AMELX Mutations and Genotype-Phenotype Correlation in X-Linked Amelogenesis Imperfecta.

AMELX mutations cause X-linked amelogenesis imperfecta (AI), known as AI types IE, IIB, and IIC in Witkop's classification, characterized by hypoplastic (reduced thickness) and/or hypomaturation (reduced hardness) enamel defects. In this study, we conducted whole exome analyses to unravel the disease-causing mutations for six AI families. Splicing assays, immunoblotting, and quantitative RT-PCR were conducted to investigate the molecular and cellular effects of the mutations. Four AMELX pathogenic variants (NM_182680.1:c.2T>C; c.29T>C; c.77del; c.145-1G>A) and a whole gene deletion (NG_012494.2:g.307534_403773del) were identified. The affected individuals exhibited enamel malformations, ranging from thin, poorly mineralized enamel with a "snow-capped" appearance to severe hypoplastic defects with minimal enamel. The c.145-1G>A mutation caused a -1 frameshift (NP_001133.1:p.Val35Cysfs*5). Overexpression of c.2T>C and c.29T>C AMELX demonstrated that mutant amelogenin proteins failed to be secreted, causing elevated endoplasmic reticulum stress and potential cell apoptosis. This study reveals a genotype-phenotype relationship for AMELX-associated AI: While amorphic mutations, including large deletions and 5' truncations, of AMELX cause hypoplastic-hypomaturation enamel with snow-capped teeth (AI types IIB and IIC) due to a complete loss of gene function, neomorphic variants, including signal peptide defects and 3' truncations, lead to severe hypoplastic/aplastic enamel (AI type IE) probably caused by "toxic" cellular effects of the mutant proteins.

Global overview on current paediatric dentistry teaching: A survey of National Member Societies of the IAPD.

BACKGROUND: Published information on the teaching and recognition of paediatric dentistry (PD) is limited worldwide. AIM: The aim of this study was to investigate the status of current teaching of undergraduate and postgraduate training in PD and identify differences by country-level economic development. DESIGN: Representatives from 80 national member societies of the International Association of Paediatric Dentistry (IAPD) were invited to complete a questionnaire on undergraduate and postgraduate PD curricula, types of postgraduate education offered, and recognition of the specialty. Country economic development level was classified according to the World Bank criteria. The chi-squared test and the Spearman correlation coefficient were used for data analysis (α = 0.005). RESULTS: Response rate was 63%. Teaching of PD at the undergraduate level was present in all countries, but PD specialization, master's, and Doctor of Philosophy (PhD) coursework were offered in 75%, 64%, and 53% of surveyed countries, respectively. Postgraduate specialization courses were offered in a significantly larger proportion of high-income countries than in upper-middle- or lower-middle-income countries (p < .01). In 20% of participating countries, PD was not an officially recognized specialty with no difference in recognition of the specialty by country's economic development level (p = .62). CONCLUSION: Paediatric dentistry is taught at the undergraduate level universally, but at the postgraduate level, significantly fewer courses are available, especially in lower-income countries.

An in vitro assessment of teething gels' effects on human gingival mesenchymal stem cells.

BACKGROUND: The aim of this study is to examine the cytotoxic effects of dental gels with different contents, which are frequently used during teething, on gingival mesenchymal stem cells (G-MSCs). METHOD: The teething gels used in this study were Dentinox, Gengigel, Osanite, and Jack and Jill. The human gingival mesenchimal stem cells (hG-MSCs) were incubated with these teething gel solutions (0.1%, 50% and 80% concentrations). Reproductive behavior of G-MSCs was monitored in real time for 72 h using the xCELLigence real-time cell analyzer (RTCA) system. Two-way repeated Anova test and post hoc Bonferroni test were used to evaluate the effect of concentration and dental gel on 0-hour and 72-hour viability. Significance was evaluated at p < 0.05 level. RESULTS: Teething gels prepared at 50% concentration are added to the G-MSC culture, the "cell index" value of G-MSCs to which Dentinox brand gel is added is significantly lower than all other groups (p = 0.05). There is a statistically significant difference between the concentrations in terms of cell index values at the 72nd hour compared to the 0th hour (p = 0.001). CONCLUSIONS: The local anesthetic dental gels used in children have a more negative effect on cell viability as concentration increases.

Phenotypic variability in LAMA3-associated amelogenesis imperfecta.

OBJECTIVE: Amelogenesis imperfecta (AI) is defined as inherited enamel malformations. LAMA3 (laminin alpha-3) encodes a critical protein component of the basement membrane (laminin-332). Individuals carrying heterozygous LAMA3 mutations have previously been shown to have localized enamel defects. This study aimed to define clinical phenotypes and to discern the genetic etiology for four AI kindreds. MATERIALS AND METHODS: Whole-exome analyses were conducted to search for sequence variants associated with the disorder, and micro-computed tomography (µCT) to characterize the enamel defects. RESULTS: The predominant enamel phenotype was generalized thin enamel with defective pits and grooves. Horizonal bands of hypoplastic enamel with chalky-white discoloration and enamel hypomineralization were also observed and demonstrated by µCT analyses of affected teeth. Four disease-causing LAMA3 mutations (NM_198129.4:c.3712dup; c.5891dup; c.7367del; c.9400G > C) were identified. Compound heterozygous MMP20 mutations (NM_004771.4:c.539A > G; c.692C > T) were also found in one proband with more severe enamel defects, suggesting a mutational synergism on disease phenotypes. Further analyses of the AI-causing mutations suggested that both α3A (short) and α3B (long) isoforms of LAMA3 are essential for enamel formation. CONCLUSIONS: Heterozygous LAMA3 mutations can cause generalized enamel defects (AI1A) with variable expressivity. Laminin-332 is critical not only for appositional growth but also enamel maturation.

FAM20A mutations and transcriptome analyses of dental pulp tissues of enamel renal syndrome.

AIM: Biallelic loss-of-function FAM20A mutations cause amelogenesis imperfecta (AI) type IG, better known as enamel renal syndrome (ERS), characterized by severe enamel hypoplasia, delayed/failed tooth eruption, intrapulpal calcifications, gingival hyperplasia and nephrocalcinosis. FAM20A binds to FAM20C, the Golgi casein kinase (GCK) and potentiates its function to phosphorylate secreted proteins critical for biomineralization. While many FAM20A pathogenic mutations have been reported, the pathogeneses of orodental anomalies in ERS remain to be elucidated. This study aimed to identify disease-causing mutations for patients with ERS phenotypes and to discern the molecular mechanism underlying ERS intrapulpal calcifications. METHODOLOGY: Phenotypic characterization and whole exome analyses were conducted for 8 families and 2 sporadic cases with hypoplastic AI. A minigene assay was performed to investigate the molecular consequences of a FAM20A splice-site variant. RNA sequencing followed by transcription profiling and gene ontology (GO) analyses were carried out for dental pulp tissues of ERS and the control. RESULTS: Biallelic FAM20A mutations were demonstrated for each affected individual, including 7 novel pathogenic variants: c.590-5T>A, c.625T>A (p.Cys209Ser), c.771del (p.Gln258Argfs*28), c.832_835delinsTGTCCGACGGTGTCCGACGGTGTC CA (p.Val278Cysfs*29), c.1232G>A (p.Arg411Gln), c.1297A>G (p.Arg433Gly) and c.1351del (p.Gln451Serfs*4). The c.590-5T>A splice-site mutation caused Exon 3 skipping, which resulted in an in-frame deletion of a unique region of the FAM20A protein, p.(Asp197_Ile214delinsVal). Analyses of differentially expressed genes in ERS pulp tissues demonstrated that genes involved in biomineralization, particularly dentinogenesis, were significantly upregulated, such as DSPP, MMP9, MMP20 and WNT10A. Enrichment analyses indicated overrepresentation of gene sets associated with BMP and SMAD signalling pathways. In contrast, GO terms related to inflammation and axon development were underrepresented. Among BMP signalling genes, BMP agonists GDF7, GDF15, BMP3, BMP8A, BMP8B, BMP4 and BMP6 were upregulated, while BMP antagonists GREM1, BMPER and VWC2 showed decreased expression in ERS dental pulp tissues. CONCLUSIONS: Upregulation of BMP signalling underlies intrapulpal calcifications in ERS. FAM20A plays an essential role in pulp tissue homeostasis and prevention of ectopic mineralization in soft tissues. This critical function probably depends upon MGP (matrix Gla protein), a potent mineralization inhibitor that must be properly phosphorylated by FAM20A-FAM20C kinase complex.

Cytotoxic effects of different detergent containing children's toothpastes on human gingival epithelial cells.

BACKGROUND: This study aimed to evaluate possible cytotoxic effects to gingival epithelial cells exposed to children toothpastes containing different detergent. METHODS: Tissues required for the isolation of human gingival epithelial cells were obtained by biopsy during the extraction of the impacted third molar tooth. Toothpaste solutions of different concentrations were prepared from five different children's toothpastes with different detergent contents. Isolated gingival epithelial cells were stimulated with experimental groups consisting of toothpaste solutions (Colgate, Sensodyne, Splat, Nenedent, Perlodent) at different concentrations and a control group consisting of complete Dulbecco's modified eagle medium. After the experiments, cell viability was evaluated using flow cytometry. 2 Way ANOVA was used to see the interaction effect of the main effects of toothpaste solution and concentration factors. Pairwise comparisons were made by Tukey post hoc tests. In the study, the significance level was taken as 0.05. RESULTS: As a result of the analysis, it was seen that the toothpaste solution and concentration factors and the interactions of these 2 factors were effective on the viable, early apoptotic, late apoptotic and necrotic cell rates. The statistically highest live cell ratios were detected in Splat's toothpaste solutions (90.14% at 0.4% concentration) after the control group (90.82%) and the group with the lowest viability values was determined in Colgate group (75.74% at 0.4% concentration) (p < 0.05). CONCLUSIONS: According to the results of the study, it was observed that toothpastes containing SLS affected the viability of cells more negatively than toothpastes with other detergent contents.

Assessment of Dental Arch Parameters in Turkish Twins.

BACKGROUND: The aim of this study is to investigate the relative contributions of genetic and environmental factors to variations in dental dimensions in a sample of Turkish twins, and to estimate heritability using dental casts. STUDY DESIGN: The study samples were selected from the twin children between 3-15 years old who referred for their first dental examination. Fifty nine monozygotic and one hundred and forty three dizygotic twin pairs were examined in the study. The alginate impression material used to create the plaster model of maxilla and mandible. Anterior arch width, posterior arch width, arch length and arch circumference were measured on models prepared from measurements taken for both maxilla and mandible with digital caliper. The similarities and differences of the measurements were compared between pairs of twins and zygocytes. Morever, the effects of bad oral habits, bruxism, a result of psychosocial factors on measurements were examined. Statistical analysis was performed using Paired T Test, Wilcoxon Test and Mann Whitney U test. RESULTS: A total of 404 dental models of 118 (29.2%) monozygotic and 286 (70.8%) dizygotic twins were evaluated. There was no statistical difference between sibling pairs in both monozygotic and dizygotic twins. The measurement similarity between twin siblings differed according to zygosity in all measurements (p<0.05). It has been observed that the finger sucking and mouth breathing affect the dental arch measurements (p<0.05). CONCLUSION: These results indicate that the differences in dental arch dimensions between monozygotic twin pairs are less than the difference between dizygotic twin pairs.

Assessment of Dental Arch Parameters in Turkish Twins.

BACKGROUND: The aim of this study is to investigate the relative contributions of genetic and environmental factors to variations in dental dimensions in a sample of Turkish twins, and to estimate heritability using dental casts. STUDY DESIGN: The study samples were selected from the twin children between 3-15 years old who referred for their first dental examination. Fifty nine monozygotic and one hundred and forty three dizygotic twin pairs were examined in the study. The alginate impression material used to create the plaster model of maxilla and mandible. Anterior arch width, posterior arch width, arch length and arch circumference were measured on models prepared from measurements taken for both maxilla and mandible with digital caliper. The similarities and differences of the measurements were compared between pairs of twins and zygocytes. Morever, the effects of bad oral habits, bruxism, a result of psychosocial factors on measurements were examined. Statistical analysis was performed using Paired T Test, Wilcoxon Test and Mann Whitney U test. RESULTS: A total of 404 dental models of 118 (29.2%) monozygotic and 286 (70.8%) dizygotic twins were evaluated. There was no statistical difference between sibling pairs in both monozygotic and dizygotic twins. The measurement similarity between twin siblings differed according to zygosity in all measurements (p<0.05). It has been observed that the finger sucking and mouth breathing affect the dental arch measurements (p<0.05). CONCLUSION: These results indicate that the differences in dental arch dimensions between monozygotic twin pairs are less than the difference between dizygotic twin pairs.

Recessive Mutations in ACPT, Encoding Testicular Acid Phosphatase, Cause Hypoplastic Amelogenesis Imperfecta.

Amelogenesis imperfecta (AI) is a heterogeneous group of genetic disorders affecting tooth enamel. The affected enamel can be hypoplastic and/or hypomineralized. In this study, we identified ACPT (testicular acid phosphatase) biallelic mutations causing non-syndromic, generalized hypoplastic autosomal-recessive amelogenesis imperfecta (AI) in individuals from six apparently unrelated Turkish families. Families 1, 4, and 5 were affected by the homozygous ACPT mutation c.713C>T (p.Ser238Leu), family 2 by the homozygous ACPT mutation c.331C>T (p.Arg111Cys), family 3 by the homozygous ACPT mutation c.226C>T (p.Arg76Cys), and family 6 by the compound heterozygous ACPT mutations c.382G>C (p.Ala128Pro) and 397G>A (p.Glu133Lys). Analysis of the ACPT crystal structure suggests that these mutations damaged the activity of ACPT by altering the sizes and charges of key amino acid side chains, limiting accessibility of the catalytic core, and interfering with homodimerization. Immunohistochemical analysis confirmed localization of ACPT in secretory-stage ameloblasts. The study results provide evidence for the crucial function of ACPT during amelogenesis.

Mutations in the pH-Sensing G-protein-Coupled Receptor GPR68 Cause Amelogenesis Imperfecta.

Amelogenesis is the process of dental enamel formation, leading to the deposition of the hardest tissue in the human body. This process requires the intricate regulation of ion transport and controlled changes to the pH of the developing enamel matrix. The means by which the enamel organ regulates pH during amelogenesis is largely unknown. We identified rare homozygous variants in GPR68 in three families with amelogenesis imperfecta, a genetically and phenotypically heterogeneous group of inherited conditions associated with abnormal enamel formation. Each of these homozygous variants (a large in-frame deletion, a frameshift deletion, and a missense variant) were predicted to result in loss of function. GPR68 encodes a proton-sensing G-protein-coupled receptor with sensitivity in the pH range that occurs in the developing enamel matrix during amelogenesis. Immunohistochemistry of rat mandibles confirmed localization of GPR68 in the enamel organ at all stages of amelogenesis. Our data identify a role for GPR68 as a proton sensor that is required for proper enamel formation.

Mutations in RELT cause autosomal recessive amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a collection of isolated (non-syndromic) inherited diseases affecting dental enamel formation or a clinical phenotype in syndromic conditions. We characterized three consanguineous AI families with generalized irregular hypoplastic enamel with rapid attrition that perfectly segregated with homozygous defects in a novel gene: RELT that is a member of the tumor necrosis factor receptor superfamily (TNFRSF). RNAscope in situ hybridization of wild-type mouse molars and incisors showed specific Relt mRNA expression by secretory stage ameloblasts and by odontoblasts. Relt-/- mice generated by CRISPR/Cas9 exhibited incisor and molar enamel malformations. Relt-/- enamel had a rough surface and underwent rapid attrition. Normally unmineralized spaces in the deep enamel near the dentino-enamel junction (DEJ) were as highly mineralized as the adjacent enamel, which likely altered the mechanical properties of the DEJ. Phylogenetic analyses showed the existence of selective pressure on RELT gene outside of tooth development, indicating that the human condition may be syndromic, which possibly explains the history of small stature and severe childhood infections in two of the probands. Knowing a TNFRSF member is critical during the secretory stage of enamel formation advances our understanding of amelogenesis and improves our ability to diagnose human conditions featuring enamel malformations.

Candidate gene sequencing reveals mutations causing hypoplastic amelogenesis imperfecta.

OBJECTIVE: Amelogenesis imperfecta (AI) is a rare hereditary disorder affecting the quality and quantity of the tooth enamel. The purpose of this study was to identify the genetic etiology of hypoplastic AI families based on the candidate gene approach. MATERIALS AND METHODS: We recruited three Turkish families with hypoplastic AI and performed a candidate gene screening based on the characteristic clinical feature to find the pathogenic genetic etiology. RESULTS: The candidate gene sequencing of the LAMB3 gene for family 1 revealed a heterozygous nonsense mutation in the last exon [c.3431C > A, p.(Ser1144*)]. FAM20A gene sequencing for families 2 and 3 identified a homozygous deletion [c.34_35delCT, p.(Leu12Alafs*67)] and a homozygous deletion-insertion (c.1109 + 3_1109 + 7delinsTGGTC) mutation, respectively. CONCLUSION: The candidate gene approach can be successfully used to identify the genetic etiology of the AI in some cases with characteristic clinical features. CLINICAL RELEVANCE: Identification of the genetic etiology of the AI will help both the family members and dentist understand the nature of the disorder. Characteristic clinical feature can suggest possible genetic causes.

A multi-ethnic genome-wide association study identifies novel loci for non-syndromic cleft lip with or without cleft palate on 2p24.2, 17q23 and 19q13.

Orofacial clefts (OFCs), which include non-syndromic cleft lip with or without cleft palate (CL/P), are among the most common birth defects in humans, affecting approximately 1 in 700 newborns. CL/P is phenotypically heterogeneous and has a complex etiology caused by genetic and environmental factors. Previous genome-wide association studies (GWASs) have identified at least 15 risk loci for CL/P. As these loci do not account for all of the genetic variance of CL/P, we hypothesized the existence of additional risk loci. We conducted a multiethnic GWAS in 6480 participants (823 unrelated cases, 1700 unrelated controls and 1319 case-parent trios) with European, Asian, African and Central and South American ancestry. Our GWAS revealed novel associations on 2p24 near FAM49A, a gene of unknown function (P = 4.22 × 10-8), and 19q13 near RHPN2, a gene involved in organizing the actin cytoskeleton (P = 4.17 × 10-8). Other regions reaching genome-wide significance were 1p36 (PAX7), 1p22 (ARHGAP29), 1q32 (IRF6), 8q24 and 17p13 (NTN1), all reported in previous GWASs. Stratification by ancestry group revealed a novel association with a region on 17q23 (P = 2.92 × 10-8) among individuals with European ancestry. This region included several promising candidates including TANC2, an oncogene required for development, and DCAF7, a scaffolding protein required for craniofacial development. In the Central and South American ancestry group, significant associations with loci previously identified in Asian or European ancestry groups reflected their admixed ancestry. In summary, we have identified novel CL/P risk loci and suggest new genes involved in craniofacial development, confirming the highly heterogeneous etiology of OFCs.

Novel FGF10 mutation in autosomal dominant aplasia of lacrimal and salivary glands.

OBJECTIVE: Aplasia of lacrimal and salivary glands (ALSG) is a rare autosomal dominant inherited disease, characterized by aplasia, atresia, or hypoplasia of the lacrimal and salivary systems with variable expressivity. The purpose of this study was to identify genetic etiology of an ALSG family. MATERIALS AND METHODS: We recruited a Turkish family with ALSG and performed a mutational analysis, based on the candidate gene approach, to clarify the molecular genetic etiology. RESULTS: The candidate gene sequencing of the FGF10 gene identified a novel heterozygous nonsense mutation (c.237G > A, p.Trp79*) in the exon 1. CONCLUSION: The identified novel mutation would result in a haploinsufficiency of the FGF10, because of nonsense-mediated mRNA decay caused by a premature stop codon. This report further confirms that ALSG is caused by the haploinsufficiency of functional FGF10. CLINICAL RELEVANCE: Identification of the genetic etiology of the ALSG will help both the family members and dentist understand the nature of the disorder. Therefore, it will positively motivate oral health care to avoid further destruction of the tooth due to the lack of salivary production.

Genetic influences on dental enamel that impact caries differ between the primary and permanent dentitions.

Clinically, primary and permanent teeth are distinct anatomically and the presentation of caries lesions differs between the two dentitions. Hence, the possibility exists that genetic contributions to tooth formation of the two dentitions are different. The purpose of this study was to test the hypothesis that genetic associations with an artificial caries model will not be the same between primary and permanent dentitions. Enamel samples from primary and permanent teeth were tested for microhardness at baseline, after carious lesion creation, and after fluoride application to verify association with genetic variants of selected genes. Associations were found between genetic variants of ameloblastin, amelogenin, enamelin, tuftelin, tuftelin interactive protein 11, and matrix metallopeptidase 20 and enamel from permanent teeth but not with enamel from primary teeth. In conclusion, our data continue to support that genetic variation may impact enamel development and consequently individual caries susceptibility. These effects may be distinct between primary and permanent dentitions.

Effect of fibroblast growth factor and enamel matrix derivative treatment on root resorption after delayed replantation.

BACKGROUND: Periodontal ligament (PDL) healing and long term prognosis of replanted avulsed teeth should rely on several factors including length of extra-oral dry time and type of the storage medium. The status of periodontal ligament is critical for the healing of replanted teeth. Different substances have been used for root surface treatment to promote formation of PDL and increase the survival of avulsed teeth submitted to replantation. AIM: The purpose of this study was to assess the effect of recombinant basic fibroblast growth factor 2 (bFGF) and enamel matrix derivative (EMD) on root resorption after delayed replantation. DESIGN: 18 freshly extracted single-rooted incisor and premolar teeth were extracted from the beagle dogs and immersed in whole bovine milk for 45 and 60 min (n = 3 each). Following storage period, sockets washed and teeth were treated with bFGF and EMD and replanted into the sockets. After 8 weeks, dogs were sacrificed, specimens processed to 4-µm thick serial sections for histopathologic examination and morphometric assessments. Thus, the proportions of the roots that exhibited signs of surface resorption, inflammatory resorption, and replacement resorption, that is, ankylosis and normal PDL were noted. RESULTS: The percentage of root resorption was in the following order: EMD>milk>bFGF for 45 min and milk>EMD>bFGF for 60 min. For all groups, teeth stored 60 min showed significantly higher incidence of PDL resorption than those stored for 45 min (P < 0.01). The highest incidence of replacement resorption was observed in teeth treated with EMD for 60 min. After 8 weeks, the least resorption was found in bFGF-treated group (P < 0.01). CONCLUSIONS: The findings of this study suggest that use of bFGF favored the formation of new periodontal ligament; prevent ankylosis and resorption process following delayed replantation of teeth while EMD shows replacement resorption, which may turn to ankylosis.

Role of estrogen related receptor beta (ESRRB) in DFN35B hearing impairment and dental decay.

BACKGROUND: Congenital forms of hearing impairment can be caused by mutations in the estrogen related receptor beta (ESRRB) gene. Our initial linkage studies suggested the ESRRB locus is linked to high caries experience in humans. METHODS: We tested for association between the ESRRB locus and dental caries in 1,731 subjects, if ESRRB was expressed in whole saliva, if ESRRB was associated with the microhardness of the dental enamel, and if ESRRB was expressed during enamel development of mice. RESULTS: Two families with recessive ESRRB mutations and DFNB35 hearing impairment showed more extensive dental destruction by caries. Expression levels of ESRRB in whole saliva samples showed differences depending on sex and dental caries experience. CONCLUSIONS: The common etiology of dental caries and hearing impairment provides a venue to assist in the identification of individuals at risk to either condition and provides options for the development of new caries prevention strategies, if the associated ESRRB genetic variants are correlated with efficacy.

The WNT10A gene in ectodermal dysplasias and selective tooth agenesis.

Mutations in the WNT10A gene were first detected in the rare syndrome odonto-onycho-dermal dysplasia (OODD, OMIM257980) but have now also been found to cause about 35-50% of selective tooth agenesis (STHAG4, OMIM150400), a common disorder that mostly affects the permanent dentition. In our random sample of tooth agenesis patients, 40% had at least one mutation in the WNT10A gene. The WNT10A Phe228Ile variant alone reached an allele frequency of 0.21 in the tooth agenesis cohort, about 10 times higher than the allele frequency reported in large SNP databases for Caucasian populations. Patients with bi-allelic WNT10A mutations have severe tooth agenesis while heterozygous individuals are either unaffected or have a mild phenotype. Mutations in the coding areas of the WNT10B gene, which is co-expressed with WNT10A during odontogenesis, and the WNT6 gene which is located at the same chromosomal locus as WNT10A in humans, do not contribute to the tooth agenesis phenotype.