MiR-153-3p inhibits osteogenic differentiation of periodontal ligament stem cells through KDM6A-induced demethylation of H3K27me3.

BACKGROUND AND OBJECTIVE: Periodontal ligament stem cells (PDLSCs) have potential for osteogenic differentiation and show a great foreground in treating bone diseases. Histone three lysine 27 (H3K27) demethylase lysine demethylase 6A (KDM6A) is a critical epigenetic modifier and plays an important role in regulating osteogenic differentiation. Multiple microRNAs have been found to play important roles in osteogenesis. The aim of this study was to explore the mechanisms underlying the roles of miR-153-3p and KDM6A in PDLSC osteogenesis. METHODS: The levels of the osteogenic markers alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteopontin (OPN) were measured by western blotting. Osteoblast activity and mineral deposition were detected by ALP and Alizarin red S (ARS) staining. The levels of miR-153-3p and KDM6A were measured by quantitative real-time PCR (qRT-PCR). A luciferase reporter assay was used to confirm the interaction between KDM6A and miR-153-3p. Gain-of-function and loss-of-function assays were performed to identify the roles of miR-153-3p and KDM6A in the osteogenic differentiation of PDLSCs. RESULTS: In osteogenic PDLSCs, the expression of KDM6A, ALP, Runx2, and OPN was upregulated, whereas that of miR-153-3p was downregulated. miR-153-3p downregulation or KDM6A overexpression promoted the osteogenic differentiation of PDLSCs, as demonstrated by increases in ALP activity, matrix mineralization, and ALP, Runx2, and OPN expression. KDM6A was confirmed to be a target of miR-153-3p, and KDM6A overexpression reversed the inhibitory effect of miR-153-3p mimic on PDLSC osteogenesis. KDM6A promoted ALP, Runx2, and OPN expression through the demethylation of H3K27me3 on the promoter regions of these genes. CONCLUSION: miR-153-3p inhibited PDLSC osteogenesis by targeting KDM6A and inhibiting ALP, Runx2, and OPN transcription. These findings provide latent hope for PDLSCs application in periodontal therapy.

Analysis of the Salivary Microbiome in Obstructive Sleep Apnea Syndrome Patients.

BACKGROUND: Oral microbiota plays an important role in oral and systemic diseases, while few reports referred to obstructive sleep apnea syndrome (OSAS). Thus, this study aimed to explore the different salivary microbiome in patients with OSAS and controls. MATERIALS AND METHODS: Saliva was collected from 15 OSAS patients and nine healthy controls, and bacterial genomic DNA was extracted for 16S rRNA amplicon sequencing based on the Illumina platform. RESULTS: The alpha and beta diversities were not significantly different between patients with OSAS and controls. The main phyla in the two groups were Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, and Fusobacteria, which accounted for 95% of the abundance. The main genera were Streptococcus, Rothia, Actinomyces, Prevotella, and Neisseria. Based on the genus and operational taxonomic units, Peptostreptococcus, Alloprevotella, and Granulicatella were enriched in controls, while only Scardovia species were significantly more abundant in patients with OSAS. CONCLUSIONS: There was no significant difference in the relative abundance of bacteria between OSAS and controls. So, further studies will need to focus on the metagenome of bacteria in OSAS patients.

A novel IRF6 mutation causing non-syndromic cleft lip with or without cleft palate in a pedigree.

Non-syndromic cleft lip with or without cleft palate (NSCLP) is the most common congenital craniofacial malformation, and its harmful influence on affected individuals is apparent. Despite many studies, the causative genes and their mechanisms are not completely clear. We recruited a Han Chinese NSCLP family and explored the causative variant in this pedigree. We performed whole-exome sequencing on two patients. Bioinformatics screening and analysis were used to identify the mutation. We also performed species conservation analysis, mutation function predictions, and homology protein modelling to evaluate the influence of the mutation. We identified a rare mutation in interferon regulatory factor 6 (IRF6) (c.26G>A; p.Arg9Gln) as a candidate of causative mutation. This mutation was predicted to be deleterious. The codon is conserved in many species. The residue change caused by this mutation would affect the structure of IRF6 to a degree. Our study suggested that the rare IRF6 variant is probably the pathogenic mutation in this family. Our result adds evidence that IRF6 variants play a role in the aetiology of orofacial clefts.

A novel PTCH1 mutation underlies nonsyndromic cleft lip and/or palate in a Han Chinese family.

OBJECTIVES: Cleft lip and/or palate (CL/P) is the most common craniofacial congenital disease, and it has a complex aetiology. This study aimed to identify the causative gene mutation of a Han Chinese family with CL/P. SUBJECTS AND METHODS: Whole exome sequencing was conducted on the proband and her mother, who exhibited the same phenotype. A Mendelian dominant inheritance model, allele frequency, mutation regions, functional prediction and literature review were used to screen and filter the variants. The candidate was validated by Sanger sequencing. Conservation analysis and homology modelling were conducted. RESULTS: A heterozygous missense mutation c.1175C>T in the PTCH1 gene predicting p.Ala392Val was identified. This variant has not been reported and was predicted to be deleterious. Sanger sequencing verified the variant and the dominant inheritance model in the family. The missense alteration affects an amino acid that is evolutionarily conserved in the first extracellular loop of the PTCH1 protein. The local structure of the mutant protein was significantly altered according to homology modelling. CONCLUSIONS: Our findings suggest that c.1175C>T in PTCH1 (NM_000264) may be the causative mutation of this pedigree. Our results add to the evidence that PTCH1 variants play a role in the pathogenesis of orofacial clefts.

LncRNA AWPPH overexpression predicts the recurrence of periodontitis.

Long non-coding RNA (LncRNA) AWPPH is a recently identified oncogenic lncRNA, while its role in other human diseases is still unknown. Blood samples from 80 periodontitis (periodontitis group) patients and 66 healthy controls (control group) who were admitted and treated by Peking University School and Hospital of Stomatology, expression levels of lncRNA AWPPH were detected by RT-PCR. In the present study, we showed that, before treatment, lncRNA AWPPH in plasma was up-regulated in periodontitis patients than in healthy controls. After treatment, expression levels of lncRNA AWPPH reduced significantly. Patients were followed up for 2 years to recorded recurrence. Compared with plasma levels of lncRNA AWPPH on the day of discharge, lncRNA AWPPH expression level increased significantly in patients with recurrence but not in patients without recurrence during follow-up. Based on Youden's index, patients were divided into high and low lncRNA AWPPH groups according to its expression level on the day of discharge. It was observed that the recurrence rate of periodontitis is significantly higher in high lncRNA AWPPH group than in low lncRNA AWPPH group. LncRNA AWPPH overexpression predicts the recurrence of periodontitis.

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.

Machine Learning Models for Genetic Risk Assessment of Infants with Non-syndromic Orofacial Cleft.

The isolated type of orofacial cleft, termed non-syndromic cleft lip with or without cleft palate (NSCL/P), is the second most common birth defect in China, with Asians having the highest incidence in the world. NSCL/P involves multiple genes and complex interactions between genetic and environmental factors, imposing difficulty for the genetic assessment of the unborn fetus carrying multiple NSCL/P-susceptible variants. Although genome-wide association studies (GWAS) have uncovered dozens of single nucleotide polymorphism (SNP) loci in different ethnic populations, the genetic diagnostic effectiveness of these SNPs requires further experimental validation in Chinese populations before a diagnostic panel or a predictive model covering multiple SNPs can be built. In this study, we collected blood samples from control and NSCL/P infants in Han and Uyghur Chinese populations to validate the diagnostic effectiveness of 43 candidate SNPs previously detected using GWAS. We then built predictive models with the validated SNPs using different machine learning algorithms and evaluated their prediction performance. Our results showed that logistic regression had the best performance for risk assessment according to the area under curve. Notably, defective variants in MTHFR and RBP4, two genes involved in folic acid and vitamin A biosynthesis, were found to have high contributions to NSCL/P incidence based on feature importance evaluation with logistic regression. This is consistent with the notion that folic acid and vitamin A are both essential nutritional supplements for pregnant women to reduce the risk of conceiving an NSCL/P baby. Moreover, we observed a lower predictive power in Uyghur than in Han cases, likely due to differences in genetic background between these two ethnic populations. Thus, our study highlights the urgency to generate the HapMap for Uyghur population and perform resequencing-based screening of Uyghur-specific NSCL/P markers.

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.

[Changes of upper airway morphology induced by mandibular advancement in patients with obstructive sleep apnea syndrome].

OBJECTIVE: To determine the changes of upper airway morphology induced by mandible position from central relation to advancement position in patients with obstructive sleep apnea syndrome. METHODS: Nineteen patients (17 males and 2 females) suffering from obstructive sleep apnea syndrome were confirmed with polysomnography. Occlusal wax record was made with mandible in advancement position. Helical computed tomography was performed on each patient in central relation and mandibular advancement position with wax record in situ respectively. On each slice, anteroposterior and transverse diameters were obtained. Airway shape was expressed as the anteroposterior/transverse (AP/T) diameter ratio. Paired-samples t test was employed to compare the measurements. RESULTS: With mandibular advancement, average and minimal diameters of glossopharynx and hypopharynx were increased significantly. Compared with it, the change of transverse diameter was more prominent. All segments of upper airway were increased significantly except hypopharynx laterally. Moreover, the change of velopharynx shape was observed on axial planes. Ellipse with transversal long axis became more compressed. CONCLUSION: Upper airway morphology of all segments was influenced by mandibular advancement whether in the sagittal or transverse plane of space, or airway shape. Not only anatomic connection but also regulation of the nerve system and other still unknown mechanism make contribution to the changes of upper airway morphology in patients with obstructive sleep apnea syndrome. The changes of upper airway morphology above mentioned constitute the rationale of treatment of obstructive sleep apnea syndrome with oral appliances.

Changes of pharyngeal airway size and hyoid bone position following orthodontic treatment of Class I bimaxillary protrusion.

OBJECTIVES: To test the hypothesis that the sagittal position of the anterior teeth has no effect on pharyngeal airway dimension or hyoid bone position and to investigate the influence of orthodontic retraction of the anterior teeth on each section of pharynx and hyoid position. MATERIALS AND METHODS: Forty-four Class I bimaxillary protrusion adults, treated with preadjusted appliances and maximum anchorage after extraction of four premolars, were divided into two groups according to their vertical craniofacial skeletal patterns. Pretreatment and posttreatment variables were compared using paired t-test, and the relationship between pharyngeal airway size and dentofacial variables was analyzed using Pearson correlation coefficient. The changes of pharyngeal airway size and hyoid position after treatment were compared between two groups using independent t-test. RESULTS: Upon retraction of the incisors, the upper and lower lips were retracted by 2.60 mm and 3.87 mm, respectively. The tip of upper incisor was retracted by 6.84 mm and lower incisor retracted by 4.95 mm. There was significant decrease in SPP-SPPW, U-MPW, TB-TPPW, V-LPW, VAL, C3H, and SH (P < .05). No statistically significant different changes were observed in the dentofacial structures, pharyngeal airway, and hyoid position between the two groups after the treatment. There was a significant correlation between the retraction distance of lower incisor and the airway behind the soft palate, uvula, and tongue. CONCLUSIONS: The pharyngeal airway size became narrower after the treatment. Extraction of four premolars with retraction of incisors did affect velopharyngeal, glossopharyngeal, hypopharyngeal, and hyoid position in bimaxillary protrusive adult patients.

[Chin morphology in subjects with different vertical skeletal craniofacial pattern].

OBJECTIVE: To evaluate whether there is difference with regard to chin morphology in subjects with different vertical skeletal craniofacial pattern and the relationship among them. METHODS: The sample was composed of 80 adolescents who denied orthodontic treatment history and presented Class I skeletal pattern, aged (12.69+/-0.70) years. They were divided into three groups according to mandibular plane angle: High angle group (21 cases, FH/MP> or = 32 degrees), average angle group(43 cases, 22 degrees

[Treatment of OSAS with modified twin-block advancement appliances].

OBJECTIVE: To introduce a new modified twin-block advancement appliance and investigate the effects on respiratory variables in patients with OSAS. METHODS: 29 patients with OSAS participated in the study and were fitted with modified twin-block appliances to hold the mandible in an anterior and inferior position. Polysomnography was performed with and without appliance insertion. And questionnaires were used for registration of patients subjective symptoms. Pair-t analysis was used to evaluate the effects of appliances in patients with OSAS. RESULTS: 26 patients responded to the appliance therapy. Apnea-hypopnea index, apnea index and hypopnea index were reduced significantly (P < 0.01). Lowest arterial oxygen saturation improved significantly (P < 0.01). Discomfort with mandibular advancement disappeared within one week. CONCLUSIONS: Modified twin-block advancement appliance is a conservative, successful treatment alternative that could benefit patients suffered from OSAS.