Amelogenesis imperfecta phenotype-genotype correlations with two amelogenin gene mutations

Amelogenin, the predominant matrix protein in developing dental enamel, is considered essential for normal enamel formation, but its exact functions are undefined. Mutations in the AMELX gene that encodes for amelogenin protein cause X-linked amelogenesis imperfecta (AI), with phenotypes characterized by hypoplastic and/or poorly mineralized enamel. Eight different AMELX deletion and substitution mutations have been reported to date. The purpose here was to evaluate the genotype and phenotype of two large kindreds segregating for X-linked AI. Phenotypically affected males in family 1 had yellowish-brown, poorly mineralized enamel; those in family 2 had thin, smooth, hypoplastic enamel. Heterozygous females in both kindreds had vertical hypoplastic grooves in their enamel. DNA was obtained from family members; exons 1-7 of AMELX were amplified and sequenced. Mutational analysis of family 1 revealed a single-base-pair change of A-->T at nucleotide 256, resulting in a His-->Leu change. Analysis of family 2 revealed deletion of a C-nucleotide in codon 119 causing a frameshift alteration of the next six codons, and a premature stop codon resulting in truncation of the protein 18 amino acids shorter than the wild-type. To date, all mutations that alter the C-terminus of amelogenin after the 157th amino acid have resulted in a hypoplastic phenotype. In contrast, other AMELX mutations appear to cause predominantly mineralization defects (e.g. the mutation seen in family 1). This difference suggests that the C-terminus of the normal amelogenin protein is important for controlling enamel thickness.     

Molecular analysis for genetic counselling in amelogenesis imperfecta

OBJECTIVE: To use molecular genetics to establish the mode of inheritance in a family with amelogenesis imperfecta. MATERIALS AND METHODS: The polymerase chain reaction was used to amplify exons of the amelogenin gene on the short arm of the X chromosome. RESULTS: A single base deletion mutation in exon 6 of the amelogenin gene was identified. This mutation was a single base deletion of a cytosine residue - 431delC - in codon 96 of exon 6, introducing a stop codon 30 codons downstream of the mutation in codon 126 of the exon. CONCLUSION: The firm establishment of an X-linked mode of inheritance affects the genetic counselling for this family.     

A novel missense mutation (p.P52R) in amelogenin gene causing X-linked amelogenesis imperfecta

Amelogenesis imperfecta (AI) is a hereditary disease with abnormal dental enamel formation. Here we report a Japanese family with X-linked AI transmitted over at least four generations. Mutation analysis revealed a novel mutation (p.P52R) in exon 5 of the amelogenin gene. The mutation was detected as heterozygous in affected females and as hemizygous in their affected father. The affected sisters exhibited vertical ridges on the enamel surfaces, whereas the affected father had thin, smooth, yellowish enamel with distinct widening of inter-dental spaces. To study the pathological cause underlying the disease in this family, we synthesized the mutant amelogenin p.P52R protein and evaluated it in vitro. Furthermore, we studied differences in the chemical composition between normal and affected teeth by x-ray diffraction analysis and x-ray fluorescence analysis. We believe that these results will greatly aid our understanding of the pathogenesis of X-linked AI.     

An amelogenin mutation leads to disruption of the odontogenic apparatus and aberrant expression of Notch1

J Oral Pathol Med (2011) 40 : 235–242 Background: Amelogenins are highly conserved proteins secreted by ameloblasts in the dental organ of developing teeth. These proteins regulate dental enamel thickness and structure in humans and mice. Mice that express an amelogenin transgene with a P70T mutation (TgP70T) develop abnormal epithelial proliferation in an amelogenin null (KO) background. Some of these cellular masses have the appearance of proliferating stratum intermedium, which is the layer adjacent to the ameloblasts in unerupted teeth. As Notch proteins are thought to constitute the developmental switch that separates ameloblasts from stratum intermedium, these signaling proteins were evaluated in normal and proliferating tissues. Methods: Mandibles were dissected for histology and immunohistochemistry using Notch1 antibodies. Molar teeth were dissected for western blotting and RT‐PCR for evaluation of Notch levels through imaging and statistical analyses. Results: Notch1 was immunolocalized to ameloblasts of TgP70TKO mice, KO ameloblasts stained, but less strongly, and wild‐type teeth had minimal staining. Cells within the proliferating epithelial cell masses were positive for Notch1 and had an appearance reminiscent of calcifying epithelial odontogenic tumor with amyloid‐like deposits. Notch1 protein and mRNA were elevated in molar teeth from TgP70TKO mice. Conclusion: Expression of TgP70T leads to abnormal structures in mandibles and maxillae of mice with the KO genetic background and these mice have elevated levels of Notch 1 in developing molars. As cells within the masses also express transgenic amelogenins, development of the abnormal proliferations suggests communication between amelogenin producing cells and the proliferating cells, dependent on the presence of the mutated amelogenin protein.     

Full length amelogenin binds to cell surface LAMP-1 on tooth root/periodontium associated cells

Objectives

Lysosome-associated membrane protein-1 (LAMP-1) has been suggested to be a cell surface receptor for a specific amelogenin isoform, leucine-rich amelogenin peptide or LRAP. However, it is unclear if LAMP-1 is an amelogenin receptor for dental mesenchymal cells. The goal of this study was to determine if LAMP-1 serves as a cell surface binding site for full length amelogenin on tooth root/periodontium associated mesenchymal cells.

Design

Murine dental follicle cells and cementoblasts (OCCM-30) were cultured for 2 days followed by addition of full length recombinant mouse amelogenin, rp(H)M180. Dose-response (0-100 μg/ml) and time course (0-120 min) assays were performed to determine the optimal conditions for live cell surface binding using immunofluorescent microscopy. A competitive binding assay was performed to determine binding specificity by adding Emdogain^® (1 mg/ml) to the media. An antibody against LAMP-1 was used to detect the location of LAMP-1 on the cell surface and the pattern was compared to cell surface bound amelogenin. Both amelogenin and cell surface LAMP-1 were immuno-co-localized to compare the amount and distribution pattern.

Results

Maximum surface binding was achieved with 50 μg/ml of rp(H)M180 for 120 min. This binding was specific as demonstrated by competitive inhibition (79% lower) with the addition of Emdogain^®. The binding pattern for rp(H)M180 was similar to the distribution of surface LAMP-1 on dental follicle cells and cementoblasts. The high co-localization coefficient (0.92) for rp(H)M180 and LAMP-1 supports rp(H)M180 binding to cell surface LAMP-1.

Conclusions

The data from this study suggest that LAMP-1 can serve as a cell surface binding site for amelogenin on dental follicle cells and cementoblasts.     

Tissue distribution and nucleotide sequence of bovine mRNA for salivary proline-rich protein P-B

The tissue distribution of P-B was investigated to obtain information on the physiological significance of this proline-rich protein. To design primers and probes for a tissue distribution analysis, a polymerase chain reaction (PCR)-based cloning of bovine P-B cDNA was performed using tooth germ and the nucleotide sequence was determined. The cloned bovine P-B cDNA was composed of 356 bp and included the region corresponding to the mature P-B protein and part of the 3' non-coding sequence. This part of the sequence is identical to the corresponding region of human P-B cDNA from the submaxillary gland. DNA corresponding to the P-B mRNA was amplified by PCR using cDNAs from various bovine tissues including tooth germ, submaxillary gland, parotid gland, lachrymal gland, heart, liver, stomach, pancreas, spleen, kidney, adrenal, and ovary. A quantitative analysis indicated the heart, submaxillary gland, tooth germ and kidney to be major sites of P-B expression. The ubiquitous distribution of P-B mRNA among bovine tissues together with findings of the presence of genes hybridizable with a DNA probe for P-B among species such as human, bovine, rat, mouse, and yeast as reported previously suggested a fundamental physiological role for this protein.     

配戴可摘义齿对口腔白色念珠菌的检出及基因分型的影响

目的：研究配戴活动义齿患者口腔中自色念珠菌的检出及基因分型。方法：研究对象共56例,分两组：实验组30例和对照组26例。含漱液浓缩法取样,CHROMagar Candida鉴定培养基分离鉴定白色念珠菌,PCR（聚合酶链式反应polymerase chain reaction,PCR）方法鉴定并进行基因分型,PCR产物经测序验证。结果：实验组与对照组白色念珠菌的基因型均以A型为主,其检出率分别为46．67％,7．69％,两组间差异有显著性。年龄大于60岁的患者及配戴义齿时间长于10年的患者,白色念珠菌及其它念珠菌的检出率显著增高。结论：配戴活动义齿影响口腔白色念珠菌的检出。     

Human enamel phenotype associated with amelogenesis imperfecta and a kallikrein-4 (g.2142G>A) proteinase mutation

Kallikrein-4 is known to be highly expressed during the maturation stage of enamel formation and is thought to be critical for the final phase of crystallite growth. The purpose of this study was to evaluate the enamel phenotype in humans with a known KLK-4 mutation (g.2142G>A). Primary teeth from two individuals with a known KLK-4 mutation were evaluated using amino acid analysis and light and electron microscopy. Light microscopy showed the enamel was of normal thickness but opaque throughout its width compared with normal enamel. Electron microscopy showed enamel affected by the KLK-4 mutation had a normal prismatic structure and generally had a well-organized and discernable crystallite composition. In some areas, globular structures were present where crystallites were not discernable or appeared to have an altered morphology. The KLK-4 mutant enamel had an increased protein content compared with normal enamel. Human enamel formed with a lack of functioning KLK-4 proteinase is altered primarily in the completeness of crystallite growth, while enamel thickness and prism structure remains essentially normal. Collectively, these studies suggest that the KLK-4 proteinase is essential for the final crystallite growth of enamel but is not critical for crystallite orientation, prism formation or enamel thickness.     

A novel autosomal-recessive mutation, whitish chalk-like teeth, resembling amelogenesis imperfecta, maps to rat chromosome 14 corresponding to human 4q21

A rat mutant, whitish chalk-like teeth (wct), with white, chalk-like abnormal incisors, was discovered and morphologically and genetically characterized. The mutant rats showed tooth enamel defects that were similar to those of human amelogenesis imperfecta. The wct mutation was found to disturb the morphological transition of ameloblasts from secretory to maturation stages and to induce cyst formation. This mutation also disturbs the transfer of iron into the enamel, resulting in the whitish chalk-like incisors. A genetic linkage study indicated that the wct locus maps to a specific interval of rat chromosome 14 between D14Got13 and D14Wox2. Interestingly, the human chromosomal region orthologous to wct, a 5.5-Mb interval in human chromosome 4q21, is a critical region for the locus of human amelogenesis imperfecta AIH2. These results strongly suggest that this wct mutant is a useful model for the identification of genes responsible for amelogenesis imperfecta and molecular mechanisms of tooth development.     

Japanese workers with long leisure time have deteriorated periodontal condition: A cross-sectional study

Objectives

The relationship between length of leisure time and periodontal condition is unknown. The aim of this cross-sectional study was to clarify the association between leisure time and periodontal states.

Effect of the phosphodiesterase 4 inhibitor,rolipram, on retinoic acid-increased alkaline phosphatase activity in the mouse fibroblastic C3H10T1/2 cell line

We have evaluated effects of a phosphodiesterase (PDE) 4 inhibitor on retinoic acid-increased alkaline phosphatase activity in the mouse fibroblastic C3H10T1/2 clone 8 (10T1/2) cell line. 10T1/2 cells were cultured in minimum essential medium (MEM) and 10% fetal bovine serum with or without 1 microM retinoic acid and/or the PDE 4 inhibitor, rolipram, and harvested at specific intervals before measurement of alkaline phosphatase activity, cAMP production in response to parathyroid hormone, osteocalcin synthesis and expression, and phosphodiesterase activity. Retinoic acid-increased alkaline phosphatase activity, and slightly enhanced cAMP production in response to parathyroid hormone. However, it did not affect osteocalcin synthesis and expression. In the presence of retinoic acid, PDE 4 activity was not changed. A PDE 4 inhibitor, rolipram, and cAMP analog, 8-bromo-cAMP dramatically increased retinoic acid's ability to induce alkaline phosphatase activity. This is the first report that PDE 4 may be involved in regulation of retinoic acid-increased alkaline phosphatase activity.     

WNT10A rs147680216 G>A mutation indicates a higher risk for non-syndromic oral cleft in a northeastern Chinese population

Non-syndromic oral cleft lip and palate is a heterogeneous group of congenital malformations that consist of cleft palate, and cleft lip with or without cleft palate. The members of the wingless type mouse mammary tumour virus (MMTV) integration site family (Wnts) regulate various developmental processes including craniofacial development, and have a role in that of cleft lip and palate. We aimed to identify the potential polymorphisms in the Wnt10a gene, and to explore the association between the variations in the gene and the risk of development of cleft palate. A total of 198 affected patients (cleft lip, n = 67; cleft palate, n = 48; and both, n = 83) together with 187 healthy controls were enrolled (all from the Chinese Han population in NE China). A fragment of 316 bp was amplified from the blood genome of each participant by polymerase chain reaction (PCR) using specific primers that targeted the Homo sapiens Wnt10a gene. By using the restriction enzyme AluI, the population analysed were classified into three genotypes, GG (316 bp), GA (316 bp, 117bp, 199bp) and AA (117bp, 199bp) based on the rs147680216 G/A polymorphism (Gly>Ser mutation at position 213 of Wnt10a protein) of theWnt10a gene. The frequency of allele A in the affected group was significantly higher (14.1% compared with 3.2% in the control group). The allele G with an odds ratio (OR) of 0.201 and 95% CI of 0.445 to 0.091 was not a risk factor for the condition in the affected group. However, the distribution of the genotype did affect its occurrence in the affected group (p < 0.001), but not the classification of types (p = 0.901). In conclusion we found an rs147680216 G>A mutation that was associated with non-syndromic cleft lip and palate in the Wnt10a gene.     

Quantitative Assessment of the Efficacy of Two Different Single-file Systems in Reducing the Bacterial load in Oval-Shaped Canals: A Clinical Study

IntroductionThis randomized clinical study compared the in vivo antibacterial efficacy of Reciproc Blue (RB), XP-endo Shaper (XP-S), and XP-endo Shaper associated with XP-endo Finisher (XP-F) systems in infected oval-shaped root canals with primary apical periodontitis.MethodsIn this study, 28 human teeth with a single root and a single canal were randomly assigned to 2 groups according to the instrumentation technique: group 1, RB (n = 14) and group 2, XP-endo (XP-S and XP-F, n = 14). The single-rooted teeth were prepared by reciprocating and rotary nickel-titanium instruments with 5.25% sodium hypochlorite irrigation. Samples were collected from the canal at the baseline (S1), after chemomechanical preparation (S2), and after XP-F instrumentation (S3). The DNA extracts were subjected to quantitative analysis for total bacterial counts by quantitative real-time polymerase chain reaction. The data were analyzed using the analysis of variance test, and the level of significance was set at 5%.ResultsAll samples tested positive for the presence of bacteria at baseline, and the bacterial counts substantially reduced after treatment procedures (P < .01). The results showed no statistical difference between RB and XP-S instrumentation with respect to the bacterial reduction (P > .05). A marked bacterial reduction was observed after the use of the XP-F instrument (P < .01).ConclusionsThe XP-S and RB systems sharply reduced the bacterial load in oval-shaped root canals with primary apical periodontitis. XP-F used as a supplementary instrument to chemomechanical preparation promoted a significantly higher bacterial reduction.     

Epstein-Barr virus (EBV) genomes and c-myc oncogene in oral Burkitt's lymphomas

In addition to Burkitt's lymphomas, tentative evidence suggests the involvement of Epstein-Barr virus (EBV) in malignant lymphomas of T-cell origin. The c-myc proto-oncogene is strongly associated with the development of lymphoid neoplasias. In the present study, a series of 38 biopsies of oral lymphomas (29 Burkitt's lymphomas, 9 malignant lymphomas of other type) obtained from patients in Tanzania were studied using in situ hybridization (ISH) and polymerase chain reaction (PCR) for detection of EBV DNA and c-myc oncogene. In ISH applied on formalin-fixed, paraffin wax-embedded biopsies, the Bam HI W fragment of EBV DNA was used as the probe. Amplification of c-myc oncogene was studied by PCR with a primer set from Exon II area. As an internal standard (β-globin gene was simultaneously amplified. EBV DNA was disclosed by ISH in five Burkitt's lymphomas only. Using the PCR, 20 of the 29 cases (70%) of Burkitt's lymphomas showed amplification for EBV DNA. Of the other EBV-positive lymphomas, two were of the lymphocytic type (large non-cleaved cell), one histiocytic and one Burkitt's-like lymphoma. All EBV-positive cases found on the agarose gel were positive also with the dot blot, when hybridized with the ³²P-labeled EBV Bam HI W-fragment probe. All lymphomas showed similar bands on the gel for c-myc and β-globin indicating that no amplification of c-myc was present.     

C‐deletion mutation of the p53 gene at exon 4 of codon 63 in the saliva of oral squamous cell carcinoma in central India: a preliminary study

Aim: The purpose of this study was to detect the C‐deletion mutation of the p53 gene at exon 4 of codon 63 in the saliva of oral squamous cell carcinoma in central India. Methods: The study was carried out in 30 oral squamous cell carcinoma cases and five healthy controls with no habit of betel nut and tobacco chewing. The C‐deletion mutation of the p53 gene at exon 4 of codon 63 was detected in the saliva samples by using polymerase chain reaction. Results: In this study, C‐deletion at exon 4 of codon 63 was detected in 28 of 30 oral squamous cell carcinoma cases (93.33%), but was negative in all five healthy controls and two oral squamous cell carcinoma cases. Conclusions: The results indicated that C‐deletion mutation at exon 4 of codon 63 of the p53 gene in the saliva might be a plausible molecular marker for oral squamous cell carcinoma patients with a habit of betel nut and tobacco lime quid chewing. The results further emphasize the presence of p53 gene mutation in patients with oral squamous cell carcinoma, which can be detected in the saliva through polymerase chain reaction.