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ABSTRACT: Dentin sialophosphoprotein (DSPP) gene mutations have been identified in isolated hereditary dentin defects; however, the genotype-phenotype correlations are poorly understood. We performed in vitro splicing assays to test the hypothesis that DSPP mutations in splice junctions as well as proposed missense/nonsense mutations experimentally result in aberrant pre-mRNA splicing.
The genomic fragment of the human DSPP gene was cloned into the pSPL3 splicing vector, and previously reported as well as informative de novo mutations were then introduced by PCR mutagenesis. The COS-7 cells were transfected with each plasmid vector, and total RNA was isolated. RT-PCR result was analyzed, and the band intensity of the product was calibrated using ImageJ.
The predictions by others of exon 3 skipping in specific DSPP mutations have been validated and a cryptic splicing donor site has been identified. However, the degree of mutational effect on pre-mRNA splicing varied considerably depending on the changed nucleotide.
The predictions of exon 3 skipping in specific DSPP mutations have been validated, and a cryptic splicing donor site has been identified. Our data may provide insight into the contribution of DSPP mutations in the pathogenesis and genotype-phenotype correlations of hereditary dentin defects.
Oral Diseases 06/2011; 17(7):690-5. · 2.49 Impact Factor
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ABSTRACT: The dentin sialophosphoprotein (DSPP) gene encodes the most abundant non-collagenous protein in tooth dentin and DSPP protein is cleaved into several segments including the highly phosphorylated dentin phosphoprotein (DPP). Mutations in the DSPP gene have been solely related to non-syndromic form of hereditary dentin defects. We recruited three Korean families with dentinogenesis imperfecta (DGI) type II and sequenced the exons and exon-intron boundaries of the DSPP gene based on the candidate gene approach. Direct sequencing of PCR products and allele-specific cloning of the highly repetitive exon 5 revealed novel single base pair (bp) deletional mutations (c.2688delT and c.3560delG) introducing hydrophobic amino acids in the hydrophilic repeat domain of the DPP coding region. All affected members of the three families showed exceptionally rapid pulp chambers obliteration, even before tooth eruption. Individuals with the c.3560delG mutation showed only mild, yellowish tooth discoloration, in contrast to the affected individuals from two families with c.2688delT mutation. We believe that these results will help us to understand the molecular pathogenesis of DGI type II as well as the normal process of dentin biomineralization.
Clinical Genetics 04/2011; 79(4):378-84. · 3.13 Impact Factor
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ABSTRACT: Mutations in a family with sequence similarity 83 member H (FAM83H) cause autosomal-dominant hypocalcification amelogenesis imperfecta (ADH CAI). All FAM83H ADHCAI-causing mutations terminate translation or shift the reading frame within the specific exon 5 segment that encodes from Ser(287) to Glu(694). Mutations near Glu(694) cause a milder, more localized phenotype. We identified disease-causing FAM83H mutations in two families with ADHCAI: family 1 (g.3115C>T, c.1993 C>T, p.Q665X) and family 2 (g.3151C>T, c.2029 C>T, p.Q677X). We also tested the hypothesis that truncation mutations alter the intracellular localization of FAM83H. Wild-type FAM83H and p.E694X mutant FAM83H fused to green fluorescent protein (GFP) localized in the cytoplasm of HEK293T cells, but the mutant FAM83H proteins (p.R325X, p.W460X, and p.Q677X) fused to GFP localized mainly in the nucleus with slight expression in the cytoplasm. We conclude that nuclear targeting of the truncated FAM83H protein contributes to the severe, generalized enamel phenotype.
Journal of dental research 03/2011; 90(3):377-81. · 3.46 Impact Factor
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ABSTRACT: Hereditary dentin defects can be grouped into three types of dentinogenesis imperfecta (DGI) and two types of dentin dysplasia. Tooth enamel is considered normal in patients with hereditary dentin defects, but is easily worn down and fractured due to DSPP mutation-induced altered dentin properties. The purposes of this study were to identify genetic cause of a family with type II DGI and enamel defects.
We identified a family with type II DGI and a unique form of hypoplastic enamel defect affecting occlusal third of the crown. Family members were recruited for the genetic analysis and DNA was obtained from peripheral whole blood.
Mutational analysis revealed a T to A transversion in exon 3 of the DSPP (c.53T>A, p.V18D). Haplotype analysis showed that the same mutation arose separately in two different families having DGI with similar enamel defects, indicating that this phenotype is associated with this specific DSPP mutation. Clinical features suggest that enamel formation was affected in the affected individuals during early amelogenesis, in addition to the dentin defect.
We observed that a DSPP gene mutation not only influences dentinogenesis but also affects early stage amelogenesis.
Oral Diseases 10/2010; 17(3):314-9. · 2.49 Impact Factor
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S-K Lee,
F Seymen, K-E Lee,
H-Y Kang,
M Yildirim,
E Bahar Tuna,
K Gencay,
Y-H Hwang,
K H Nam,
R J De La Garza,
J C-C Hu,
J P Simmer,
J-W Kim
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ABSTRACT: The proven candidate genes for amelogenesis imperfecta (AI) are AMELX, ENAM, MMP20, KLK4, FAM83H, and WDR72. We performed mutation analyses on seven families with hypomaturation AI. A novel WDR72 dinucleotide deletion mutation (g.57,426_57,427delAT; c.1467_ 1468delAT; p.V491fsX497) was identified in both alleles of probands from Mexico and Turkey. Haplotype analyses showed that the mutations arose independently in the two families. The disease perfectly segregated with the genotype. Only persons with both copies of the mutant allele were affected. Their hypomineralized enamel suffered attrition and orange-brown staining following eruption. Expression of WDR72 fused to green fluorescent protein showed a cytoplasmic localization exclusively and was absent from the nucleus. We conclude that WDR72 is a cytoplasmic protein that is critical for dental enamel formation.
Journal of dental research 10/2010; 89(12):1378-82. · 3.46 Impact Factor
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ABSTRACT: Chitinases are produced in significant quantities by hosts defending against infections with chitin-containing organisms. However, little is known about the immune response of exogenous chitinase in human epithelial cells. IL-8 has been suggested to have a role in the pathogenesis of the allergenic inflammation of bronchial asthma. We examined whether Streptomyces griseus (S. griseus) chitinase-induced IL-8 on airway epithelium and identified the involvement of intracellular signalling pathways. H292 cells were treated with S. griseus chitinase with different concentrations and times. The IL-8 levels were determined by specific human IL-8 enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction. Using a series of pharmacological inhibitors, we examined the upstream signalling pathway responsible for IL-8 expression in response to S. griseus chitinase. Cells exposed to S. griseus chitinase showed higher level of IL-8 protein production and mRNA expression. Cells stimulated by S. griseus chitinase resulted in the activation of protein kinase C (PKC), extracellular signal-regulated kinase (ERK) and nuclear factor kappa-B (NF-kB) pathways. Inhibitors of Ca(2+)-dependent PKC (Ro-31-8220, calphostin C and Go6976) significantly abolished chitinase-induced expression of IL-8. However, Ca(2+)-independent PKC inhibitor (rottlerin) did not inhibit IL-8 expression. Through ERK inhibitor (U0126) and NF-kB inhibitor (caffeine acid phenethyl ester) treatment, it was proven that ERK and NF-kB regulated chitinase-induced IL-8 expression. We concluded that S. griseus chitinase-induced IL-8 expression was regulated by the activation of Ca(2+/-)-dependent PKC, ERK and NF-kB in human airway epithelial cells.
Scandinavian Journal of Immunology 07/2010; 72(1):15-21. · 2.23 Impact Factor
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ABSTRACT: Proteolytic enzymes serve important functions during dental enamel formation, and mutations in the kallikrein 4 (KLK4) and enamelysin (MMP20) genes cause autosomal-recessive amelogenesis imperfecta (ARAI). So far, only 1 KLK4 and 3 MMP20 mutations have been reported in ARAI kindreds. To determine whether ARAI in a family with a hypomaturation-type enamel defect is caused by mutations in the genes encoding enamel proteolytic enzymes, we performed mutational analysis on candidate genes. Mutational and haplotype analyses revealed an ARAI-causing point mutation (c.910G>A, p.A304T) in exon 6 of MMP20 that results in a single amino acid substitution in the hemopexin domain. Western blot analysis showed decreased expression of the mutant protein, but zymogram analysis demonstrated that this mutant was a functional protein. The proband and an affected brother were homozygous for the mutation, and both unaffected parents were carriers. The enamel of newly erupted teeth had normal thickness, but was chalky white and became darker with age.
Journal of dental research 12/2009; 89(1):46-50. · 3.46 Impact Factor
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ABSTRACT: To determine the underlying molecular genetic aetiology of a family with the hypocalcified form of amelogenesis imperfecta and to investigate the hardness of the enamel and dentine of a known FAM83H mutation.
Mutational screening of the FAM83H on the basis of candidate gene approach was performed. All exons and exon-intron boundaries was amplified and sequenced. A microhardness test was performed to measure the Vickers microhardness value.
A novel nonsense mutation (c.1354C>T, p.Q452X) was identified in the last exon of FAM83H, which resulted in soft, uncalcified enamel. The affected enamel was extremely soft (about 17% of the normal control), but the underlying dentine was as hard as the normal control.
Mutational analysis revealed a novel mutation in FAM83H gene. Hardness of dentine was not affected by the mutation, whilst the enamel was extremely soft.
International Endodontic Journal 11/2009; 42(11):1039-43. · 2.18 Impact Factor
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ABSTRACT: Mutations in the RUNX2 gene, a master regulator of bone formation, have been identified in cleidocranial dysplasia (CCD) patients. CCD is a rare autosomal-dominant disease characterized by the delayed closure of cranial sutures, defects in clavicle formation, and supernumerary teeth. The purposes of this study were to identify genetic causes of two CCD nuclear families and to report their clinical phenotypes.
We identified two CCD nuclear families and performed mutational analyses to clarify the underlying molecular genetic etiology.
Mutational analysis revealed a novel nonsense mutation (c.273T>A, p.L93X) in family 1 and a de novo missense one (c.673C>T, p.R225W) in family 2. Individuals with a nonsense mutation showed maxillary hypoplasia, delayed eruption, multiple supernumerary teeth, and normal stature. In contrast, an individual with a de novo missense mutation in the Runt domain showed only one supernumerary tooth and short stature.
Mutational and phenotypic analyses showed that the severity of mutations on the skeletal system may not necessarily correlate with that of the disruption of tooth development.
Oral Diseases 09/2009; 16(1):55-60. · 2.49 Impact Factor
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ABSTRACT: Amelogenesis imperfecta (AI) is a genetically and phenotypically heterogeneous genetic disorder affecting tooth enamel without other non-oral syndromic conditions. Based on a review of the literature, the authors constructed a candidate-gene-based mutational analysis strategy. To test the strategy, they identified two Turkish families with hypoplastic enamel without any other non-oral syndromic phenotype. The authors analyzed all exons and exon/intron boundaries of the enamelin (ENAM) gene for family 1 and the DLX3 and ENAM genes for family 2, to identify the underlying genetic etiology. The analysis revealed 2 ENAM mutations (autosomal-dominant g.14917delT and autosomal-recessive g.13185-13186insAG mutations). A single T deletion in exon 10 is a novel deletional mutation (g.14917delT, c.2991delT), which is predicted to result in a frameshift with a premature termination codon (p.L998fsX1062). This result supports the use of a candidate-gene-based strategy to study the genetic basis for AI.
Journal of dental research 04/2009; 88(3):266-9. · 3.46 Impact Factor
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ABSTRACT: Hereditary dentin defects are divided into dentinogenesis imperfecta and dentin dysplasia. We identified a family segregating severe dentinogenesis imperfecta. The kindred spanned four generations and showed an autosomal-dominant pattern of inheritance. The proband was a child presenting with a severely affected primary dentition, with wide-open pulp chambers and multiple pulp exposures, resembling a DGI type III (DGI-III) pattern. We hypothesized that a mutation in the DSPP gene is responsible for this severe phenotype. Mutational analyses revealed a novel mutation (c.53T>A, p.V18D) near the intron-exon boundary in the third exon of the DSPP gene. We analyzed the effect of the mutation by means of an in vitro splicing assay, which revealed that the mutation did not affect pre-mRNA splicing. Further studies are needed for a better understanding of the nature of the disease and the development of an appropriate treatment strategy.
Journal of dental research 02/2009; 88(1):51-5. · 3.46 Impact Factor
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ABSTRACT: To assess the efficacy and safety of covered retrievable nitinol stents in oesophageal cancer patients with airway involvement.
Under fluoroscopic guidance, covered retrievable nitinol airway stents were placed in 23 oesophageal cancer patients with airway stricture and/or oesophagorespiratory fistula (ERF) over a long period of 12 years. Six patients only had aspiration by ERF and three patients had both airway stricture and asymptomatic ERF. Technical aspects, dyspnoea improvement, and/or resolution of ERF symptoms, complications, reinterventions, and survival data were evaluated.
A total of 27 airway stents (14 tracheal, 11 bronchial, and two hinged) were placed successfully in 23 patients with airway stricture or ERF. Dyspnoea score decreased significantly after stent placement (p<0.001). ERF were sealed off in all nine patients. Complications included stent migration or expectoration (n=4), haemoptysis (n=2), sputum retention (n=7), and tumour overgrowth (n=1). All three migrated stents were easily removed. Twenty-one patients died, with the median survival period of 76 days (range 2-197 days).
Placement of covered retrievable expandable nitinol stents was safe and effective for the palliative treatment of airway strictures and/or ERF, with a reasonable range of complications, in patients with advanced oesophageal cancer.
Clinical radiology 02/2009; 64(2):133-41. · 1.65 Impact Factor
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ABSTRACT: Cockroaches have been known as a cause of respiratory allergies such as asthma. IL-8 plays an integral role in the coordination and persistence of the inflammatory process in the chronic inflammation of the airways in asthma.
We investigated the mechanism by which German cockroach extract (GCE) triggers IL-8 release from human airway epithelial cells.
Chemical inhibitors were pretreated before addition of GCE for promoter activity and protein synthesis of IL-8. The Transcriptional activity of IL-8 promoter was analysed by mutational, deletional anaylsis and electrophoretic mobility shift assay (EMSA).
Stimulation of H292 cells with GCE resulted in a time- and concentration-dependent induction of IL-8 transcription and protein synthesis. IL-8 promoter deletion analysis indicated that position -132 to +41 was essential for GCE-induced IL-8 transcription, and mutants with substitutions in activator protein (AP)-1, nuclear factor (NF)-IL6 and NF-kappaB-binding sites revealed a requirement for NF-kappaB and NF-IL6, but not AP-1, in GCE-induced activation of the IL-8 promoter. The DNA-binding activities of NF-kappaB and NF-IL6 were induced by GCE, as determined by EMSA. The chemical inhibition of extracellular signal-regulated kinase (ERK) attenuated GCE-induced transcriptional activity and protein synthesis. In addition, through aprotinin treatment and PAR2 small interfering RNA transfection, it was proven that protease of GCE is consistent with the regulation of GCE-induced IL-8.
We conclude that GCE with protease activity-induced IL-8 expression is regulated by transcriptional activation of NF-kappaB and NF-IL6 coordinating with the ERK pathway in human airway epithelial cells.
Clinical & Experimental Allergy 10/2007; 37(9):1364-73. · 5.03 Impact Factor
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ABSTRACT: Islets were isolated from the pancreata of Sprague-Dawley rats and transplanted into streptozotocin-induced diabetic outbred Wistar rats. The effect of transplantation of islets into the cisterna magna on the diabetic state of the recipients was compared with that of the conventional transplantation of islets into liver via the portal vein. After successful intraportal (IP) transplantation, rejection took place between days 7 and 15 in all diabetic recipients. All of the eleven rats surviving after stereotaxic implantation of islets into the cisterna magna returned to normoglycemia within 7 days after transplantation. Nine of the recipients with intra-cisterna magna (IM) islet allografts were still normoglycemic at 210 days after transplantation. The glucose disappearance rate of the IM transplant rats was slower than that of the IP transplant rats, and blood glucose returned to the normal basal level within 5 hr following glucose administration. Although the insulin levels were almost undetectable in cerebrospinal fluid before IM transplantation, the insulin levels were markedly increased after IM transplantation and twice as great in CSF than blood. Thus, these findings indicate that the cisterna magna can serve as an immunologically privileged site for implantation of allogeneic pancreatic islets, and islets in CSF can regulate and maintain normal glucose homeostasis via secretion of insulin across the blood-brain barrier.
Transplantation 04/1992; 53(3):513-6. · 4.00 Impact Factor
