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*

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*

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 investigate the polymorphisms of interleukin-1B (IL-1B) promoter region -511C/T and interleukin-1 receptor antagonist (RN) gene, the relationship between the genotype of IL-1B and IL-1RN, and the susceptibility to chronic hepatitis B in Chinese population.

METHODSGenomic DNA was extracted from the peripheral blood of 190 patients with chronic hepatitis B and 249 normal controls and then was subjected to PCR amplification. The PCR product was digested by restriction endonuclease Ava I. The product of digestion was subjected to 2% gel-electrophoresis and ethidium bromide staining.

RESULTSThe IL-1B -511C allele was detected in 0.50 of normal controls and 0.48 of patients, while the IL-1B -511T allele was detected in 0.50 of normal controls and 0.52 of patients. The frequency of CC genotype was 0.26 (65/249) and in normal controls 0.24 (45/190) in patients. The frequency of CT genotype was 0.47 (118/249) in normal controls and 0.49 (94/190) in patients. The frequency of TT genotype was 0.27 (66/249) in normal controls and 0.27 (51/190) in patients. The HBV-DNA copies in chronic hepatitis B with CC genotype were significantly decreased, compared with controls (P<0.05). Only four (1/1, 1/2, 2/2 and 1/4) of the five kinds of polymorphism of IL-1RN were found in this study. The frequencies of 1/1, 1/2, 2/2 and 1/4 were 0.88, 0.09, 0.01 and 0.02 in chronic hepatitis B patients respectively, while in controls were 0.81, 0.16, 0.01 and 0.02. The IL-1 RN*1 allele was detected in 0.94 of chronic hepatitis B patients and in 0.90 of controls, while the IL-1RN*2 allele was detected in 0.05 of patients and in 0.09 of normal controls. The frequencies of 1/2 genotype and IL-1RN*2 allele were lower in chronic hepatitis B than in controls (P<0.05).

CONCLUSIONThe polymorphisms of the promoter region -511C/T of IL-1B gene and IL-1RN intron 2 gene are associated with the development of chronic hepatitis B. The people with IL-1RN*2 allele may be protected against HBV infection, and the IL-1B -511 CC genotype may be linked to HBV-DNA copy.

Adolescent ; Adult ; Aged ; Child ; Female ; Genotype ; Hepatitis B, Chronic ; genetics ; Humans ; Interleukin 1 Receptor Antagonist Protein ; Interleukin-1 ; genetics ; Male ; Middle Aged ; Polymorphism, Genetic ; Sialoglycoproteins ; genetics

OBJECTIVETo clone and sequence the upstream of mouse dentin sialophosphoprotein promoter.

METHODSGenomic DNA was obtained from Balb/c mouse blood. The upstream of mouse dentin sialophosphoprotein promoter segments was obtained by PCR. Then the segments were inserted into T-vector. The plasmids were identified by digestion with the restriction enzyme analysis. The positive clone was sequenced and compared with Genebank.

RESULTSThe upstream of mouse dentin sialophosphoprotein promoter was divided into three sequences and three different target segments with 997 bp, 1004 bp and 674 bp in length were obtained. After identified, sequenced and compared with Genebank, the sequences of the segments were consistent with those displayed on Genebank by 99%.

CONCLUSIONThe clone of the upstream of mouse dentin sialophosphoprotein promoter was successful. This work will help to study the regulation of DSPP expression.

Animals ; Cloning, Molecular ; Extracellular Matrix Proteins ; genetics ; Mice ; Mice, Inbred BALB C ; Phosphoproteins ; genetics ; Promoter Regions, Genetic ; Sequence Analysis, DNA ; Sialoglycoproteins ; genetics

OBJECTIVETo investigate interleukin-1 receptor antagonist (IL-1ra) genotype and its association with the susceptibility of chronic periodontitis in Uighur patients of Xinjiang.

METHODSGenomic DNA was obtained from buccal swabs of 41 subjects with severe chronic periodontitis (CP), 43 subjects with moderate CP, 49 subjects with mild CP and 92 ethnically matched healthy control individuals. Genotypes of IL-1RN intron 2 VNTR was analyzed by SSP-PCR method. Then compared the differences in distribution of each genotype.

RESULTSA significant over-representation of IL-1RN intron 2 VNTR allele 2 was found in severe chronic periodontitis group.

CONCLUSIONIL-1RN intron 2 VNTR allele 2 may be a risk indicator for the susceptibility of severe chronic periodontitis in Uighur patients of Xinjiang.

Adult ; Aged ; Chronic Disease ; Female ; Genetic Predisposition to Disease ; Genotype ; Humans ; Interleukin 1 Receptor Antagonist Protein ; Male ; Middle Aged ; Minisatellite Repeats ; Periodontitis ; genetics ; Sialoglycoproteins ; genetics

OBJECTIVETo evaluate the expression of dentin sialophosphoprotein (DSPP) in transfected rat bone marrow mesenchymal stem cells (BM-MSC) and the influence of the transfection.

METHODSPlasmid containing mice dentin DSPP was constructed by using the cytomegalovirus (CMV) promoter and then transfected the cultured BM-MSC by lipofectamine; The expression of Pax-9 and dentin matrix protein 1 (DMP1) gene of transfected BM-MSC were detected by RT-PCR. The expression of DSPP was examined by immunocytochemical staining, and the formation ratio of mineralized nodules of transfected BM-MSC was compared with untransfected ones after mineralized induction.

RESULTSThe constructed pcDNA3.1(+)/mDSPP could produced 3.0 kb and 5.4 kb fragments, DSPP gene and Pax9 gene were expressed 24 h and 48 h respectively, after BM-MSC were transfected Pax-9 gene was exprssed, but DMP1 gene was not; Immunohistochemical staining showed that DSPP was positive in transfected BM-MSC; The formation ratio of mineralized nodules of transfected BM-MSC was higher than that of untransfected ones after mineralized induction.

CONCLUSIONSThe expression of mice DSPP in BM-MSC by gene transfection can induce the expression of tooth development-associated gene Pax9 and enhance the formation of mineralized nodules, which suggests that DSPP gene might induce odontogenic differentiation of BM-MSC.

Animals ; Bone Marrow Cells ; metabolism ; Cells, Cultured ; Extracellular Matrix Proteins ; Genetic Vectors ; Mesenchymal Stromal Cells ; metabolism ; Mice ; Phosphoproteins ; Protein Precursors ; genetics ; Rats ; Sialoglycoproteins ; Transfection

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 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

Conditions of disuse such as bed rest, space flight, and immobilization result in decreased mechanical loading of bone, which is associated with reduced bone mineral density and increased fracture risk. Mechanisms involved in this process are not well understood except the suppression of osteoblast function. To investigate the effect of simulated weightlessness on mRNA level of extracellular matrix proteins, osteoblasts were rotated in horizontal plane as a model of simulated microgravity. Primordial osteoblasts of rats were grown for 2 d and then rotated for 24, 48 and 72 h, respectively. After isolating total RNA in cells, reverse transcription polymerase chain reaction (PT-PCR) was made to examine the mRNA level of osteopontin (OPN) and osteonectin (ON). Meanwhile, the levels of alkaline phosphatase (ALP) and osteocalcin (BGP) in the cultured medium were measured to evaluate the calcific function of cell. The expression of OPN and ON mRNA fell significantly after rotating for 24, 48 and 72 h, respectively. The contents of BGP descended significantly, meanwhile, the activity of ALP also showed a degressive tendency. Horizontal rotation decreased the expression of ON and OPN as well as diminished the secretion of BGP and ALP, which affected the calcific function of osteoblast. The results obtained suggest that depression of extracellular matrix proteins expression plays a key role in bone loss during weightlessness.
Animals ; Animals, Newborn ; Cells, Cultured ; Extracellular Matrix Proteins ; biosynthesis ; genetics ; Osteoblasts ; cytology ; metabolism ; Osteonectin ; biosynthesis ; genetics ; Osteopontin ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Rotation ; Sialoglycoproteins ; biosynthesis ; genetics ; Skull ; cytology ; Weightlessness Simulation