Koray Gencay

ENAM Mutations in Autosomal-dominant Amelogenesis Imperfecta

To date, 4 unique enamelin gene (ENAM) defects have been identified in kindreds with amelogenesis imperfecta. To improve our understanding of the roles of enamelin in normal enamel formation, and to gain information related to possible genotype/phenotype correlations, we have identified 2 ENAM mutations in kindreds with hypoplastic ADAI, 1 novel (g.4806A>C, IVS6-2A>C) and 1 previously identified (g.8344delG), and have characterized the resulting enamel phenotypes. The IVS6-2A>C mutation caused a severe enamel phenotype in the proband, exhibiting horizontal grooves of severely hypoplastic enamel. The affected mother had several shallow hypoplastic horizontal grooves in the lower anterior teeth. In the case of the g.8344delG mutation, the phenotype was generalized hypoplastic enamel with shallow horizontal grooves in the middle 1/3 of the anterior teeth. In general, mutations in the human enamelin gene cause hypoplastic enamel, often with horizontal grooves, but the severity of the enamel defects is variable, even among individuals with the same mutation.

Dental Implant Systems

Among various dental materials and their successful applications, a dental implant is a good example of the integrated system of science and technology involved in multiple disciplines including surface chemistry and physics, biomechanics, from macro-scale to nano-scale manufacturing technologies and surface engineering. As many other dental materials and devices, there are crucial requirements taken upon on dental implants systems, since surface of dental implants is directly in contact with vital hard/soft tissue and is subjected to chemical as well as mechanical bio-environments. Such requirements should, at least, include biological compatibility, mechanical compatibility, and morphological compatibility to surrounding vital tissues. In this review, based on carefully selected about 500 published articles, these requirements plus MRI compatibility are firstly reviewed, followed by surface texturing methods in details. Normally dental implants are placed to lost tooth/teeth location(s) in adult patients whose skeleton and bony growth have already completed. However, there are some controversial issues for placing dental implants in growing patients. This point has been, in most of dental articles, overlooked. This review, therefore, throws a deliberate sight on this point. Concluding this review, we are proposing a novel implant system that integrates materials science and up-dated surface technology to improve dental implant systems exhibiting bio- and mechano-functionalities.

Genes expressed in dental enamel development are associated with molar-incisor hypomineralization

Genetic disturbances during dental development influence variation of number and shape of the dentition. In this study, we tested if genetic variation in enamel formation genes is associated with molar-incisor hypomineralization (MIH), also taking into consideration caries experience. DNA samples from 163 cases with MIH and 82 unaffected controls from Turkey, and 71 cases with MIH and 89 unaffected controls from Brazil were studied. Eleven markers in five genes [ameloblastin (AMBN), amelogenin (AMELX), enamelin (ENAM), tuftelin (TUFT1), and tuftelin-interacting protein 11 (TFIP11)] were genotyped by the TaqMan method. Chi-square was used to compare allele and genotype frequencies between cases with MIH and controls. In the Brazilian data, distinct caries experience within the MIH group was also tested for association with genetic variation in enamel formation genes. The ENAM rs3796704 marker was associated with MIH in both populations (Brazil: p=0.03; OR=0.28; 95% C.I.=0.06-1.0; Turkey: p=1.22e-012; OR=17.36; 95% C.I.=5.98-56.78). Associations between TFIP11 (p=0.02), ENAM (p=0.00001), and AMELX (p=0.01) could be seen with caries independent of having MIH or genomic DNA copies of Streptococcus mutans detected by real time PCR in the Brazilian sample. Several genes involved in enamel formation appear to contribute to MIH.

STIM1 and SLC24A4 Are Critical for Enamel Maturation

Dental enamel formation depends upon the transcellular transport of Ca2+ by ameloblasts, but little is known about the molecular mechanism, or even if the same process is operative during the secretory and maturation stages of amelogenesis. Identifying mutations in genes involved in Ca2+ homeostasis that cause inherited enamel defects can provide insights into the molecular participants and potential mechanisms of Ca2+ handling by ameloblasts. Stromal Interaction Molecule 1 (STIM1) is an ER transmembrane protein that activates membrane-specific Ca2+ influx in response to the depletion of ER Ca2+ stores. Solute carrier family 24, member 4 (SLC24A4), is a Na+/K+/Ca2+ transporter that exchanges intracellular Ca2+ and K+ for extracellular Na+. We identified a proband with syndromic hypomaturation enamel defects caused by a homozygous C to T transition (g.232598C>T c.1276C>T p.Arg426Cys) in STIM1, and a proband with isolated hypomaturation enamel defects caused by a homozygous C to T transition (g.124552C>T; c.437C>T; p.Ala146Val) in SLC24A4. Immunohistochemistry of developing mouse molars and incisors showed positive STIM1 and SLC24A4 signal specifically in maturation-stage ameloblasts. We conclude that enamel maturation is dependent upon STIM1 and SLC24A4 function, and that there are important differences in the Ca2+ transcellular transport systems used by secretory- and maturation-stage ameloblasts.

Fracture resistance of immature teeth filled with BioAggregate, mineral trioxide aggregate and calcium hydroxide

UNLABELLED Abstract -  Background: The aim of this in vitro study was to assess the long-term fracture resistance of human immature permanent teeth filled with BioAggregate (BA), mineral trioxide aggregate (MTA) and calcium hydroxide (CH). MATERIALS AND METHODS  The study consisted of single rooted premolar teeth with immature root formation extracted for orthodontic reasons. A total of 28 immature premolars with average root length of 10.7 mm and apical diameter of 3 mm were included in the study. The pulps were extirpated and the canals were prepared using an apical approach. The teeth were randomly assigned to four groups: Group I: DiaRoot(®) BA (DiaDent, Burnaby, BC, Canada), Group II: Angelus MTA (MTA-A; Angelus, Londrina, Brazil), Group III: ProRoot(®) MTA (MTA-PR; Dentsply, Tulsa, OK, USA), Group IV: CH (Sultan Chemists Inc., Englewood, NJ, USA). The teeth were placed in saline solution at 4°C for 1 year. The root of each tooth was then embedded in an acrylic resin block. All specimens were loaded at a crosshead speed of 1 mm min(-1) in an Instron testing machine and the peak loads up to fracture were recorded. Data were analysed statistically by Kruskal-Wallis and Mann-Whitney U-tests. RESULTS  Mean (±SD) failure loads (MPa) were: 37.69 ± 14.43 for BA group, 32.94 ± 8.15 for MTA-A group, 28.74 ± 9.49 for MTA-PR group and 23.18 ± 8.48 for CH group. The BA group exhibited the highest fracture resistance and the CH group showed the lowest resistance to fracture. Significant differences (P < 0.05) in fracture resistance were found between the DiaRoot-BA and CH groups, and also between the MTA-A and CH groups. CONCLUSION  Within the limitations of this study, data suggest that DiaRoot-BA-filled immature teeth demonstrate higher fracture resistance than other groups at 1 year. Considering the long-term risk of cervical root fracture associated with immature teeth, the use of DiaRoot-BA as a root canal filling material appears to be the most advantageous of the materials tested.

MMP20 Hemopexin Domain Mutation in Amelogenesis Imperfecta

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.

Novel FAM20A mutations in hypoplastic amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a genetically and clinically heterogeneous group of inherited dental enamel defects without any other nonoral symptoms. Recently, a disease‐causing nonsense mutation (c.406C>T) in a novel gene, FAM20A, was identified in a large consanguineous family affected by AI with gingival hyperplasia. We performed mutational analyses on nine AI families with similar phenotypes and identified three homozygous mutations (c.34_35delCT, c.813‐2A>G, c.1175_1179delGGCTC) in three families and a compound heterozygous mutation (c.[590‐2A>G] + [c.826C>T]) in one family. An in vitro splicing assay with a minigene confirmed the mutations located in the splicing acceptor site caused the deletion of exons 3 and 6, respectively. Taking into consideration the locations of the nonsense and frameshift mutations, the mutant transcripts are most likely degraded by nonsense‐mediated mRNA degradation and it results in a loss of the FAM20A protein. This study confirms the importance of the FAM20A protein in enamel biomineralization as well as tooth eruption. Hum Mutat 33:91–94, 2012.

LAMB3 Mutations Causing Autosomal-dominant Amelogenesis Imperfecta

Amelogenesis imperfecta (AI) can be either isolated or part of a larger syndrome. Junctional epidermolysis bullosa (JEB) is a collection of autosomal-recessive disorders featuring AI associated with skin fragility and other symptoms. JEB is a recessive syndrome usually caused by mutations in both alleles of COL17A1, LAMA3, LAMB3, or LAMC2. In rare cases, heterozygous carriers in JEB kindreds display enamel malformations in the absence of skin fragility (isolated AI). We recruited two kindreds with autosomal-dominant amelogenesis imperfecta (ADAI) characterized by generalized severe enamel hypoplasia with deep linear grooves and pits. Whole-exome sequencing of both probands identified novel heterozygous mutations in the last exon of LAMB3 that likely truncated the protein. The mutations perfectly segregated with the enamel defects in both families. In Family 1, an 8-bp deletion (c.3446_3453del GACTGGAG) shifted the reading frame (p.Gly 1149Glufs*8). In Family 2, a single nucleotide substitution (c.C3431A) generated an in-frame translation termination codon (p.Ser1144*). We conclude that enamel formation is particularly sensitive to defects in hemidesmosome/basement-membrane complexes and that syndromic and non-syndromic forms of AI can be etiologically related.

Novel WDR72 Mutation and Cytoplasmic Localization

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.

Exonal Deletion of SLC24A4 Causes Hypomaturation Amelogenesis Imperfecta

Amelogenesis imperfecta is a heterogeneous group of genetic conditions affecting enamel formation. Recently, mutations in solute carrier family 24 member 4 (SLC24A4) have been identified to cause autosomal recessive hypomaturation amelogenesis imperfecta. We recruited a consanguineous family with hypomaturation amelogenesis imperfecta with generalized brown discoloration. Sequencing of the candidate genes identified a 10-kb deletion, including exons 15, 16, and most of the last exon of the SLC24A4 gene. Interestingly, this deletion was caused by homologous recombination between two 354-bp-long homologous sequences located in intron 14 and the 3′ UTR. This is the first report of exonal deletion in SLC24A4 providing confirmatory evidence that the function of SLC24A4 in calcium transport has a crucial role in the maturation stage of amelogenesis.