Systemic Dietary Hesperidin Modulation of Osteoclastogenesis, Bone Homeostasis and Periodontal Disease in Mice.

This study aimed to evaluate the effects of hesperidin (HE) on in vitro osteoclastogenesis and dietary supplementation on mouse periodontal disease and femoral bone phenotype. RAW 264.7 cells were stimulated with RANKL in the presence or absence of HE (1, 100 or 500 µM) for 5 days, and evaluated by TRAP, TUNEL and Western Blot (WB) analyses. In vivo, C57BL/6 mice were given HE via oral gavage (125, 250 and 500 mg/kg) for 4 weeks. A sterile silk ligature was placed between the first and second right maxillary molars for 10 days and microcomputed tomography (µCT), histopathological and immunohistochemical evaluation were performed. Femoral bones subjected or not to dietary HE (500 mg/kg) for 6 and 12 weeks were evaluated using µCT. In vitro, HE 500 µM reduced formation of RANKL-stimulated TRAP-positive(+) multinucleated cells (500 µM) as well as c-Fos and NFATc1 protein expression (p < 0.05), markers of osteoclasts. In vivo, dietary HE 500 mg/kg increased the alveolar bone resorption in ligated teeth (p < 0.05) and resulted in a significant increase in TRAP+ cells (p < 0.05). Gingival inflammatory infiltrate was greater in the HE 500 mg/kg group even in the absence of ligature. In femurs, HE 500 mg/kg protected trabecular and cortical bone mass at 6 weeks of treatment. In conclusion, HE impaired in vitro osteoclastogenesis, but on the contrary, oral administration of a high concentration of dietary HE increased osteoclast numbers and promoted inflammation-induced alveolar bone loss. However, HE at 500 mg/kg can promote a bone-sparing effect on skeletal bone under physiological conditions.

Roles of autophagy in orthodontic tooth movement.

INTRODUCTION: Orthodontic tooth movement (OTM) relies on efficient remodeling of alveolar bone. While a well-controlled inflammatory response is essential during OTM, the mechanism regulating inflammation is unknown. Autophagy, a conserved catabolic pathway, has been shown to protect cells from excess inflammation in disease states. We hypothesize that autophagy plays a role in regulating inflammation during OTM. METHODS: A split-mouth design was used to force load molars in adult male mice, carrying a GFP-LC3 transgene for in vivo detection of autophagy. Confocal microscopy, Western blot, and quantitative polymerase chain reaction analyses were used to evaluate autophagy activation in tissues of loaded and control molars at time points after force application. Rapamycin, a Food and Drug Administration-approved immunosuppressant, was injected to evaluate induction of autophagy. RESULTS: Autophagy activity increases shortly after loading, primarily on the compression side of the tooth, and is closely associated with inflammatory cytokine expression and osteoclast recruitment. Daily administration of rapamycin, an autophagy activator, led to reduced tooth movement and osteoclast recruitment, suggesting that autophagy downregulates the inflammatory response and bone turnover during OTM. CONCLUSIONS: This is the first demonstration that shows that autophagy is induced by orthodontic loading and plays a role during OTM, likely via negative regulation of inflammatory response and bone turnover. Exploring roles of autophagy in OTM holds great promise, as aberrant autophagy is associated with periodontal disease and its related systemic inflammatory disorders.

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.

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.

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.

Genetic analysis: Wnt and other pathways in nonsyndromic tooth agenesis.

Tooth agenesis (TA) is one of the most common developmental anomalies that affects the number of teeth. An extensive analysis of publicly accessible databases revealed 15 causative genes responsible for nonsyndromic TA, along with their signaling pathways in Wnt/ß-catenin, TGF-ß/BMP, and Eda/Edar/NF-κB. However, genotype-phenotype correlation analysis showed that most of the causal genes are also responsible for syndromic TA or other conditions. In a total of 198 different mutations of the 15 genes responsible for nonsyndromic TA, 182 mutations (91.9%) are derived from seven genes (AXIN2, EDA, LRP6, MSX1, PAX9, WNT10A, and WNT10B) compared with the remaining 16 mutations (8.1%) identified in the remaining eight genes (BMP4, DKK1, EDAR, EDARADD, GREM2, KREMEN1, LTBP3, and SMOC2). Furthermore, specificity analysis in terms of the ratio of nonsyndromic TA mutations versus syndromic mutations in each of the aforementioned seven genes showed a 98.2% specificity rate in PAX9, 58.9% in WNT10A, 56.6% in MSX1, 41.2% in WNT10B, 31.4% in LRP6, 23.8% in AXIN2%, and 8.4% in EDA. These findings underscore an important role of the Wnt and Wnt-associated pathways in the genetic etiology of this heterozygous disease and shed new lights on the discovery of novel molecular mechanisms associated with tooth agenesis.

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.

A novel non-stop mutation in MSX1 causing autosomal dominant non-syndromic oligodontia.

Oligodontia, which is the congenital absence of six or more permanent teeth, excluding the third molars, may contribute to masticatory dysfunction, speech alteration, aesthetic problems and malocclusion. Msh homeobox 1 (MSX1) was the first gene identified as causing non-syndromic oligodontia. In this study, we identified a novel heterozygous non-stop mutation (c.910_911dupTA, p.*304Tyrext*48) in MSX1 in a Chinese family with autosomal dominant non-syndromic oligodontia. This novel mutation substitutes the stop codon with a tyrosine residue, potentially adding 48 amino acids to the C-terminus of MSX1. Further in vitro study found that mutant MSX1 could be expressed but had lost its ability to enter the nucleus. This is the first report indicating that a non-stop mutation in MSX1 is responsible for oligodontia. This study broadens the mutation spectrum for MSX1 and provides a new way to clarify the mechanism of MSX1 in tooth agenesis.

Occlusal Rehabilitation in a Patient with Oligodontia and Microdontia using Implants and Full-Ceramic Restorations: A Clinical Report.

Oligodontia is the agenesis of six or more permanent teeth, excluding the third molars. Multidisciplinary dental treatments should be performed sequentially due to the restoration requirements for good oral function, aesthetics and self-confidence of patients. In this study, we report a case using dental implants and full-ceramic prostheses to restore the absent and malformed teeth in a patient with agenesis of 18 permanent teeth and with some primary teeth retained. The dental sequential treatments began when she was 16 years old, and she wore removable partial dentures for 4 years with unsatisfying restoration outcome. When she became an adult, dental implants and full-ceramic prostheses were used to restore the absent and malformed teeth. Finally, the patient was very satisfied with the functional and aesthetic outcomes of the prosthetic treatment.

BMP4 mutations in tooth agenesis and low bone mass.

OBJECTIVE: To identify an uncommon genetic cause of tooth agenesis (TA) by utilizing whole exome sequencing (WES) and targeted Sanger sequencing in a cohort of 120 patients with isolated TA. DESIGN: One deleterious mutation in the gene encoding bone morphogenetic protein 4 (BMP4) was identified in 6 unrelated patients with TA by WES. After that, the coding exons of BMP4 were examined in 114 TA patients using Sanger sequencing. Dual-energy X-ray absorptiometry (DEXA) was used to measure the bone mineral density of patients who carried a BMP4 mutation. Finally, preliminary functional studies of two BMP4 mutants were performed. RESULTS: We detected 3 novel missense mutations (c.58 G > A: p.Gly20Ser, c.326 G > T: p.Arg109Leu and c.614 T > C: p.Val205Ala) and 1 reported mutation in the BMP4 gene among 120 TA probands. The previously reported BMP4 mutation (c.751C > T: p.His251Tyr) was associated with urethra and eye anomalies. By extending the pedigrees, we determined that the tooth phenotypes had an autosomal dominant inheritance pattern, as individuals carrying a BMP4 mutation exhibit different types of dental anomalies. Interestingly, we observed that patients harboring a BMP4 mutation manifested early onset osteopenia or osteoporosis. Further in vitro functional assays demonstrated that two BMP4 mutants resulted in a decreased activation of Smad signaling. Therefore, a loss-of-function in BMP4 may contribute to the clinical phenotypes seen in this study. CONCLUSIONS: We identified 4 mutations in the BMP4 gene in 120 TA patients. To our knowledge, this is the first study to describe human skeletal diseases associated with BMP4 mutations.

Tooth defects of EEC and AEC syndrome caused by heterozygous TP63 mutations in three Chinese families and genotype-phenotype correlation analyses of TP63-related disorders.

BACKGROUND: Ectrodactyly-Ectodermal dysplasia-Cleft lip/palate (EEC) syndrome and Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) syndrome belong to p63 syndromes, a group of rare disorders exhibiting a wide variety of clinical manifestations. TP63 mutations have been reported to be associated with both EEC and AEC. METHODS: Analysis of whole exome sequencing (WES) from patients with EEC or AEC syndrome and Sanger sequencing from family members. RESULTS: We confirmed that three Chinese pedigrees affected with EEC or AEC harboring a distinct TP63 mutation, and described novel clinical phenotypes of EEC and AEC, including the presence of cubitus valgus deformity and taurodontism, which were discordant to their classical disease features. We also analyzed the genotype-phenotype correlation based on our findings. CONCLUSION: We reported that the cubitus valgus deformity in patients with EEC and severe taurodontism in a patient with AEC had not been mentioned previously. Our study expands the phenotypic spectrum of EEC and AEC syndrome.

A novel inhibitor of nuclear factor kappa-B kinase subunit gamma mutation identified in an incontinentia pigmenti patient with syndromic tooth agenesis.

OBJECTIVE: To explore the gene mutation in an incontinentia pigmenti (IP) patient with syndromic tooth agenesis. METHODS: Long-range polymerase chain reaction (PCR) and Sanger sequencing were used to detect inhibitor of nuclear factor kappa-B kinase subunit gamma (IKBKG) mutation in the IP patient. We used the nuclear factor kappa B (NF-κB) reporter gene to assess activation of NF-κB, after transfecting an empty vector, wild-type, or mutant NF-κB essential modulator (NEMO) plasmid into IKBKG-deficient HEK293T cells, respectively. Furthermore, we performed immunoprecipitation and immunoblotting to describe the polyubiquitination of NEMO. Lastly, we detected the interactions between mutant NEMO and I kappa B kinase alpha (IKKα), I kappa B kinase beta (IKKß), TNF receptor associated factor 6 (TRAF6), HOIL-1-interacting protein (HOIP), hemo-oxidized iron regulatory protein 2 ligase 1 (HOIL-1), and SHANK-associated RH domain interactor (SHARPIN). RESULTS: A de novo nonsense mutation in IKBKG (c.924C > G; p.Tyr308*) was observed. The Tyr308* mutation inhibited activation of the NF-κB pathway by reducing K63-linked polyubiquitination and linear polyubiquitination. The mutant NEMO was not able to interact with TRAF6, HOIL-1, or SHARPIN. CONCLUSIONS: We identified a novel nonsense IKBKG mutation (c.924C > G; p.Tyr308*) in an IP patient with syndromic tooth agenesis. This research enriches the mutation spectrum of the IKBKG gene.

Senescence: novel insight into DLX3 mutations leading to enhanced bone formation in Tricho-Dento-Osseous syndrome.

The homeodomain transcription factor distal-less homeobox 3 gene (DLX3) is required for hair, tooth and skeletal development. DLX3 mutations have been found to be responsible for Tricho-Dento-Osseous (TDO) syndrome, characterized by kinky hair, thin-pitted enamel and increased bone density. Here we show that the DLX3 mutation (c.533 A>G; Q178R) attenuates osteogenic potential and senescence of bone mesenchymal stem cells (BMSCs) isolated from a TDO patient, providing a molecular explanation for abnormal increased bone density. Both DLX3 mutations (c.533 A>G and c.571_574delGGGG) delayed cellular senescence when they were introduced into pre-osteoblastic cells MC3T3-E1. Furthermore, the attenuated skeletal aging and bone loss in DLX3 (Q178R) transgenic mice not only reconfirmed that DLX3 mutation (Q178R) delayed cellular senescence, but also prevented aging-mediated bone loss. Taken together, these results indicate that DLX3 mutations act as a loss of function in senescence. The delayed senescence of BMSCs leads to increased bone formation by compensating decreased osteogenic potentials with more generations and extended functional lifespan. Our findings in the rare human genetic disease unravel a novel mechanism of DLX3 involving the senescence regulation of bone formation.

Simultaneous Occurence of an Autosomal Dominant Inherited MSX1 Mutation and an X-linked Recessive Inherited EDA Mutation in One Chinese Family with Non-syndromic Oligodontia.

OBJECTIVE: To describe the simultaneous occurence of an autosomal dominant inherited MSX1 mutation and an X-linked recessive inherited EDA mutation in one Chinese family with nonsyndromic oligodontia. METHODS: Clinical data of characteristics of tooth agenesis were collected. MSX1 and EDA gene mutations were detected in a Chinese family of non-syndromic oligodontia. RESULTS: Mild hypodontia in the parents and severe oligodontia in the son was recorded. A novel missense heterozygous mutation c.517C>A (p.Arg173Ser) was detected in the MSX1 gene in the boy and the father. A homozygous missense mutation c.1001G>A (p.Arg334His) was detected in the EDA gene in the boy and the same mutant occurred heterozygously in the mother. CONCLUSION: Simultaneous occurence of two different gene mutations with different inheritence patterns, which both caused oligodontia, which occurred in one subject and in one family, was reported.