The classification as well as the clinical manifestations of hereditary malformations of dentin are of great concern and have been deeply elucidated. The understanding of its genetic basis also increases progressively. Dentin sialophosphoprotein (DSPP) is the pathogenic gene of dentinogenesis imperfecta type Ⅱ, dentinogenesis imperfecta type Ⅲ and dentin dysplasia type Ⅱ. In this article, the classification of DSPP mutations as well as the resultant dysfunction of the mutant DSPP are summarized respectively and the corresponding clinical manifestations are analyzed. This work will provide a reference for the diagnosis and treatment of hereditary malformations of dentin.
Humans ; Dentinogenesis Imperfecta/pathology* ; Mutation ; Extracellular Matrix Proteins/genetics* ; Phosphoproteins/genetics* ; Sialoglycoproteins/genetics* ; Dentin/pathology*

OBJECTIVE: To explore the clinical and genetic characteristics of a Chinese pedigree affected with hereditary dentinogenesis imperfecta (DGI) type II. METHODS: Clinical data of the pedigree members were collected. Genomic DNA was extracted from peripheral blood samples and subjected to whole exome sequencing. RESULTS: Clinical characteristics of the affected family members have included amber teeth along with significant attrition, constricted roots and dentine hypertrophy leading to pulpal obliteration, which were suggestive of DGI type II. All of the affected members were found to have harbored a novel heterozygous c.2837delA (p.Asp946Valfs*368) variant of the DSPP gene which was predicted to be likely pathogenic. CONCLUSION The c.2837delA variant of the DSPP gene probably underlay the disease in this pedigree. Above finding has expanded the variant spectrum of DSPP gene and provided a basis for molecular diagnosis and genetic counseling for this pedigree.
Dentinogenesis Imperfecta/genetics* ; Extracellular Matrix Proteins/genetics* ; Humans ; Mutation ; Pedigree ; Phosphoproteins/genetics* ; Sialoglycoproteins/genetics*

OBJECTIVE: To verify the effect of the mutant gene vps4b on the expression of tooth development-related proteins, dentin sialophosphoprotein (DSPP) and collagenⅠ (COL-Ⅰ). METHODS: Paraffin tissue sections of the first molar tooth germ were obtained from the heads of fetal mice at the embryonic stages of 13.5, 14.5, and 16.5 days and from the mandibles of larvae aged 2.5 and 7 days after birth. The immunohistochemical method was used to detect the expression and location of DSPP and COL-Ⅰ in wild-type mouse and vps4b knockout mouse. RESULTS: DSPP and COL-Ⅰ were not found in the bud and cap stages of wild-type mouse molar germ. In the bell stage, DSPP was positively expressed in the inner enamel epithelium and dental papilla, whereas COL-Ⅰ was strongly expressed in the dental papilla and dental follicle. During the secretory and mineralized periods, DSPP and COL-Ⅰ were intensely observed in ameloblasts, odontoblasts, and dental follicles, but COL-Ⅰ was also expressed in the dental papilla. After vps4b gene knockout, DSPP was not expressed in the dental papilla of the bell stage and in the dental papilla and dental follicle of the secretory phase. The expression position of COL-Ⅰ in the bell and mineralization phase was consistent with that in the wild-type mice. Moreover, the expression of COL-Ⅰ in the dental papilla changed in the secretory stage. CONCLUSIONS Gene vps4b plays a significant role in the development of tooth germ. The expression of DSPP and COL-Ⅰ may be controlled by gene vps4b and regulates the development of tooth dentin and cementum together with vps4b.
ATPases Associated with Diverse Cellular Activities ; genetics ; Animals ; Collagen ; metabolism ; Endosomal Sorting Complexes Required for Transport ; genetics ; Extracellular Matrix Proteins ; metabolism ; Mice ; Mice, Knockout ; Molar ; Odontoblasts ; Phosphoproteins ; metabolism ; Sialoglycoproteins ; metabolism ; Tooth Germ

OBJECTIVETo identify the causative mutation in a Chinese family affected with dentinogenesis imperfecta shields type II (DGI-II).

METHODSWith informed consent obtained from all participants, peripheral blood or chorionic villi samples were collected from the family members. Genomic DNA was extracted using a standard SDS-proteinase K-phenol/chloroform method. The whole coding region and exon/intron boundaries of the DSPP gene were amplified with polymerase chain reaction (PCR) and subjected to Sanger sequencing. To confirm the pathogenicity of the identified mutation, an Alu I recognition sequence was introduced into the mutant allele using mismatch primers by semi-nested PCR. Restriction fragment length polymorphism (RFLP) analysis was then carried out for all family members and 60 unrelated healthy controls. Meanwhile, mini-DSPP constructs were conducted to confirm the effect of the mutation in vitro.

RESULTSA splicing site mutation, c.52-1G>A, which was located upstream of exon 3, was found in all three patients and the fetus of the proband. Restriction analysis confirmed that all unaffected individuals and the 60 healthy controls did not carry the same mutation. The expression of minigene showed that the exon 3 of the DSPP gene was skipped during the transcription.

CONCLUSIONA novel pathogenic splicing-mutation c.52-1G>A has been detected in a Chinese family affected with DGI-II, which enabled prenatal diagnosis for the fetus of the proband.

Adult ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child, Preschool ; Dentinogenesis Imperfecta ; genetics ; Exons ; Extracellular Matrix Proteins ; genetics ; Female ; Humans ; Male ; Molecular Sequence Data ; Pedigree ; Phosphoproteins ; genetics ; Point Mutation ; RNA Splicing ; Sialoglycoproteins ; genetics

The expression of dentin sialophosphoprotein (DSPP) is the marker for cells differentiated into odontoblasts. This study attempted to analyze the DSPP promoter and build the reporter LacZ expression system driven by this promoter, which allows convenient and quick detection of odontoblast cells. First, we separated the human dental mesenchymal cells in which the expression of DSPP can be effectively induced by dexamethasone. Second, four 5' flanking regions of human DSPP gene (- 4 000-+54, -2 500-+54, -1 447-+54 and -1 027-+54) were analyzed, the results showed that the highest promoter activity lied in the -2 500-+54 region. The promoter activity is reduced when the 5' flanking region was extended from -2 500 to -4 000 bp upstream from the transcription start site; The promoter activity are also decreased when the 5' flanking regions were shorted from -2 500 to -1 447 bp and from -1 447 to -1 027 bp, indicating that potential suppresser elements are lied in the region between -4 000 and -2 500 bp and potential activator elements are lied in the region between -2 500 and -1 027 bp. Then we constructed the lentiviral report vector phDSPP-LacZ containing the -2 500-+ 54 promoter region in front of the LacZ gene. The expression of LacZ was detected using X-Gal staining in both human dental mesenchymal cells and immortalized human dental mesenchymal cells infected with phDSPP-LacZ. The phDSPP-LacZ lentiviral vector may provide a more convenient method to detect the expression of DSPP in human odontogenic differentiation, tooth development and tooth regeneration studies.
Cell Differentiation ; Extracellular Matrix Proteins ; genetics ; Genes, Reporter ; Humans ; Lac Operon ; Odontoblasts ; cytology ; Phosphoproteins ; genetics ; Promoter Regions, Genetic ; Sialoglycoproteins ; genetics

OBJECTIVETo study the expression of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in children with tetralogy of Fallot (TOF), and to investigate the role of histone acetylation and acetylation-related enzymes in the pathogenesis of TOF.

METHODSMyocardial tissue samples in the TOF group were obtained from 46 children with TOF who underwent radical operation, and myocardial tissue samples in the control group were obtained from 16 children who suffered accidental deaths and had no cardiac anomalies as shown by autopsy. The acetylation of H3K9, H3K18 and H3K27 was evaluated by immunohistochemistry. The mRNA expression of HATs and HDACs in the myocardium was measured by real-time PCR. The correlation between mRNA expression of HATs and HDACs and histone acetylation was analyzed.

RESULTSCompared with the control group, the TOF group showed significantly increased acetylation of H3K9 (P=0.0165) and significantly decreased acetylation of H3K18 (P=0.0048) and H3K27 (P=0.0084). As to 4 HATs and 6 HDACs, the mRNA expression of EP300 and CBP was significantly higher in the TOF group than in the control group (P=0.025; P=0.017), and there was no significant difference in the mRNA expression of other HATs and HDACs between the two groups. The correlation analysis revealed a positive correlation between H3K9 acetylation and mRNA expression of EP300 (r=0.71, P<0.01) and CBP (r=0.72, P<0.01).

CONCLUSIONSUpregulated mRNA expression of EP300 and CBP may be associated with increased H3K9 acetylation, suggesting that EP300 and CBP might affect cardiac development by regulating H3K9 acetylation.

Acetylation ; E1A-Associated p300 Protein ; genetics ; Female ; Histone Acetyltransferases ; genetics ; Histone Deacetylases ; genetics ; Histones ; metabolism ; Humans ; Infant ; Male ; Myocardium ; metabolism ; Peptide Fragments ; genetics ; RNA, Messenger ; analysis ; Sialoglycoproteins ; genetics ; Tetralogy of Fallot ; metabolism

OBJECTIVETo detect the effect of connective tissue growth factor (CTGF) on podocalyxin expression in mouse podocytes exposed to high glucose in vitro and explore the possible pathway involved.

METHODSThe expression vector carrying a small interfering RNA (siRNA) targeting CTGF was transfected into mouse podocytes cultured in the presence of 1 g/L glucose (normal control), 4.5 g/L glucose (high glucose group), 1 g/L glucose + 3.5 g/L mannitol (iso-osmolar control group). The changes in the protein expression levels of podocalyxin, CTGF and ERK1/2 in the cells in response to the treatments were investigated using Western blotting.

RESULTSHigh glucose exposure for 24 and 48 h resulted in significantly decreased expression of podocalyxin and increased CTGF in the podocytes (P<0.05). Phosphorylation of ERK1/2 occurred as early as 30 min after the exposure, and the activation was maintained till 24 h. Transfection of the cells with siRNA targeting CTGF significantly inhibited these changes.

CONCLUSIONCTGF is an important mediator of high glucose-induced podocyte damage and decreases the protein level of podocalyxin by the ERK1/2 pathway. CTGF-specific siRNA can alleviate high glucose-induced podocyte injury, suggesting its potential value in treatment of diabetic nephropathy.

Animals ; Cells, Cultured ; Connective Tissue Growth Factor ; metabolism ; Diabetic Nephropathies ; Glucose ; adverse effects ; MAP Kinase Signaling System ; drug effects ; Mice ; Podocytes ; cytology ; drug effects ; metabolism ; RNA, Small Interfering ; genetics ; Sialoglycoproteins ; metabolism

OBJECTIVETo study the genetic etiology of an autosomal dominant dentinogenesis imperfecta in a Chinese family.

METHODSThe molecular change of the disease in the family was analyzed through the clinical examination, linkage analysis, mutational screening of the DSPP gene and restriction fragment length polymorphism analysis.

RESULTSThe disease related gene was completely linked with microsatellite marker D4S1534. We found a novel mutation in the first exon of the DSPP gene (c.49C>T, p.Pro17Ser). All patients in the family had the mutation, while this mutation was not observed in the normal individuals of this family and 100 unrelated controls.

CONCLUSIONThe p.Pro17Ser identified in the family was a new pathogenic mutation. Our finding provided further understanding of the molecular mechanism of dentinogenesis imperfecta.

Amino Acid Sequence ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Dentinogenesis Imperfecta ; genetics ; Exons ; Extracellular Matrix Proteins ; genetics ; Female ; Humans ; Male ; Microsatellite Repeats ; Molecular Sequence Data ; Mutation ; Pedigree ; Phosphoproteins ; Sialoglycoproteins ; Young Adult

BACKGROUNDTissue engineering techniques combined with gene therapy have been recently used to improve osteogenesis. NEL-like molecule-1 (Nell-1), a novel growth factor, has been reported to have specificity for osteochondral lineage. The study assessed the osteogenic differentiation of rat bone marrow stromal cells (bMSCs) after Nell-1 gene modification and examined its ectopic bone formation ability in a nude mice model with tissue engineering technique.

METHODSbMSCs obtained from Fischer 344 rats were transduced with either AdNell-1 (Nell-1 group) or Ad-beta-galactosidase (AdLacZ, LacZ group) or left untransduced (untransduced group). The expression of Nell-1 protein was determined by Western blotting and transfer efficiency was assessed. mRNA expressions of osteopontin (OP), bone sialoprotein (BSP) and osteocalcin (OC) were assessed by real-time PCR 0, 3, 7, 14, and 21 days after gene transfer. Alkaline phosphatase (ALP) activity was measured and von Kossa test was also conducted. Finally, with a tissue engineering technique, gene transduced bMSCs, combining with beta-tricalcium phosphate (beta-TCP) at a concentration of 2 x 10(7) cells/ml, were implanted at subcutaneous sites on the back of nude mice. Four weeks after surgery, the implants were evaluated with histological staining and computerized analysis of new bone formation.

RESULTSUnder current transduction conditions, gene transfer efficiency reached (57.9 +/- 6.8)%. Nell-1 protein was detected in Nell-1 group but not in untransduced group and LacZ group. Induced by Nell-1, BSP and OP expression were increased at intermediate stage and OC expression was increased at later stage. ALP activity and the number of calcium nodules were highest in Nell-1 group. Four weeks after implanted into nude mice subcutaneously, the percentage of new bone area in Nell-1 group was (18.1 +/- 5.0)%, significantly higher than those of untransduced group (11.3 +/- 3.2)% and LacZ group (12.3 +/- 3.1)% (P < 0.05).

CONCLUSIONSThis study has demonstrated the ability of Nell-1 to induce osteogenic differentiation of rat bMSCs in vitro and to enhance bone formation with a tissue engineering technique. The results suggest that Nell-1 may be a potential osteogenic gene to be used in bone tissue engineering.

Alkaline Phosphatase ; metabolism ; Animals ; Blotting, Western ; Bone Marrow Cells ; cytology ; metabolism ; ultrastructure ; Integrin-Binding Sialoprotein ; Male ; Mice ; Mice, Nude ; Microscopy, Electron, Scanning ; Nerve Tissue Proteins ; metabolism ; Osteocalcin ; genetics ; Osteogenesis ; Osteopontin ; genetics ; Rats ; Rats, Inbred F344 ; Reverse Transcriptase Polymerase Chain Reaction ; Sialoglycoproteins ; genetics ; Stromal Cells ; cytology ; metabolism ; ultrastructure ; Tissue Engineering

BACKGROUNDOsteoarthritis (OA) is a chronic and incurable disease, lacking effective treatment. Gene therapy offers a radical different approach to the treatment of arthritis. Even though the etiology of OA remains unclear, there is now considerable evidence to suggest that interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) are the main mediators in the pathogenesis of OA. The goal of this study was to determine the efficacy of local expression of interleukin-1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor-alpha receptor type I (sTNF-RI) by direct adenoviral-mediated intra-articular gene delivery in the rabbit model of osteoarthritis.

METHODSAdenoviral vectors containing IL-1Ra or sTNF-RI genes were constructed. OA was induced in both hind knees of 12 New Zealand white rabbits by the excision of the medial collateral ligament plus medial meniscectomy. Five days after surgery, approximately 1 x 10(8) plaque-forming units (pfu) of adenovirus were injected into the joint space of the knee through the patellar tendon. A total of 12 operated rabbits were divided into four groups. Three experimental rabbit groups received 1 x 10(8) pfu of adenovirus encoding either IL-1Ra (3 rabbits), sTNF-RI (3 rabbits) or IL-1Ra and sTNF-RI in combination (3 rabbits), into both knee joints respectively. An inflamed control group of 3 rabbits received approximately 1 x 10(8) pfu of Ad-GFP into both joints. Three days after injection of the adenovirus, both knees of each rabbit were lavaged with 1 ml of saline solution through the patellar tendon. At day 7, the rabbits were sacrificed, and the knees were lavaged, dissected and analyzed for effects of transgene expression. Levels of IL-1Ra and sTNF-RI expression in recovered lavage fluids were measured using a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were fixed, embedded, sectioned and stained with hematoxylin and eosin (cartilage and synovium) and toluidine blue (cartilage). The samples were examined by light microscopy and quantitatively evaluated.

RESULTSIntra-articular delivery of IL-1Ra resulted in a significant inhibition of cartilage degradation, but did not affect synovial changes. In contrast, rabbit knee joints receiving sTNF-RI alone showed no detectable reduction in cartilage degradation. However, double gene transfer of IL-1Ra and sTNF-RI resulted in a higher suppression of the cartilage degradation and an observable reduction in synovitis. These data add to and confirm that IL-1Ra has good chondroprotective properties, but TNF-alpha blockade has little effect on joint destruction.

CONCLUSIONThe enhanced therapeutic effects of both antagonists in combination suggest inhibition of multiple inflammatory cytokines may be more efficacious than blockade of either cytokine alone in treating OA.

Adenoviridae ; genetics ; Animals ; Arthritis, Experimental ; genetics ; therapy ; Cartilage ; metabolism ; pathology ; Cartilage, Articular ; metabolism ; pathology ; Cell Line ; Cells, Cultured ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Humans ; Interleukin 1 Receptor Antagonist Protein ; Osteoarthritis ; genetics ; therapy ; Rabbits ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; physiology ; Sialoglycoproteins ; genetics ; physiology ; Synovial Fluid ; metabolism ; Synovial Membrane ; cytology ; metabolism ; Transfection ; methods