Rare loss-of-function variants in FLNB cause non-syndromic orofacial clefts.

Orofacial clefts (OFCs) are the most common congenital craniofacial disorders, of which the etiology is closely related to rare coding variants. Filamin B (FLNB) is an actin-binding protein implicated in bone formation. FLNB mutations have been identified in several types of syndromic OFCs and previous studies suggest a role of FLNB in the onset of non-syndromic OFCs (NSOFCs). Here, we report two rare heterozygous variants (p.P441T and p.G565R) in FLNB in two unrelated hereditary families with NSOFCs. Bioinformatics analysis suggests that both variants may disrupt the function of FLNB. In mammalian cells, p.P441T and p.G565R variants are less potent to induce cell stretches than wild type FLNB, suggesting that they are loss-of-function mutations. Immunohistochemistry analysis demonstrates that FLNB is abundantly expressed during palatal development. Importantly, Flnb-/- embryos display cleft palates and previously defined skeletal defects. Taken together, our findings reveal that FLNB is required for development of palates in mice and FLNB is a bona fide causal gene for NSOFCs in humans.

Inclination of mandibular incisors and symphysis in severe skeletal class III malocclusion.

OBJECTIVE: The aim of this study was to systematically explore the inclination of the lower central incisor and symphysis in alveolar bone in severe skeletal class III patients. MATERIALS AND METHODS: A total of 198 severe skeletal class III patients (ANB ≤ -4°) who underwent combined orthodontic and orthognathic treatment were divided into three groups based on the mandibular plane angle (MP-SN). Pretreatment lateral cephalograms were analysed and compared among the three groups. We also assessed cone-beam computed tomography (CBCT) images of 11 samples to investigate the reliability of the cephalometric analysis. RESULTS: ANOVA showed no statistically significant differences in the angle between the long axis of the mandibular symphysis and the long axis of the lower central incisor (MIA) among the low-angle, normal-angle and high-angle groups (P > 0.05), while significant differences were found in the angle between the axis of the lower incisor and the mandibular plane (IMPA) among the three groups (P < 0.001). The mean IMPA decreased with increasing MP-SN in the 198 patients. The mean MIA in the low-angle and normal-angle groups was 3.70° and 3.52°, respectively, while the value (2.33°) was smaller in the high-angle group. Paired-samples t test showed no statistically significant differences between the cephalometric and CBCT measurements of the MP-SN, the angle between the mandibular plane and the Frankfort plane (FH-MP) and the MIA (P > 0.05). CONCLUSIONS: In severe skeletal class III patients, the long axis of the lower central incisor was highly consistent with the long axis of the mandibular symphysis, which was more obvious in the high-angle subjects. The MIA reflects the physiological inclination of the lower central incisor better than the IMPA.

Time-series expression profiles of mRNAs and lncRNAs during mammalian palatogenesis.

OBJECTIVES: Mammalian palatogenesis is a highly regulated morphogenetic process to form the intact roof of the oral cavity. Long noncoding RNAs (lncRNAs) and mRNAs participate in numerous biological and pathological processes, but their roles in palatal development and causing orofacial clefts (OFC) remain to be clarified. METHODS: Palatal tissues were separated from ICR mouse embryos at four stages (E10.5, E13.5, E15, and E17). Then, RNA sequencing (RNA-seq) was used. Various analyses were performed to explore the results. Finally, hub genes were validated via qPCR and in situ hybridization. RESULTS: Starting from E10.5, the expression of cell adhesion genes escalated in the following stages. Cilium assembly and ossification genes were both upregulated at E15 compared with E13.5. Besides, the expression of cilium assembly genes was also increased at E17 compared with E15. Expression patterns of three lncRNAs (H19, Malat1, and Miat) and four mRNAs (Cdh1, Irf6, Grhl3, Efnb1) detected in RNA-seq were validated. CONCLUSIONS: This study provides a time-series expression landscape of mRNAs and lncRNAs during palatogenesis, which highlights the importance of processes such as cell adhesion and ossification. Our results will facilitate a deeper understanding of the complexity of gene expression and regulation during palatogenesis.

Two rare variants reveal the significance of Grainyhead-like 3 Arginine 391 underlying non-syndromic cleft palate only.

OBJECTIVES: Non-syndromic cleft palate only (NSCPO) is one of the most common craniofacial birth defects with largely undetermined genetic etiology. It has been established that Grainyhead-like 3 (GRHL3) plays an essential role in the pathogenesis of NSCPO. This study aimed to identify and verify the first-reported GRHL3 variant underlying NSCPO among the Chinese cohort. METHODS: We performed whole-exome sequencing (WES) on a Chinese NSCPO patient and identified a rare variant of GRHL3 (p.Arg391His). A validated deleterious variant p.Arg391Cys was introduced as a positive control. Zebrafish embryos injection, reporter assays, live-cell imaging, and RNA sequencing were conducted to test the pathogenicity of the variants. RESULTS: Zebrafish embryos microinjection demonstrated that overexpression of the variants could disrupt the normal development of zebrafish embryos. Reporter assays showed that Arg391His disturbed transcriptional activity of GRHL3 and exerted a dominant-negative effect. Interestingly, Arg391His and Arg391Cys displayed distinct nuclear localization patterns from that of wild-type GRHL3 in live-cell imaging. Bulk RNA sequencing suggested that the two variants changed the pattern of gene expression. CONCLUSIONS: In aggregate, this study identified and characterized a rare GRHL3 variant in NSCPO, revealing the critical role of Arginine 391 in GRHL3. Our findings will help facilitate understanding and genetic counseling of NSCPO.

Accuracy of integration of dental cast and cephalograms compared with cone-beam computed tomography: a comparative study.

This study proposes a method that integrates maxillary dental cast and cephalograms and evaluates its accuracy compared with cone-beam computed tomography (CBCT) scans. The study sample comprised 20 adult patients with records of dental casts, cephalograms, and craniofacial CBCT scans. The maxillary dental cast was integrated with lateral and frontal cephalograms based on best-fit registration of palatal and dental outline curves from dental cast with cephalogram tracings. Linear measurement was conducted to assess the intra- and inter-examiner reproducibility of the proposed integration method using intraclass correlation coefficients; linear and angular measurements were conducted to assess its accuracy with CBCT scans as a standard reference. Paired t test, one sample t test, and mean ± standard deviation of the absolute value of difference were used to compare the integrated images and CBCT. The integration method showed good intra- and inter-examiner reproducibility (intraclass correlation coefficients > 0.98). The differences in linear and angular measurements between the integrated images and CBCT were not statistically significant but with a large deviation. When absolute value of difference was computed, the linear distance error was 0.51 ± 0.34 mm, the tooth point coordinate errors in X, Y and Z axes were 0.22 ± 0.22, 0.38 ± 0.32 and 0.21 ± 0.21 mm, respectively; the angular error in pitch, roll and yaw of the dental cast was 0.82 ± 0.51, 0.92 ± 0.59 and 0.80 ± 0.41 degree, respectively. The proposed method for integration of dental cast and cephalograms showed good reproducibility and acceptable accuracy compared with CBCT. It could be helpful for researchers to study three-dimensional tooth growth changes using the existing craniofacial growth data especially cephalograms.

Dynamic Change in Oral Microbiota of Children With Cleft Lip and Palate After Alveolar Bone Grafting.

To investigate the longitudinal influence of alveolar bone grafting on the oral microbiota of children with cleft lip and palate (CLP).Twenty-eight children with nonsyndromic CLP were recruited and underwent secondary alveolar bone grafting at the first time. Unstimulated saliva and plaque samples were collected from the subjects preoperatively and at 2 days, 1 month, and 3 months postoperatively. The v3-v4 hypervariable regions of the 16S rRNA gene from bacterial DNA were sequenced using the Illumina MiSeq sequencing platform.The alpha diversity of the saliva and plaque microbiota was significantly decreased at 2 days postoperatively and then increased at 1 and 3 months postoperatively. The saliva and plaque microbiota compositions at 2 days postoperatively differed from those at the other time points, and the microbiota compositions at 1 and 3 months postoperatively showed a gradual shift toward the preoperative composition. The saliva, but not plaque, microbiota composition 3 months postoperatively was similar to that preoperatively.The effect of secondary alveolar bone grafting on the plaque microbiota in children with CLP lasted longer than the saliva microbiota. Alveolar bone grafting altered the saliva microbiota in children with CLP within 3 months postoperatively.

Apolipoprotein E is an effective biomarker for orthodontic tooth movement in patients treated with transmission straight wire appliances.

INTRODUCTION: Orthodontic tooth movement (OTM) is the core component of orthodontic treatment and is increasingly popular for treating malocclusions. In this study, we aimed to investigate the role of apolipoprotein E (ApoE) in OTM. METHODS: Thirty patients treated with transmission straight wire technology were selected and longitudinally tracked at 2 different stages of orthodontic treatment (initial 2 months and 12 months of orthodontic treatment). Total saliva was collected and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blotting was used to detect the difference in ApoE expression in the saliva samples of the 2 groups. The expression of ApoE was further verified by immunohistochemical staining in a mouse model of tooth movement. RESULTS: The results of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry showed significant differences in the components of the salivary peptides in the 2 groups and peptides with a molecular weight of 2010.7 Da were predicted to be ApoE by database analysis. Western blotting further verified a significant difference in the expression of salivary ApoE in the 2 groups. In addition, an OTM model was successfully constructed in mice. The immunohistochemical staining results showed that ApoE expression significantly increased after force loading in the OTM model. CONCLUSIONS: This study indicated that ApoE participated in and played a role during OTM in patients treated with transmission straight wire technology. This relationship might be related to alveolar bone reconstruction and root resorption. The results provide new ideas for research on the mechanism of tooth movement using precision medicine based on saliva detection.

A Novel PAX3 Variant in a Chinese Pedigree with Nonsyndromic Cleft Lip With or Without Palate.

Objectives: Nonsyndromic cleft lip with or without palate (NSCL/P) is a common congenital orofacial defect, which is associated with severe disruption of orofacial development. The present study was designed to identify potential underlying gene variants in a Chinese pedigree with NSCL/P, in which the proband and the proband's father were affected. Methods: DNA was extracted from the participants' peripheral venous blood, and whole-exome sequencing was performed on the proband and the proband's parents. Results: After filtering, a paired box gene 3 (PAX3) missense variant (c.92C>G_p.Thr31Ser) was identified, which was verified by Sanger sequencing. This variant, which was not present in 113 unrelated healthy individuals or in a Chinese public database, may affect the transcription inhibition domain of the PAX3 protein. Conservation analysis and in silico predictions suggested that this variant may be evolutionarily conserved and potentially deleterious. In addition, it was reported that mice with PAX3 variants show cleft palates. Thus, the PAX3 missense variant (c.92C>G_p.Thr31Ser) is a candidate causative variant in this family. Conclusions: To the best of our knowledge, the present study is the first to report on a PAX3 variant in a pedigree with NSCL/P. The present study further suggests that PAX3 may be associated with CL/P etiology.

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).

A novel FZD6 mutation revealed the cause of cleft lip and/or palate in a Chinese family.

Cleft lip and/or palate (CL/P) is a most common craniofacial birth defect which has multifactorial etiology. In our study, we aimed to discover the underlying etiological gene variation in a Chinese family diagnosed as non-syndromic CL/P (NSCL/P). The blood sample of the proband and her parents were detected by whole exome sequencing. The Mendelian inheritance pattern, allele frequency, variation location, function analysis and literature search were applied to filtrate and screen the mutation. Besides, the candidates were confirmed by Sanger sequencing. We meanwhile explored the conservative analysis and protein homology simulation. As a result, a start-lost mutation c.1A > GAtg/Gtg in the Frizzled-6 (FZD6) gene predicting p.Met1 was detected. The variation has not been reported before and was predicted to be harmful. The alteration caused missing of two starting amino acids that are evolutionarily conserved for FZD6 protein. Moreover, the specific structure of the mutant protein obviously changed according to the results of the homologous model. In conclusion, the results suggest c.1A > GAtg/Gtg in the FZD6 (NM_001164616) might be the genetic etiology for non-syndromic CL/P in this pedigree. Furthermore, this finding provided new etiologic information, supplementing the evidence that FZD6 is a strong potential gene for CL/P.

Biological Safe Gold Nanoparticle-Modified Dental Aligner Prevents the Porphyromonas gingivalis Biofilm Formation.

Oral microbiology could directly influence overall health. Porphyromonas gingivalis (P. gingivalis) is a highly pathogenic bacterium that causes periodontitis and other related systematic diseases, including Alzheimer's disease. Orthodontic devices (e.g., invisalign aligner) is commonly used in populations with periodontitis who are also at a high risk of systematic diseases. In this study, newly explored antibacterial 4,6-diamino-2-pyrimidinethiol-modified gold nanoparticles (AuDAPT) were coated onto aligners. The coated aligners showed favorable antibacterial activity against P. gingivalis. In the presence of the coated aligner, the number of planktonic cells was decreased, and biofilm formation was prevented. This material also showed favorable biocompatibility in vivo and in vitro. This study reveals a new method for treating oral P. gingivalis by coating aligners with AuDAPT, which has typical advantages compared to other treatments for both periodontitis and related systematic diseases.

Functional Characterization of a Novel IRF6 Frameshift Mutation From a Van Der Woude Syndrome Family.

BACKGROUND: Loss-of-function mutations in interferon regulatory factor-6 (IRF6) are responsible for about 70% of cases of Van Der Woude Syndrome (VWS), an autosomal dominant developmental disorder characterized by pits and/or sinuses of the lower lip and cleft lip, cleft palate, or both. METHODS: We collected a Chinese Han VWS pedigree, performed sequencing and screening for the causal gene mutant. Initially, species conservation analysis and homology protein modeling were used to predict the potential pathogenicity of mutations. To test whether a VWS family-derived mutant variant of IRF6 retained function, we carried out rescue assays in irf6 maternal-null mutant zebrafish embryos. To assess protein stability, we overexpressed reference and family-variants of IRF6 in vitro. RESULTS: We focused on a VWS family that includes a son with bilateral lip pits, uvula fissa and his father with bilateral cleft lip and palate. After sequencing and screening, a frameshift mutation of IRF6 was identified as the potential causal variant (NM.006147.3, c.1088-1091delTCTA; p.Ile363ArgfsTer33). The residues in this position are strongly conserved among species and homology modeling suggests the variant alters the protein structure. In irf6 maternal-null mutant zebrafish embryos the periderm differentiates abnormally and the embryos rupture and die during gastrulation. Injection of mRNA encoding the reference variant of human IRF6, but not of the frame-shift variant, rescued such embryos through gastrulation. Upon overexpression in HEK293FT cells, the IRF6 frame-shift mutant was relatively unstable and was preferentially targeted to the proteasome in comparison to the reference variant. CONCLUSION: In this VWS pedigree, a novel frameshift of IRF6 was identified as the likely causative gene variant. It is a lost function mutation which could not rescue abnormal periderm phenotype in irf6 maternal-null zebrafish and which causes the protein be unstable through proteasome-dependent degradation.

Hydrogel-Coated Dental Device with Adhesion-Inhibiting and Colony-Suppressing Properties.

Bacterial infection is the main cause of implantation failure worldwide, and the importance of antibiotics on medical devices has been undermined because of antibiotic resistance. Antimicrobial hydrogels have emerged as a promising approach to combat infections associated with medical devices and wound healing. However, hydrogel coatings that simultaneously possess both antifouling and antimicrobial attributes are scarce. Herein, we report an antimicrobial hydrogel that incorporates adhesion-inhibiting polyethylene glycol (PEG) and colony-suppressing chitosan (CS) as a dressing to combat bacterial infections. These two polymers have important environmentally benign characteristics including low toxicity, low volatility, and biocompatibility. Although hydrogels containing PEG and CS have been reported for applications in the fields of wound dressing, tissue repair, water purification, drug delivery, and scaffolds for bone regeneration, there still has been no report on the application of CS/PEG hydrogel coatings in dental applications. Herein, this biointerface shows superior activity in early-stage adhesion inhibition (98.8%, 5 h) and displays remarkably long-lasting colony-suppression activity (93.3%, 7 d). Thus, this novel nanomaterial, which has potential as a dual-functional platform with integrated antifouling and antimicrobial functions with excellent biocompatibility, might be used as a safe and effective antimicrobial coating in biomedical device applications.

Posteroanterior cephalometric analysis of White-American and Chinese adolescents: a cross-sectional study.

Objective: To characterize ethnic differences between Chinese and White-Americans between 8.5 and 17.5 years of age, with respect to transverse cephalometric characteristics and to establish transverse craniofacial normative values for Chinese adolescents. Methods: Two-hundred fifty-seven and 547 posteroanterior cephalograms were selected from 35 White-Americans and 157 Chinese with individual normal occlusions. Transverse measurements were obtained and compared between ethnicities to guide determination of normative values for Chinese adolescents. Student's t-test or one-way analysis of variance was used, as appropriate. Results: Chinese girls demonstrated significantly larger measurements than White-American girls at all ages, with the exception of nasal width. Chinese boys exhibited larger measurements than White-American boys at different ages. Chinese boys had larger measurements than girls for most measurements. These data established normative values for Chinese adolescents. Discussion: Ethnic differences existed between Chinese and White-American adolescents with respect to transverse craniofacial measurements. Transverse normative values were established for Chinese adolescents.

Three GLI2 mutations combined potentially underlie non-syndromic cleft lip with or without cleft palate in a Chinese pedigree.

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial birth defect. Its etiology is complex and it has a lifelong influence on affected individuals. Despite many studies, the pathogenic gene alleles are not completely clear. Here, we recruited a Chinese NSCL/P family and explored the candidate causative variants in this pedigree. METHODS: We performed whole-exome sequencing on two patients and two unaffected subjects of this family. Variants were screened based on bioinformatics analysis to identify the potential etiological alleles. Species conservation analysis, mutation function prediction, and homology protein modeling were also performed to preliminarily evaluate the influence of the mutations. RESULTS: We identified three rare mutations that are located on a single chromatid (c.2684C > T_p.Ala895Val, c.4350G > T_p.Gln1450His, and c.4622C > A_p.Ser1541Tyr) in GLI2 as candidate causative variants. All of these three mutations were predicted to be deleterious, and they affect amino acids that are conserved in many species. The mutation c.2684C > T was predicted to affect the structure of the GLI2 protein. CONCLUSION: Our results further demonstrate that GLI2 variants play a role in the pathogenesis of NSCL/P, and the three rare missense mutations combined are probably the potential disease-causing variants in this family.

Longitudinal Quantitation of Tooth Displacement in Chinese Adolescents with Normal Occlusion.

This longitudinal study aims to analyze the different modes of the maxillary and mandibular tooth displacement in subjects, who were aged 12.5-17.5 years (150-210 months), with untreated normal (Class I) occlusion. Longitudinal lateral cephalograms for a set of 10 subjects (7 females and 3 males) at consecutive annual time points were selected and monitored. Data were analyzed on the basis of the superimpositions of serial tracings of lateral cephalograms on stable anterior cranial base, the anatomies of the maxillary and mandibular structures. The horizontal and vertical displacements of the first molar and incisor were assessed by t-test. The local and the secondary tooth displacements with growth contributed to the total horizontal and vertical displacements of the molars and incisors of the subjects. In the total tooth displacement, the horizontal growth of maxilla and mandible had the same contribution as the local tooth displacements. The vertical maxillary growth played a smaller role than the local drift, and mandibular remodeling went in a reverse direction with the local tooth drift. The first molars moved more forward than the incisors in the upper and lower arches. Both the upper and lower first molars showed forward tipping. The analysis of tooth displacement may be utilized in making orthodontic treatment plan, including anchorage or torque control.