Identification of rare PTCH1 nonsense variant causing orofacial cleft in a Chinese family and an up-to-date genotype-phenotype analysis.

The Patched 1 (PTCH1) gene encodes a membrane receptor involved in the Hedgehog (Hh) signaling pathway, an abnormal state of which may result in congenital defects or human tumors. In this study, we conducted whole-exome sequencing on a three-generation Chinese family characterized with variable penetrance of orofacial clefts. A rare heterozygous variant in the PTCH1 gene (c.2833C > T p.R945X) was identified as a disease-associated mutation. Structural modeling revealed a truncation starting from the middle of the second extracellular domain of PTCH1 protein. This may damage its ligand recognition and sterol transportation abilities, thereby affecting the Hh signaling pathway. Biochemical assays indicated that the R945X protein had reduced stability compared to the wild-type in vitro. In addition, we reviewed the locations and mutation types of PTCH1 variants in individuals with clefting phenotypes, and analyzed the associations between clefts and locations or types of variants within PTCH1. Our findings provide further evidence that PTCH1 variants result in orofacial clefts, and contributed to genetic counseling and clinical surveillance in this family.

Functional identification of a rare vascular endothelial growth factor a (VEGFA) variant associating with the nonsyndromic cleft lip with/without cleft palate.

Vascular endothelial growth factor A (VEGFA) is a crucial growth factor, which participates in multiple processes of human growth and development, such as angiogenesis and osteogenesis and is also necessary for development of palate. The purpose of this study was to investigate the effect of a rare VEGFA mutation (NM_001025366.2 773 T > C p.Val258Ala) on the cell functions and osteogenesis. Here, we found that the VEGFA mutation has adverse effects on the function of human embryonic palatal plate mesenchymal (HEPM) cells, and may affect the development of palate. The VEGFA mutation has adverse effects on promoting cell proliferation and migration and inhibiting apoptosis in HEPM and HEK-293 cells. In addition, the mutant VEGFA allele has a negative influence on osteogenesis. Taken together, the rare variant of the VEGFA gene had an adverse effect on cell functions and osteogenesis, which may impact the development of the palate. And these findings suggested that VEGFA mutation (c.773 T > C) may lead to nonsyndromic cleft lip with or without cleft palate and also provide a new insight into the mechanism of VEGFA gene in osteogenesis and palatogenesis.

Analysis of facial features and prediction of lip position in skeletal class III malocclusion adult patients undergoing surgical-orthodontic treatment.

OBJECTIVES: This study presents a retrospective study aimed to analyze the facial features at each stage of surgical-orthodontic treatment for skeletal class III malocclusion, and predict the changes in the lips after treatment. MATERIALS AND METHODS: There were 49 skeletal class III malocclusion patients treated with bimaxillary surgery and orthodontic treatment enrolled in this study. Lateral cephalograms were obtained before treatment (T0), 1 month before surgery (T1), 1 month after surgery (T2), and after debonding (T3) for cephalometric measurements. After the measurement of the required variables, paired t-test, Pearson's correlation analysis, and multiple linear regression were performed using SPSS 19.0. RESULTS: The main factors associated with changes in the upper lip included ΔUIE-V, ΔA-V, ΔU1A-V, and ΔL1A-V, and those associated with changes in the lower lip included ΔLIE-V, ΔL1A-V, ΔB-V, ΔPog-V, and Δfacial angle. The predicted regression equation for the horizontal change in the upper lip was represented as ΔUL-vertical reference line (VRL) = 9.430 + 0.779 (ΔUIE-VRL) - 0.542(VULT) (P < 0.05) with a mean error of 1.04 mm; the corresponding equation for the lower lip was ΔLL-VRL = -1.670 + 0.530 (ΔB-VRL) + 0.360 (Ls-E) + 0.393 (ΔLIE-VRL) (P < 0.05), with a mean error of 1.51 mm. CONCLUSIONS: This study explored the relationship between orthognathic surgery and changes in the lips and obtained the predictive equations of lip position after treatment by using multiple linear regression, which likely offers a reference for prediction of soft tissue changes before surgical-orthodontic treatment in patients with skeletal class III malocclusion. CLINICAL RELEVANCE: The findings can help dentists to rapidly predict the lip changes after surgical-orthodontic treatment in patients with skeletal class III malocclusion. The study has been registered with the Chinese Clinical Trial Registration (No: ChiCTR1800017694).

Nell-1-ΔE, a novel transcript of Nell-1, inhibits cell migration by interacting with enolase-1.

NELL-1 is a secreted protein that was originally found to be upregulated in pathologically fusing and fused sutures in non-syndromic unilateral coronal synostosis patients. Apart from the ability of NELL-1 to promote osteogenesis in long and craniofacial bones, NELL-1 reportedly inhibits the formation of several benign and malignant tumors. We previously identified a novel transcript of Nell-1 that lacked a calcium-binding epidermal growth factor (EGF)-like domain compared with full-length Nell-1; this new transcript was named Nell-1-ΔE. Three obvious structural differences between these two isoforms were revealed by homology modeling. Furthermore, the recombinant Nell-1-ΔE protein, but not the full-length Nell-1 protein, inhibited cell migration in vitro. However, full-length Nell-1 and Nell-1-ΔE proteins were present in similar subcellular locations and displayed similar expression patterns in both the intracellular and extracellular spaces. The results from the co-immunoprecipitation and liquid chromatography/tandem mass spectrometry analyses using two cell lines demonstrated that Nell-1-ΔE but not full-length Nell-1 interacted with enolase-1 in the extracellular spaces of both cell lines. The results of wound healing assays using ENO-1-overexpressing cells treated with full-length Nell-1/Nell-1-ΔE suggested that Nell-1-ΔE inhibited cell migration by interacting with ENO-1. Our study indicated that the novel transcript Nell-1-ΔE, but not full-length Nell-1, might be a candidate tumor suppressor factor for basic research and clinical practice.

Salivary exosomal PSMA7: a promising biomarker of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is an intestinal immune-dysfunctional disease worldwide whose prevalence increasing in Asia including China. It is a chronic disease of the gastrointestinal tract with unknown cause. Exosomes are small vesicles in various body fluids. They have diameters of 40-120 nm, and one of their functions is long-distance transfer of various substances. In this study, we investigated the contents of salivary exosomes in patients with IBD and in healthy controls to explore a new biomarker in patients with IBD. In this study, whole saliva was obtained from patients with IBD (ulcerative colitis (UC), n = 37; Crohn's disease (CD), n = 11) and apparently healthy individuals (HC, n = 10). Salivary exosomes were extracted from samples, and the proteins within the exosomes were identified by liquid chromatograph-mass spectrometer (LC-MS/MS). The results showed that more than 2000 proteins were detected in salivary exosomes from patients with IBD. Through gene ontology analysis, we found that proteasome subunit alpha type 7 (PSMA7) showed especially marked differences between patients with IBD and the healthy controls, in that its expression level was much higher in the CD and UC groups. This exosomal protein is related to proteasome activity and inflammatory responses. So we conclude that in this research, salivary exosomal PSMA7 was present at high levels in salivary exosomes from subjects with IBD. It can be a very promising biomarker to release the patients from the pain of colonoscopy.

Novel Mutation of Cleidocranial Dysplasia-related Frameshift Runt-related Transcription Factor 2 in a Sporadic Chinese Case.

BACKGROUND: Cleidocranial dysplasia (CCD) is an autosomal dominant disease that affects the skeletal system. Common symptoms of CCD include hypoplasia or aplasia of the clavicles, delayed or even absent closure of the fontanels, midface hypoplasia, short stature, and delayed eruption of permanent and supernumerary teeth. Previous studies reported a connection between CCD and the haploinsufficiency of runt-related transcription factor 2 (RUNX2). Here, we report a sporadic Chinese case presenting typical symptoms of CCD. METHODS: We made genetic testing on this sporadic Chinese case and identified a novel RUNX2 frameshift mutation: c.1111dupT. In situ immunofluorescence microscopy and osteocalcin promoter luciferase assay were performed to compare the functions of the RUNX2 mutation with those of wild-type RUNX2. RESULTS: RUNX2 mutation was observed in the perinuclear region, cytoplasm, and nuclei. In contrast, wild-type RUNX2 was confined in the nuclei, which indicated that the subcellular compartmentalization of RUNX2 mutation was partially perturbed. The transactivation function on osteocalcin promoter of the RUNX2 mutation was obviously abrogated. CONCLUSIONS: We identified a sporadic CCD patient carrying a novel insertion/frameshift mutation of RUNX2. This finding expanded our understanding of CCD-related phenotypes.

Subgingival microbiome in patients with healthy and ailing dental implants.

Dental implants are commonly used to replace missing teeth. However, the dysbiotic polymicrobial communities of peri-implant sites are responsible for peri-implant diseases, such as peri-implant mucositis and peri-implantitis. In this study, we analyzed the microbial characteristics of oral plaque from peri-implant pockets or sulci of healthy implants (n = 10), peri-implant mucositis (n = 8) and peri-implantitis (n = 6) sites using pyrosequencing of the 16S rRNA gene. An increase in microbial diversity was observed in subgingival sites of ailing implants, compared with healthy implants. Microbial co-occurrence analysis revealed that periodontal pathogens, such as Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia, were clustered into modules in the peri-implant mucositis network. Putative pathogens associated with peri-implantitis were present at a moderate relative abundance in peri-implant mucositis, suggesting that peri-implant mucositis an important early transitional phase during the development of peri-implantitis. Furthermore, the relative abundance of Eubacterium was increased at peri-implantitis locations, and co-occurrence analysis revealed that Eubacterium minutum was correlated with Prevotella intermedia in peri-implantitis sites, which suggests the association of Eubacterium with peri-implantitis. This study indicates that periodontal pathogens may play important roles in the shifting of healthy implant status to peri-implant disease.

[Indication of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) in gingival crevicular fluid to remodeling of alveolar bone during retention].

OBJECTIVE: To investigate the level of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) in gingival crevicular fluid (GCF) during orthodontic retention and corresponding expression of OPG and RANKL in periodontal tissues. METHODS: Fifteen male Wistar rats (age, 6 weeks) were divided into three groups with 5 rats for each. GCF samples were collected at the baseline, 14 days after orthodontic force application, and 14 days after orthodontic force removal. enzyme linked immunosorbent assay (ELISA) was used to quantify the concentration of OPG and sRANKL (soluble receptor activator of nuclear factor kappa B ligand) in GCF. Immunohistochemical staining was performed to identify the OPG and RANKL expression in periodontal tissues. RESULTS: The concentration of sRANKL in GCF increased statistically significant from baseline to T2 (P<0.05) while decreased significantly from T2 to T3 (P<0.05). The concentration of OPG had minimum fluctuation from baseline to T3 without any statistical significance (P>0.05). The sRANKL/OPG ratio in GCF and that in periodontal tissues during the three time points were similar which showed a prominent increase in T2 (P<0.05 and P<0.01, respectively) and sharp decrease in T3 (P<0.05). CONCLUSION: sRANKL/OPG ratio may be one of the predictors reflecting the remodeling of periodontal tissues in orthodontic retention.

Local osteoprotegerin gene transfer inhibits relapse of orthodontic tooth movement.

INTRODUCTION: In orthodontic treatment, teeth can relapse after tooth movement without retention. The aim of this study was to evaluate the inhibition effects of local osteoprotegerin (OPG) gene transfer on orthodontic relapse. METHODS: Eighteen male Wistar rats were divided into 3 groups. The maxillary right first molars of all animals were subjected to orthodontic force and moved mesially. Three weeks later, the force was removed, and the teeth relapsed. During the 2-week relapse period, the 3 groups of rats received local OPG gene transfer (experimental group), mock vector transfer (mock group), and no injections (control group). Tooth movement and relapse were measured by using palatal superimpositions of 3-dimensional digital models. Histomorphometric analysis was used to quantify osteoclasts, and microcomputed tomography analysis was done to quantify the alveolar bone and the tibia. RESULTS: Relapse was significantly inhibited and the number of osteoclasts was reduced in the experimental group. On the other hand, bone mineral density and bone volume fraction of alveolar bone were significantly increased. Bone mineral density and bone volume fraction of the tibia showed no significant difference between the groups. CONCLUSIONS: Local OPG gene transfer to periodontal tissues could inhibit relapse after orthodontic tooth movement, through the inhibition of osteoclastogenesis.

Longitudinal study of relative growth rates of the maxilla and the mandible according to quantitative cervical vertebral maturation.

INTRODUCTION: The purpose of this study was to investigate the relative growth rates (RGR) of the maxilla and the mandible according to quantitative cervical vertebral maturation (QCVM) of adolescents with normal occlusion. METHODS: Mixed longitudinal data were used. The sample included 87 adolescents (32 boys, 55 girls) from 8 to 18 years of age with normal occlusion, selected from 901 candidates. Sequential lateral cephalograms and hand-wrist films were taken once a year for 6 consecutive years. The growth magnitude (GM) and RGR of the maxilla and the mandible were measured and analyzed. RESULTS: GM and RGR were not always consistent, because subjects had different periods of time between the QCVM stages. GM was not as reliable as RGR. RGR had no significant sex differences in the maxilla and the mandible, in spite of different decelerating curves. However, statistically significant sex differences were found in the GM of mandibular measurements. The greatest growth potentials were not synchronized between the maxilla and the mandible. For both sexes, the greatest RGR of maxillary length and height was in QCVM stage I; then, deceleration occurred. The greatest RGR of mandibular length and height was in QCVM stage II, and the next largest was in QCVM stage I. CONCLUSIONS: Understanding the RGR can provide references for orthodontic treatment and orthognathic surgery.

[Preliminary investigation of lower second molar extraction in correction of severe skeletal class III malocclusion].

OBJECTIVE: To evaluate dento-skeletal and soft-tissue profile changes after extraction of lower second molars and treatment using fixed appliances in severe class III subjects. METHODS: Fourteen patients with severe skeletal class III malocclusion (male 4, female 10, age rang 12.0 - 17.1 years old, mean age 13.3 +/- 0.8), diagnosed as requiring orthognathic surgery, but rejected surgical therapy were included in the study. Lateral cephalometric films taken at the beginning and end of treatment were analyzed using Pancherz analysis and a traditional cephalometric analysis. RESULTS: After active treatment, inclination of lower incisors was decreased 11.7 degrees when measured to the mandibular plane (P < 0.001). A negative value of the distance between upper and lower lip position to SnPg' at the beginning of treatment changed to a positive value (P < 0.001). CONCLUSIONS: The results of the current preliminary study suggest that success in the treatment of some severe class III deformity in the permanent dentition can be achieved with fixed appliances and extraction of lower second molars. A remarkable soft-tissue change was noted after the treatment and concave facial profiles changed to straight profiles.

Lower second molar extraction in correction of severe skeletal class III malocclusion.

The purpose of this study was to evaluate dentoskeletal and soft-tissue profile changes after extraction of lower second molars and treatment using the Tip-Edge technique in severe Class III subjects. Thirteen patients with severe skeletal Class III malocclusion (four males, nine females), diagnosed as requiring orthognathic surgery, but who rejected surgical therapy, were included in the study. The average age was 13.2 +/- 0.8 years. Lateral cephalometric films taken at the beginning and the end of treatment were analyzed using the Pancherz analysis and a traditional cephalometric analysis. The arithmetic mean and standard deviation were calculated for each variable. Paired t-test was performed to evaluate significant treatment change. After active treatment, dramatic overjet change was noted, with an average value of 5.5 mm (P < .001). Inclination of lower incisors was decreased 12.0 degrees when measured to the mandibular plane (P < .001). Inclination of upper incisors was increased by 2.1 degrees to the SN plane (P > .05). A negative value of the distance between upper and lower lip position to Sn-Pg' at the beginning of treatment changed to a positive value (P < .001). The results of this preliminary study suggest that success in the treatment of some severe Class III deformity in the permanent dentition can be achieved with fixed appliances and extraction of lower second molars. A remarkable soft-tissue change was noted after the treatment, and concave facial profiles changed to straight profiles.

[Retinoic acid induced cell cycle arrest and apoptosis in mouse embryonic palatal mesenchymal cells].

OBJECTIVES: To investigate the effect of all-trans retinoic acid (atRA) on proliferation activity and cell cycle distribution in mouse embryonic palatal mesenchymal (MEPM) cells and the underlying molecular mechanisms. METHODS: MEPM cells were prepared from palate shelves of mouse fetal on gestation day 13. Cell viability was determined by MTI assay. Cell cycle distribution and subdiploid population were analyzed by cytometry. The expression of cyclin D and E and phosphorylation of retinoblastoma protein was examined using Western-blot. RESULTS: atRA remarkably inhibited the growth of MEPM cells in a dose-dependent manner. atRA also caused an increase in the proportion of cells in G0/G1 and a decrease in the proportion of cells in S phase. atRA inhibited expression of cyclins D and E at protein level. Furthermore, atRA treatment reduced phosphorylated Rb. CONCLUSION: These data suggested that atRA had antiproliferative activity by modulating G1/S cell cycle regulators and by inhibition of Rb phosphorylation in MEPM cells, which might account for the pathogenesis of cleft palate induced by retinoic acid.

Induction of cell-cycle arrest by all-trans retinoic acid in mouse embryonic palatal mesenchymal (MEPM) cells.

all-trans retinoic acid (atRA), the oxidative metabolite of vitamin A, is essential for normal embryonic development. Also, high levels of atRA are teratogenic in many species and can effectively induce cleft palate in the mouse. Most cleft palate resulted from the failed fusion of secondary palate shelves, and maintenance of the normal cell proliferation is important in this process of shelf growth. To clarify the mechanism by which atRA causes cleft palate, we investigated the effect of atRA on proliferation activity and cell cycle distribution in mouse embryonic palatal mesenchymal (MEPM) cells. atRA inhibited the growth of MEPM cells by inducing apoptosis in a dose-dependent manner. atRA also caused a G1 block in the cell cycle with an increase in the proportion of cells in G0/G1 and a decrease in the proportion of cells in S phase, as determined by flow cytometry. We next investigated the effects of atRA on molecules that regulate the G1 to S phase transition. These studies demonstrated that atRA inhibited expression of cyclins D and E at the protein level. Furthermore, atRA treatment reduced phosphorylated Rb and decreased cdk2 and cdk4 kinase activity. These data suggest that atRA had antiproliferative activity by modulating G1/S cell cycle regulators and by inhibition of Rb phosphorylation in MEPM cells, which might account for the pathogenesis of cleft palate induced by retinoic acid.

[Preliminary study of non-surgical treatment of severe Class III malocclusion in 18 patients of 12-20 years old].

OBJECTIVE: To analyze effects of non-surgical treatment on subjects of 12-20 years old with severe skeletal Class III deformity and to directly evaluate dental and facial profile changes. METHODS: Eighteen patients with severe skeletal Class III malocclusion (male 5, female 13), diagnosed as indication for orthognathic surgery, were included in the study. The average age was 14.6 +/- 2.5 years old., with age range from 12 years old to 20 years old. 12 cases were treated with Tip-Edge straight-wire technique and 6 cases with Begg light wire technique. The average treatment time was 2.5 +/- 0.8 years. The selection criteria included: (1) mesial or superior mesial Class III molar relationship, with maxillary first molar occlude the buccal groove of the mandibular second molars (2) no mandibular shift (3) ANB < -1.5 degrees (4) high angle cases with average value of SN-MP 34.9 degrees +/- 5.9 degrees, 4 cases present with openbite (5) concave facial profile (6) originally classified as surgery cases. Lateral cephalometric films taken at the beginning and the end of the treatment were analyzed with traditional cephalometric analysis. The arithmetic mean (Mean) and standard deviation (SD) were calculated for each variable. Paired t-test was performed to evaluate the significant treatment change. RESULTS: Normal overjet and overbite were established with proclination of upper incisors and retroclination of lower incisors. Inclination of upper incisors was increased 5.9 degrees when measured with the angle of upper incisor to SN plane (P < 0.01). Inclination of lower incisors was decreased 6.6 degrees when measured with the angle of lower incisor to mandibular plane (P < 0.001). Negative value of the distance difference of upper lip and lower lip to Sn-Pg' at the beginning of treatment changed to positive value with significant difference (P < 0.001). CONCLUSION: Successful treatment effects could be obtained with non-surgical therapy in severe skeletal Class III malocclusion in permanent dentition. Remarkable soft-tissue change was noted after the treatment and concave facial profile changed to straight profile.

Preliminary investigation of nonsurgical treatment of severe skeletal Class III malocclusion in the permanent dentition.

The purpose of the study was to analyze the effects of nonsurgical treatment on subjects with a severe skeletal Class III deformity and to directly evaluate dental and facial profile changes. Eighteen patients with severe skeletal Class III malocclusions (5 males, 13 females), diagnosed as indication for orthognathic surgery, were included in the study. The average age was 13.7 +/- 2.5 years. All the cases were treated with Tip-Edge straight-wire technique or Begg light wire technique. Lateral cephalometric films taken at the beginning and at the end of treatment were analyzed with the Pancherz analysis and traditional cephalometric analysis. The arithmetic mean and standard deviation were calculated for each variable and paired t-test was performed. A mean reduction of 6.5 mm in the overjet was noted (P < .001), with skeletal changes and dental changes contributing 20% and 80% to the overjet correction, respectively. The inclination of the upper incisors to the SN plane was increased 5.9 degrees (P < .01). The inclination of the lower incisors to the mandibular plane was decreased 6.6 degrees (P < .001). The difference between the distance of the upper lip and lower lip to Sn-Pg' at the beginning of treatment changed from a negative value to a positive value with a significant difference (P < .001). Successful treatment effects can be obtained with nonsurgical therapy in severe skeletal Class III malocclusions in the permanent dentition. A remarkable soft tissue change was noted after the treatment, and the concave facial profile changed to a straight profile (see two case reports).