>A, c.43G>A, c.1072C>T, and c.1109T>C) and two known mutations (c.319A>G and c.1138A>C). Genotype-phenotype correlation analysis demonstrated that the EDAR-related tooth agenesis pattern was markedly different from EDA. The mandibular second premolars were most frequently missing (57.69%) in EDAR-mutated patients. Our results provide new evidence for the genotypic study of nonsyndromic oligodontia and suggest that EDAR haploinsufficiency results in nonsyndromic tooth agenesis. Furthermore, the distinct pattern between EDAR- and EDA-related tooth agenesis can be used as a guide for mutation screening during the clinical genetic diagnosis of this genetic disorder.

The Effectiveness of Waist-Shaped and Straight-Shaped Interdental Brushes in Cleaning Implant Overdenture Attachments: A Self-Controlled Clinical Trial.

Elderly patients often find it challenging to remove plaque accumulated on the attachments of implant overdentures (IODs) using conventional cleaning instruments. Further, excessive plaque accumulation can lead to peri-implant diseases and occasionally to respiratory diseases. Therefore, here, we aimed to compare the effectiveness of waist-shaped interdental brushes (WIBs) with that of straight-shaped interdental brushes (SIBs) in plaque removal from the locator attachments of IODs. Twenty participants with 2 locator attachments retaining mandibular IODs participated in this study. After the baseline cleaning, the participants refrained from oral hygiene maintenance for 3 days. A dentist cleaned 1 of the attachments using the WIB and the other attachment using the SIB. The pre- and post-cleaning modified plaque index (mPLI) scores were recorded. After another 3 days free from oral hygiene maintenance, the trained participants repeated the same cleaning procedure using the WIB and SIB. Pre- and post-cleaning mPLI scores were recorded. Regardless of the type of brush used, the post-cleaning mPLI scores were lower than the pre-cleaning scores. After the cleaning procedure, the overall mean mPLI score was lower in the WIB group than in the SIB group. The post-cleaning mPLI scores at the line angles and on the axial surfaces of the attachments were also lower in the WIB group than in the SIB group. There was no difference in the cleaning effectiveness between the dentist and participants when they used the same type of interdental brush. The WIB was significantly more efficient in plaque removal than the SIB, especially at the line-angle sites.

Mutations in WNT10B Are Identified in Individuals with Oligodontia.

Tooth agenesis is one of the most common developmental anomalies in humans. Oligodontia, a severe form of tooth agenesis, is genetically and phenotypically a heterogeneous condition. Although significant efforts have been made, the genetic etiology of dental agenesis remains largely unknown. In the present study, we performed whole-exome sequencing to identify the causative mutations in Chinese families in whom oligodontia segregates with dominant inheritance. We detected a heterozygous missense mutation (c.632G>A [p.Arg211Gln]) in WNT10B in all affected family members. By Sanger sequencing a cohort of 145 unrelated individuals with non-syndromic oligodontia, we identified three additional mutations (c.569C>G [p.Pro190Arg], c.786G>A [p.Trp262(∗)], and c.851T>G [p.Phe284Cys]). Interestingly, analysis of genotype-phenotype correlations revealed that mutations in WNT10B affect the development of permanent dentition, particularly the lateral incisors. Furthermore, a functional assay demonstrated that each of these mutants could not normally enhance the canonical Wnt signaling in HEPG2 epithelial cells, in which activity of the TOPFlash luciferase reporter was measured. Notably, these mutant WNT10B ligands could not efficiently induce endothelial differentiation of dental pulp stem cells. Our findings provide the identification of autosomal-dominant WNT10B mutations in individuals with oligodontia, which increases the spectrum of congenital tooth agenesis and suggests attenuated Wnt signaling in endothelial differentiation of dental pulp stem cells.

DLX3 regulates osteogenic differentiation of bone marrow mesenchymal stem cells via Wnt/ß-catenin pathway mediated histone methylation of DKK4.

OBJECTIVE: Distal-less homeobox 3 (DLX3) is an important transcription factor involved in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the underlying mechanism is not clear. This study investigated the underlying mechanism of DLX3 in osteogenic differentiation. METHODS: DLX3 overexpression and knockdown in cells were achieved using lentiviruses. The osteogenic differentiation of BMSCs was detected using alkaline phosphatase expression, alizarin red staining, real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, and chromatin immunoprecipitation (ChIP) assays. RESULTS: DLX3 overexpression promoted the osteogenic differentiation of BMSCs, whereas DLX3 knockdown reduced the osteogenic differentiation of BMSCs. RT-qPCR and Western blotting assays showed that DLX3 modulated osteogenic differentiation via the Wnt/ß-catenin pathway. ChIP-qPCR showed that DLX3 knockdown promoted DKK4 expression by decreasing the enrichment of histone H3 lysine 27 trimethylation (H3K27me3) in the promotor region of DKK4. CONCLUSION: Our data implied that DLX3 regulated Wnt/ß-catenin pathway through histone modification of DKK4 during the osteogenic differentiation of BMSCs.

Distinct impacts of bi-allelic WNT10A mutations on the permanent and primary dentitions in odonto-onycho-dermal dysplasia.

Odonto-onycho-dermal dysplasia (OODD) is a rare autosomal recessive syndrome characterized by multiple ectodermal abnormalities. Mutations of the wingless-type MMTV integration site family member 10A (WNT10A) gene have been associated with OODD. To date, only 11 OODD-associated WNT10A mutations have been reported. In this report, we Characterized the clinical manifestations with focusing on dental phenotypes in four unrelated OODD patients. By Sanger sequencing, we identified five novel mutations in the WNT10A gene, including two homozygous nonsense mutations c.1176C>A (p.Cys392*) and c.742C>T (p.Arg248*), one homozygous frame-shift mutation c.898-899delAT (p.Ile300Profs*126), and a compound heterozygous mutation c.826T>A (p.Cys276Ser) and c.949delG (p.Ala317Hisfs*121). Our findings confirmed that bi-allelic mutations of WNT10A were responsible for OODD and greatly expanded the mutation spectrum of OODD. For the first time, we demonstrated that bi-allelic WNT10A mutations could lead to anodontia of permanent teeth, which enhanced the phenotypic spectrum of WNT10A mutations. Interestingly, we found that bi-allelic mutations in the WNT10A gene preferentially affect the permanent dentition rather the primary dentition, suggesting that the molecular mechanisms regulated by WNT10A in the development of permanent teeth and deciduous teeth might be different.

mmu-miR-1963 negatively regulates the ameloblast differentiation of LS8 cell line by directly targeting Smoc2 3'UTR.

RUNX2 is a key regulator of osteogenic differentiation and odontoblastic differentiation. RUNX2 mutations could cause Cleidocranial dysplasia (CCD; OMIM119600), which is featured by abnormal development of bone and teeth. By using microRNA array, we identified a large number of microRNAs that showed different expression between wild-type Runx2 group and mutant groups. The aim of this study is to find out the effect of mmu-miR-1963, which was downregulated in all mutant Runx2 groups, on the ameloblast differentiation of LS8 cells. qPCR and Western Blot results showed the suppressive effect of mmu-miR-1963 on ameloblast differentiation of LS8 cell line. We further confirmed Smoc2 as one direct target of mmu-miR-1963. For the first time, we showed that mmu-miR-1963 could regulate the ameloblast differentiation of LS8 by targeting Smoc2. This study suggests the suppressive role of mmu-miR-1963 on ameloblast differentiation of LS8 via directly targeting the 3'UTR of Smoc2. We also demonstrated that Smoc2 itself could promote the ameloblast differentiation of LS8 for the first time. Our results indicate a novel explanation to the enamel hypoplasia phenotype in part of CCD patients.

miR-675 promotes odontogenic differentiation of human dental pulp cells by epigenetic regulation of DLX3.

In a previous study, we showed that microRNA-675 (miR-675) was significantly down-regulated in patients with tricho-dento-osseous (TDO) syndrome. One of the main features of TDO syndrome is dentin hypoplasia. Thus, we hypothesize that miR-675 plays a role in dentin development. In this study, we determined the role of miR-675 in the odontogenic differentiation of human dental pulp cells (hDPCs). Stable overexpression and knockdown of miR-675 in hDPCs were performed using recombinant lentiviruses containing U6 promoter-driven miR-675 and short hairpin-miR675 expression cassettes, respectively. Alkaline phosphatase (ALP) assay, Alizarin red staining assay, quantitative polymerase chain reaction (qPCR), Western blot analysis, and immunofluorescent staining revealed the promotive effects of miR-675 on the odontogenic differentiation of hDPCs. Further, we found that miR-675 facilitates the odontogenic differentiation process of hDPCs by epigenetic regulation of distal-less homeobox (DLX3). Thus, for the first time, we determined that miR-675 regulates the odontogenic differentiation of hDPCs by inhibiting the DNA methyltransferase 3 beta (DNMT3B)-mediated methylation of DLX3. Our findings uncover an unanticipated regulatory role for miR-675 in the odontogenic differentiation of hDPCs by epigenetic changes in DLX3 and provide novel insight into dentin hypoplasia feature in TDO patients.

Novel PITX2 mutations identified in Axenfeld-Rieger syndrome and the pattern of PITX2-related tooth agenesis.

OBJECTIVES: To investigate the mutations in patients with Axenfeld-Rieger syndrome (ARS) and the pattern of PITX2-related tooth agenesis. METHODS: Whole-exome sequencing (WES) and copy number variation (CNV) array were used to screen the mutations in four ARS probands. After Sanger sequencing and quantitative polymerase chain reaction (qPCR) validation, secondary structure prediction and dual-luciferase assay were employed to investigate the functional impact. Eighteen PITX2-mutated patients with definite dental records were retrieved from our database and literatures, and the pattern of PITX2-related tooth agenesis was analyzed. RESULTS: A novel de novo segmental deletion of chromosome 4q25 (GRCh37/hg19 chr4:111, 320, 052-111, 754, 236) encompassing PITX2 and three novel PITX2 mutations c.148C > T, c.257G > A, and c.630insCG were identified. Preliminary functional studies indicated the transactivation capacity of mutant PITX2 on Distal-less homeobox 2 (DLX2) promoter was compromised. The maxillary teeth showed significantly higher rate of agenesis (57.94%) than the mandibular teeth (44.05%). The most often missing teeth were upper lateral incisors (83.33%) and upper second premolars (69.44%). Teeth with the least agenesis rate were the lower second molars (19.44%) and lower first molars (8.33%). CONCLUSIONS: We identified a novel 4q25 microdeletion including PITX2 and three novel PITX2 mutations, and statistically analyzed the PITX2-related tooth agenesis pattern.

DLX3 promotes bone marrow mesenchymal stem cell proliferation through H19/miR-675 axis.

The underlying molecular mechanism of the increased bone mass phenotype in Tricho-dento-osseous (TDO) syndrome remains largely unknown. Our previous study has shown that the TDO point mutation c.533A>G, Q178R in DLX3 could increase bone density in a TDO patient and transgenic mice partially through delaying senescence in bone marrow mesenchymal stem cells (BMSCs). In the present study, we provided a new complementary explanation for TDO syndrome: the DLX3 (Q178R) mutation increased BMSCs proliferation through H19/miR-675 axis. We found that BMSCs derived from the TDO patient (TDO-BMSCs) had stronger proliferation ability than controls by clonogenic and CCK-8 assays. Next, experiments of overexpression and knockdown of wild-type DLX3 via lentiviruses in normal BMSCs confirmed the results by showing its negative role in cell proliferation. Through validated high-throughput data, we found that the DLX3 mutation reduced the expression of H19 and its coexpression product miR-675 in BMSCs. Function and rescue assays suggested that DLX3, long noncoding RNA H19, and miR-675 are negative factors in modulation of BMSCs proliferation as well as NOMO1 expression. The original higher proliferation rate and the expression of NOMO1 in TDO-BMSCs were suppressed after H19 restoration. Collectively, it indicates that DLX3 regulates BMSCs proliferation through H19/miR-675 axis. Moreover, the increased expression of NOMO1 and decreased H19/miR-675 expression in DLX3 (Q178R) transgenic mice, accompanying with accrual bone mass and density detected by micro-CT, further confirmed our hypothesis. In summary, we, for the first time, demonstrate that DLX3 mutation interferes with bone formation partially through H19/miR-675/NOMO1 axis in TDO syndrome.

Functional analysis of novel RUNX2 mutations in cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is a rare autosomal dominant skeletal disorder caused by mutation of runt-related transcription factor 2 (RUNX2) gene. The purpose of this study was to explore novel RUNX2 mutations in seven individuals with CCD and investigate the function of the mutant RUNX2 proteins. DNA samples were prepared from the peripheral blood of the CCD individuals, and then subjected to DNA sequencing. Conservation and secondary structure analysis were performed based on RUNX2 sequencing results. pEGFP-C1 plasmids containing GFP-tagged wild-type RUNX2 and three novel RUNX2 mutations expression cassettes were constructed, and then transfected into HEK293T cells. Cell fluorescence, luciferase assay and western blotting were used to analyse the subcellular distribution and function of the mutant RUNX2 proteins. Three novel mutations (R193G, 258fs, Y400X) were found in the seven CCD patients. Conservation and structure analysis show one novel mutation (R193G) in Runt domain and two novel mutations (258fs and Y400X) in PST domain of RUNX2. Western blotting confirmed that the 258fs and Y400X mutations produced truncated proteins. Fluorescence detection showed that the three novel mutants localised exclusively in the nucleus. However, luciferase assay indicated all mutants severely impaired the transactivation activities of RUNX2 on osteocalcin promoter. Our results broaden the spectrum of RUNX2 mutations in CCD individuals and demonstrated that loss of function in RUNX2 is responsible for CCD.

DLX3 negatively regulates osteoclastic differentiation through microRNA-124.

Homeodomain gene Distal-less-3 (DLX3) plays an essential role in the development of bones. Mutations of DLX3 are closely associated with Tricho-Dento-Osseous (TDO) syndrome featured with increased bone formation. However, the mechanism regarding whether DLX3 regulates osteoclastogenesis remains largely unknown. In this study, we firstly examined the expression of DLX3 mounting during osteoclastic differentiation process, and then established stably expressing wild type DLX3 (WT-DLX3), a novel mutant DLX3 (Q178R) found in our laboratory recently (MT-DLX3) and Dlx3 knockdown cell lines (Dlx3-shRNA) in Raw 264.7 cells using corresponding lentiviruses. Next, we investigated the influence of DLX3 on these stable cells in the process of osteoclastogenesis. The results showed that the expression of osteoclastogenesis-related genes as well as tartrate-resistant acid phosphatase-positive multinucleated cells were lower in WT-DLX3 and MT-DLX3, but higher in Dlx3-shRNA compared with control cells. Besides, the microRNA-124 expression was higher in WT-DLX3 and MT-DLX3 but lower in Dlx3-shRNA. Moreover, the microRNA-124 expression level positively correlated with DLX3, negatively with osteoclastogenesis-related gene NFATc1. Our results indicate that DLX3 negatively regulates osteoclastic differentiation through microRNA-124, which is partially responsible for the increased bone density in TDO patient. DLX3 may be exploited for osteoclastogenesis regulator and potential therapeutic target of osteoporosis in future.

Will wearing dentures affect edentulous patients' breathing during sleep?

PURPOSES: The purpose of this study is to investigate the effect of wearing dentures on obstructive sleep apnea and hypopnea among completely edentulous patients. METHODS AND MATERIALS: A self-controlled study was conducted among 30 edentulous patients. Polysomnograms were recorded in the sleep laboratory on two consecutive nights. Participants slept with their dentures in one night and without dentures in the other. The apnea and hypopnea index (AHI), lowest oxygen saturation (L-SpO2), and morning blood pressure (MBP) were collected for statistical analysis. RESULTS: Among the edentulous participants, 24 showed a higher AHI when sleeping with dentures. The average AHI for all 30 participants was significantly higher when they slept with dentures than without dentures (16.3 ± 14.7 vs 13.4 ± 14.0/h, P < 0.05). Participants in the non-obstructive sleep apnea-hypopnea syndrome (non-OSAHS) subgroup (AHI <5 when sleeping without dentures) had a significant increase in AHI when sleeping with dentures, and nearly half of them (5 out of 11) reached the diagnostic standard for OSAHS (AHI >5). A higher morning diastolic blood pressure was recorded when participants slept with dentures (P < 0.05), while no significant difference was found in the L-SpO2 score and morning systolic blood pressure. CONCLUSIONS: Wearing dentures can lead to significant increase of AHI and diastolic MBP among edentulous people. Hence, we suggest that Chinese edentulous people should remove their dentures before sleep. TRIAL REGISTRATION: ChiCTR-IOR-16008404.