Heterozygous COL17A1 variants are a frequent cause of amelogenesis imperfecta.

BACKGROUND: Collagen XVII is most typically associated with human disease when biallelic COL17A1 variants (>230) cause junctional epidermolysis bullosa (JEB), a rare, genetically heterogeneous, mucocutaneous blistering disease with amelogenesis imperfecta (AI), a developmental enamel defect. Despite recognition that heterozygous carriers in JEB families can have AI, and that heterozygous COL17A1 variants also cause dominant corneal epithelial recurrent erosion dystrophy (ERED), the importance of heterozygous COL17A1 variants causing dominant non-syndromic AI is not widely recognised. METHODS: Probands from an AI cohort were screened by single molecule molecular inversion probes or targeted hybridisation capture (both a custom panel and whole exome sequencing) for COL17A1 variants. Patient phenotypes were assessed by clinical examination and analyses of affected teeth. RESULTS: Nineteen unrelated probands with isolated AI (no co-segregating features) had 17 heterozygous, potentially pathogenic COL17A1 variants, including missense, premature termination codons, frameshift and splice site variants in both the endo-domains and the ecto-domains of the protein. The AI phenotype was consistent with enamel of near normal thickness and variable focal hypoplasia with surface irregularities including pitting. CONCLUSION: These results indicate that COL17A1 variants are a frequent cause of dominantly inherited non-syndromic AI. Comparison of variants implicated in AI and JEB identifies similarities in type and distribution, with five identified in both conditions, one of which may also cause ERED. Increased availability of genetic testing means that more individuals will receive reports of heterozygous COL17A1 variants. We propose that patients with isolated AI or ERED, due to COL17A1 variants, should be considered as potential carriers for JEB and counselled accordingly, reflecting the importance of multidisciplinary care.

SAXS of murine amelogenin identifies a persistent dimeric species from pH 5.0 to 8.0.

Amelogenin is an intrinsically disordered protein essential to tooth enamel formation in mammals. Using the latest, advanced small angle X-ray scattering (SAXS) capabilities at synchrotrons and computational models, we revisited measuring the quaternary structure of murine amelogenin as a function of pH and phosphorylation at Ser-16. The SAXS data shows that at the pH extremes, amelogenin exists as an extended monomer at pH 3.0 (Rg = 38.4 Å) and nanospheres at pH 8.0 (Rg = 84.0 Å), consistent with multiple previous observations. At pH 5.0 and above there was no evidence for a significant population of monomeric species. Instead, at pH 5.0 ∼ 80% of the population is a heterogenous dimeric species that increases to ∼ 100% at pH 5.5. The dimer population was observed at all pH > 5 conditions in dynamic equilibrium with a species in the pentamer range at pH < 6.5 and nanospheres at pH 8.0. At pH 8.0 ∼ 40% of the amelogenin remained in the dimeric state. In general, serine-16 phosphorylation of amelogenin appears to modestly stabilize the population of the dimeric species.

Digenic inheritance accounts for phenotypic variability in amelogenesis imperfecta.

Amelogenesis imperfecta (AI) represents a group of clinically and genetically heterogeneous disorders that affect enamel formation and mineralization. Although AI is commonly considered a monogenic disorder, digenic inheritance is rarely reported. In this study, we recruited two nonconsanguineous Chinese families exhibiting diverse phenotypes of enamel defects among affected family members. Digenic variants were discovered in both probands. In family 1, the proband inherited a paternal frameshift variant in LAMA3 (NM_198129.4:c.3712dup) and a maternal deletion encompassing the entire AMELX gene. This resulted in a combined hypoplastic and hypomineralized AI phenotype, which was distinct from the parents' manifestations. In family 2, whole-exome sequencing analysis revealed the proband carried a maternal heterozygous splicing variant in COL17A1 (NC_000010.11 (NM_000494.3): c.4156 + 2dup) and compound heterozygous variants in RELT (paternal: NM_032871.4:c.260A > T; maternal: NM_032871.4:c.521 T > G). These genetic changes caused the abundant irregular enamel defects observed in the proband, whereas other affected family members carrying heterozygous variants in both COL17A1 and RELT displayed only horizontal grooves as their phenotype. The pathogenicity of the novel COL17A1 splice site variant was confirmed through RT-PCR and minigene assay. This study enhances our understanding by highlighting the potential association between the co-occurrence of variants in two genes and variable phenotypes observed in AI patients.

Developmental Defects of Enamel: A Bibliometric Analysis of the Top 100 Most-Cited Papers.

BACKGROUND: Developmental defects of enamel (DDE) are a result of disturbances during formation and maturation of the enamel. Evaluating the most-cited DDE papers can provide important tools that point to the gaps and strengths of this important topic in dentistry. SUMMARY: This bibliometric study analyzed the 100 most-cited papers on DDE. Using a combined keyword search strategy, the 100 most-cited papers were selected in the Web of Science Core Collection. Papers that addressed any type of DDE were included. The extracted data were title, number of citations, study theme, authorship, journal, type of DDE, type of dentition (primary or permanent), type of diagnosis, study design, year, and country of publication. The bibliometric networks were generated through VOSviewer software. The 100 papers had a range from 78 to 459 citations. The main themes of studies were etiopathogenesis (53%), prevalence and incidence (22%), and diagnosis (8%). The authors with the highest number of citations were Goodman AH and Rose JC (459 citations). Most articles were published in dental journals (47%). The most studied types of DDE were fluorosis and amelogenesis imperfecta in the permanent dentition (47%). Observational (24%) and non-systematic reviews (24%) were the most common study designs and ranged from 1977 to 2019. The country with the highest number of publications was the USA (41%). KEY MESSAGES: Most of the top 100 DDE papers were about fluorosis and amelogenesis imperfecta, with top papers from three continents with English as the native language. This topic is of great importance in dentistry, and the need for further studies is highlighted, especially regarding the diagnosis and treatment of some DDEs.

In-depth investigation of FAM20A insufficiency effects on deciduous dental pulp cells: Altered behaviours, osteogenic differentiation, and inflammatory gene expression.

AIM: Loss-of-function mutations in FAM20A result in amelogenesis imperfecta IG (AI1G) or enamel-renal syndrome, characterized by hypoplastic enamel, ectopic calcification, and gingival hyperplasia, with some cases reporting spontaneous tooth infection. Despite previous reports on the consequence of FAM20A reduction in gingival fibroblasts and transcriptome analyses of AI1G pulp tissues, suggesting its involvement in mineralization and infection, its role in deciduous dental pulp cells (DDP) remains unreported. The aim of this study was to evaluate the properties of DDP obtained from an AI1G patient, providing additional insights into the effects of FAM20A on the mineralization of DDP. METHODOLOGY: DDP were obtained from a FAM20A-AI1G patient (mutant cells) and three healthy individuals. Cellular behaviours were examined using flow cytometry, MTT, attachment and spreading, colony formation, and wound healing assays. Osteogenic induction was applied to DDP, followed by alizarin red S staining to assess their osteogenic differentiation. The expression of FAM20A-related genes, osteogenic genes, and inflammatory genes was analysed using real-time PCR, Western blot, and/or immunolocalization. Additionally, STRING analysis was performed to predict potential protein-protein interaction networks. RESULTS: The mutant cells exhibited a significant reduction in FAM20A mRNA and protein levels, as well as proliferation, migration, attachment, and colony formation. However, normal FAM20A subcellular localization was maintained. Additionally, osteogenic/odontogenic genes, OSX, OPN, RUNX2, BSP, and DSPP, were downregulated, along with upregulated ALP. STRING analysis suggested a potential correlation between FAM20A and these osteogenic genes. After osteogenic induction, the mutant cells demonstrated reduced mineral deposition and dysregulated expression of osteogenic genes. Remarkably, FAM20A, FAM20C, RUNX2, OPN, and OSX were significantly upregulated in the mutant cells, whilst ALP, and OCN was downregulated. Furthermore, the mutant cells exhibited a significant increase in inflammatory gene expression, that is, IL-1ß and TGF-ß1, whereas IL-6 and NFκB1 expression was significantly reduced. CONCLUSION: The reduction of FAM20A in mutant DDP is associated with various cellular deficiencies, including delayed proliferation, attachment, spreading, and migration as well as altered osteogenic and inflammatory responses. These findings provide novel insights into the biology of FAM20A in dental pulp cells and shed light on the molecular mechanisms underlying AI1G pathology.

Salivary Molecular Spectroscopy with Machine Learning Algorithms for a Diagnostic Triage for Amelogenesis Imperfecta.

Amelogenesis imperfecta (AI) is a genetic disease characterized by poor formation of tooth enamel. AI occurs due to mutations, especially in AMEL, ENAM, KLK4, MMP20, and FAM83H, associated with changes in matrix proteins, matrix proteases, cell-matrix adhesion proteins, and transport proteins of enamel. Due to the wide variety of phenotypes, the diagnosis of AI is complex, requiring a genetic test to characterize it better. Thus, there is a demand for developing low-cost, noninvasive, and accurate platforms for AI diagnostics. This case-control pilot study aimed to test salivary vibrational modes obtained in attenuated total reflection fourier-transformed infrared (ATR-FTIR) together with machine learning algorithms: linear discriminant analysis (LDA), random forest, and support vector machine (SVM) could be used to discriminate AI from control subjects due to changes in salivary components. The best-performing SVM algorithm discriminates AI better than matched-control subjects with a sensitivity of 100%, specificity of 79%, and accuracy of 88%. The five main vibrational modes with higher feature importance in the Shapley Additive Explanations (SHAP) were 1010 cm-1, 1013 cm-1, 1002 cm-1, 1004 cm-1, and 1011 cm-1 in these best-performing SVM algorithms, suggesting these vibrational modes as a pre-validated salivary infrared spectral area as a potential biomarker for AI screening. In summary, ATR-FTIR spectroscopy and machine learning algorithms can be used on saliva samples to discriminate AI and are further explored as a screening tool.

Fluoride Alters Gene Expression via Histone H3K27 Acetylation in Ameloblast-like LS8 Cells.

Excessive fluoride ingestion during tooth development can cause dental fluorosis. Previously, we reported that fluoride activates histone acetyltransferase (HAT) to acetylate p53, promoting fluoride toxicity in mouse ameloblast-like LS8 cells. However, the roles of HAT and histone acetylation status in fluoride-mediated gene expression remain unidentified. Here, we demonstrate that fluoride-mediated histone modification causes gene expression alterations in LS8 cells. LS8 cells were treated with or without fluoride followed by ChIP-Seq analysis of H3K27ac. Genes were identified by differential H3K27ac peaks within ±1 kb from transcription start sites. The levels of mRNA of identified genes were assessed using rea-time PCR (qPCR). Fluoride increased H3K27ac peaks associated with Bax, p21, and Mdm2 genes and upregulated their mRNA levels. Fluoride decreased H3K27ac peaks and p53, Bad, and Bcl2 had suppressed transcription. HAT inhibitors (Anacardic acid or MG149) suppressed fluoride-induced mRNA of p21 and Mdm2, while fluoride and the histone deacetylase (HDAC) inhibitor sodium butyrate increased Bad and Bcl2 expression above that of fluoride treatment alone. To our knowledge, this is the first study that demonstrates epigenetic regulation via fluoride treatment via H3 acetylation. Further investigation is required to elucidate epigenetic mechanisms of fluoride toxicity in enamel development.

Full-mouth rehabilitation with lithium disilicate ceramic crowns in hypoplastic amelogenesis imperfecta: a case report and review of literature.

BACKGROUND: Amelogenesis imperfecta (AI) is a group of genetic disorders characterized by tooth discoloration and enamel defects. Patients with AI always exhibit generalized attrition and defective tooth structure, leading to the loss of occlusal vertical dimension (OVD). Appropriate rehabilitation is challenging and essential to improve patients' aesthetics and function. CASE PRESENTATION: This case report presents a comprehensive management of a 30-year-old woman with hypoplastic AI. A 52-month follow-up revealed satisfactory full-mouth rehabilitation performances of lithium disilicate ceramic crowns after clinical crown lengthening, with increased vertical dimension. CONCLUSIONS: Patients with severe hypoplastic AI require proper full-mouth rehabilitation. Using full-crown lithium disilicate restorations to increase the OVD by 2‒4 mm is a safe and predictable recommendation for such cases. In addition, patients with AI require complex and comprehensive management. The long-term effects of full-mouth rehabilitation with lithium disilicate ceramic crowns still necessitate further follow-ups.

HMGN2 represses gene transcription via interaction with transcription factors Lef-1 and Pitx2 during amelogenesis.

The chromatin-associated high mobility group protein N2 (HMGN2) cofactor regulates transcription factor activity through both chromatin and protein interactions. Hmgn2 expression is known to be developmentally regulated, but the post-transcriptional mechanisms that regulate Hmgn2 expression and its precise roles in tooth development remain unclear. Here, we demonstrate that HMGN2 inhibits the activity of multiple transcription factors as a general mechanism to regulate early development. Bimolecular fluorescence complementation, pull-down, and coimmunoprecipitation assays show that HMGN2 interacts with the transcription factor Lef-1 through its HMG-box domain as well as with other early development transcription factors, Dlx2, FoxJ1, and Pitx2. Furthermore, EMSAs demonstrate that HMGN2 binding to Lef-1 inhibits its DNA-binding activity. We found that Pitx2 and Hmgn2 associate with H4K5ac and H3K4me2 chromatin marks in the proximal Dlx2 promoter, demonstrating Hmgn2 association with open chromatin. In addition, we demonstrate that microRNAs (miRs) mir-23a and miR-23b directly target Hmgn2, promoting transcriptional activation at several gene promoters, including the amelogenin promoter. In vivo, we found that decreased Hmgn2 expression correlates with increased miR-23 expression in craniofacial tissues as the murine embryo develops. Finally, we show that ablation of Hmgn2 in mice results in increased amelogenin expression because of increased Pitx2, Dlx2, Lef-1, and FoxJ1 transcriptional activity. Taken together, our results demonstrate both post-transcriptional regulation of Hmgn2 by miR-23a/b and post-translational regulation of gene expression by Hmgn2-transcription factor interactions. We conclude that HMGN2 regulates tooth development through its interaction with multiple transcription factors.

A tooth crown morphology framework for interpreting the diversity of primate dentitions.

Variation in tooth crown morphology plays a crucial role in species diagnoses, phylogenetic inference, and the reconstruction of the evolutionary history of the primate clade. While a growing number of studies have identified developmental mechanisms linked to tooth size and cusp patterning in mammalian crown morphology, it is unclear (1) to what degree these are applicable across primates and (2) which additional developmental mechanisms should be recognized as playing important roles in odontogenesis. From detailed observations of lower molar enamel-dentine junction morphology from taxa representing the major primate clades, we outline multiple phylogenetic and developmental components responsible for crown patterning, and formulate a tooth crown morphology framework for the holistic interpretation of primate crown morphology. We suggest that adopting this framework is crucial for the characterization of tooth morphology in studies of dental development, discrete trait analysis, and systematics.

Conditional knockout of transient receptor potential melastatin 7 in the enamel epithelium: Effects on enamel formation.

Transient receptor potential melastatin 7 (TRPM7) is a unique ion channel connected to a kinase domain. We previously demonstrated that Trpm7 expression is high in mouse ameloblasts and odontoblasts, and that amelogenesis is impaired in TRPM7 kinase-dead mice. Here, we analyzed TRPM7 function during amelogenesis in Keratin 14-Cre;Trpm7fl/fl conditional knockout (cKO) mice and Trpm7 knockdown cell lines. cKO mice showed lesser tooth pigmentation than control mice and broken incisor tips. Enamel calcification and microhardness were lower in cKO mice. Electron probe microanalysis (EPMA) showed that the calcium and phosphorus contents in the enamel were lower in cKO mouse than in control mice. The ameloblast layer in cKO mice showed ameloblast dysplasia at the maturation stage. The morphological defects were observed in rat SF2 cells with Trpm7 knockdown. Compared with mock transfectants, the Trpm7 knockdown cell lines showed lower levels of calcification with Alizarin Red-positive staining and an impaired intercellular adhesion structures. These findings suggest that TRPM7 is a critical ion channel in enamel calcification for the effective morphogenesis of ameloblasts during amelogenesis.

Molecular-based phenotype variations in amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is one of the typical dental genetic diseases in human. It can occur isolatedly or as part of a syndrome. Previous reports have mainly clarified the types and mechanisms of nonsyndromic AI. This review aimed to compare the phenotypic differences among the hereditary enamel defects with or without syndromes and their underlying pathogenic genes. We searched the articles in PubMed with different strategies or keywords including but not limited to amelogenesis imperfecta, enamel defects, hypoplastic/hypomaturation/hypocalcified, syndrome, or specific syndrome name. The articles with detailed clinical information about the enamel and other phenotypes and clear genetic background were used for the analysis. We totally summarized and compared enamel phenotypes of 18 nonsyndromic AI with 17 causative genes and 19 syndromic AI with 26 causative genes. According to the clinical features, radiographic or ultrastructural changes in enamel, the enamel defects were basically divided into hypoplastic and hypomineralized (hypomaturated and hypocalcified) and presented a higher heterogeneity which were closely related to the involved pathogenic genes, types of mutation, hereditary pattern, X chromosome inactivation, incomplete penetrance, and other mechanisms.The gene-specific enamel phenotypes could be an important indicator for diagnosing nonsyndromic and syndromic AI.

Epigenetics in developmental defects of enamel: A scoping review.

OBJECTIVES: The significant role of epigenetics has been revealed in normal enamel formation process and occurrence of developmental defects. This presented literature is aiming at summarizing the regulatory function of epigenetics in physiological amelogenesis process and reviewing the epigenetic mechanisms in occurrence of developmental defects of enamel (DDE), so as to provide biological foundation evidence to support early predication and clinical management of DDE. METHOD: An extensive literature review was conducted using electronic databases MEDLINE (through PubMed), Web of Science and EMBASE up to November 30, 2022. Studies about epigenetic effects on enamel tissue or cells associated with amelogenesis, including in vivo studies using human or animal models, and in vitro studies, are selected. RESULTS: A total of 22 studies were included. Epigenetic factors or effects specifically activate or silence certain genes, which may regulate related biological activities including cell proliferation, cell differentiation, enamel secretion, and mineralization during the process of amelogenesis. Once the status of epigenetic modification is altered, the quantity and quality of enamel may both be disturbed, which can finally result in DDE. CONCLUSION: Epigenetics plays a noteworthy role of regulating the amelogenesis process and DDE potentially by altering the expression levels of genes related to enamel formation, providing a new perspective of early predication and clinical management of DDE.

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.

Tooth ultrastructure changes induced by a nonsense mutation in the FAM83H gene: insights into the diversity of amelogenesis imperfecta.

OBJECTIVES: The current research on single-nucleotide polymorphism (SNP) mutation sites at different positions of the FAM83H gene and their phenotypic changes leading to amelogenesis imperfecta (AI) is inconsistent. We identified a previously reported heterozygous nonsense mutation c.1192C>T (p.Q398*) in the FAM83H gene and conducted a comprehensive analysis of the dental ultrastructure and chemical composition changes induced by this mutation. Additionally, we predicted the protein feature affected by this mutation site. The aim was to further deepen our understanding of the diversity of AI caused by different mutation sites in the FAM83H gene. METHODS: Whole-exome sequencing (WES) and Sanger sequencing were used to confirm the mutation sites. Physical features of the patient's teeth were investigated using various methods including cone beam computer tomography (CBCT), scanning electron microscopy (SEM), contact profilometry (roughness measurement), and a nanomechanical tester (nanoindentation measurement). The protein features of wild-type and mutant FAM83H were predicted using bioinformatics methods. RESULTS: One previously discovered FAM83H heterozygous nonsense mutation c.1192C>T (p.Q398*) was detected in the patient. SEM revealed inconsistent dentinal tubules, and EDS showed that calcium and phosphorus were lower in the patient's dentin but higher in the enamel compared to the control tooth. Roughness measurements showed that AI patients' teeth had rougher occlusal surfaces than those of the control tooth. Nanoindentation measurements showed that the enamel and dentin hardness values of the AI patients' teeth were both significantly reduced compared to those of the control tooth. Compared to the wild-type FAM83H protein, the mutant FAM83H protein shows alterations in stability, hydrophobicity, secondary structure, and tertiary structure. These changes could underlie functional differences and AI phenotype variations caused by this mutation site. CONCLUSIONS: This study expands the understanding of the effects of FAM83H mutations on tooth structure. CLINICAL RELEVANCE: Our study enhances our understanding of the genetic basis of AI and may contribute to improved diagnostics and personalized treatment strategies for patients with FAM83H-related AI.

Amelogenesis imperfecta in a Chinese family resulting from a FAM83H variation and the effect of FAM83H on the secretion of enamel matrix proteins.

OBJECTIVES: To investigate the variant of an amelogenesis imperfecta (AI) family and to explore the function of the FAM83H (family with sequence similarity 83 member H) in the enamel formation. MATERIALS AND METHODS: We investigated a five-generation Chinese family diagnosed with AI; clinical data was collected, whole-exome sequencing (WES) was conducted to explore the pathogenic gene and variants and Sanger sequencing was used to verify the variants. The three-dimensional protein structures of wild-type and mutant FAM83H were predicted using alpha fold 2. To study the possible regulatory function of Fam83h on amelogenesis, immunolocalization was performed to observe the expression of Fam83h protein in Sprague-Dawley rat postnatal incisors. The mRNA and protein level of amelogenin, enamelin, kallikrein-related peptidase-4 and ameloblastin were also detected after the Fam83h was knocked down by small interfering RNA (siRNA) in HAT-7 cells. RESULTS: A known nonsense variant (c.973 C > T) in exon 5 of FAM83H gene was found in this family, causing a truncated protein (p.R325X). Immunolocalization of Fam83h in Sprague-Dawley rat postnatal incisors showed that Fam83h protein expression was detected in presecretory and secretory stages. When Fam83h expression was reduced by siRNA, the expression of amelogenin, enamelin, kallikrein-related peptidase-4 decreased. However, the expression of ameloblastin increased. CONCLUSIONS: FAM83H gene variant (c.973 C > T) causes AI. FAM83H regulates the secretion of enamel matrix proteins and affects ameloblast differentiation. CLINICAL RELEVANCE: This study provided that FAM83H variants could influence enamel formation and provided new insights into the pathogenesis of AI.

Amelogenesis imperfecta: More than just an enamel problem.

OBJECTIVES: This article will provide an overview of the clinical presentation, treatment considerations, and sequencing of treatment for a patient with amelogenesis imperfecta (AI). The different types and subgroups of AI will be described, focusing on Type I hypoplastic form of the condition. OVERVIEW: Patients with AI all have abnormal enamel formation but some may also present with vertical dysgnathia, anterior open bite, and posterior crossbite. A case report demonstrates the sequencing and implementation of necessary orthodontic and prosthodontic treatments, beginning in the mixed dentition and ending with esthetic and functional permanent restorations in the permanent dentition. CLINICAL SIGNIFICANCE: AI is a disorder of tooth enamel formation but may also affect the face, jaw relationship, occlusion, compromised esthetics, and can potentially cause psychological damage due to the appearance of the teeth. Treatment of AI should be initiated at a young age.

Splicing mutations in AMELX and ENAM cause amelogenesis imperfecta.

BACKGROUND: Amelogenesis imperfecta (AI) is a developmental enamel defect affecting the structure of enamel, esthetic appearance, and the tooth masticatory function. Gene mutations are reported to be relevant to AI. However, the mechanism underlying AI caused by different mutations is still unclear. This study aimed to reveal the molecular pathogenesis in AI families with 2 novel pre-mRNA splicing mutations. METHODS: Two Chinese families with AI were recruited. Whole-exome sequencing and Sanger sequencing were performed to identify mutations in candidate genes. Minigene splicing assays were performed to analyze the mutation effects on mRNA splicing alteration. Furthermore, three-dimensional structures of mutant proteins were predicted by AlphaFold2 to evaluate the detrimental effect. RESULTS: The affected enamel in family 1 was thin, rough, and stained, which was diagnosed as hypoplastic-hypomature AI. Genomic analysis revealed a novel splicing mutation (NM_001142.2: c.570 + 1G > A) in the intron 6 of amelogenin (AMELX) gene in family 1, resulting in a partial intron 6 retention effect. The proband in family 2 exhibited a typical hypoplastic AI, and the splicing mutation (NM_031889.2: c.123 + 4 A > G) in the intron 4 of enamelin (ENAM) gene was observed in the proband and her father. This mutation led to exon 4 skipping. The predicted structures showed that there were obvious differences in the mutation proteins compared with wild type, leading to impaired function of mutant proteins. CONCLUSIONS: In this study, we identified two new splicing mutations in AMELX and ENAM genes, which cause hypoplastic-hypomature and hypoplastic AI, respectively. These results expand the spectrum of genes causing AI and broaden our understanding of molecular genetic pathology of enamel formation.

CRISPR/Cas9-mediated deletion of Fam83h induces defective tooth mineralization and hair development in rabbits.

Family with sequence similarity 83 members H (Fam83h) is essential for dental enamel formation. Fam83h mutations cause human amelogenesis imperfecta (AI), an inherited disorder characterized by severe hardness defects in dental enamel. Nevertheless, previous studies showed no enamel defects in Fam83h-knockout/lacZ-knockin mice. In this study, a large deletion of the Fam83h gene (900 bp) was generated via a dual sgRNA-directed CRISPR/Cas9 system in rabbits. Abnormal tooth mineralization and loose dentine were found in homozygous Fam83h knockout (Fam83h-/- ) rabbits compared with WT rabbits. In addition, reduced hair follicle counts in dorsal skin, hair cycling dysfunction and hair shaft differentiation deficiency were observed in Fam83h-/- rabbits. Moreover, X-rays and staining of bone sections showed abnormal bending of the ulna and radius and an ulnar articular surface with insufficient trabecular bone in Fam83h-/- rabbits. Taken together, these data are the first report of defective hair cycling, hair shaft differentiation and abnormal bending of the ulna and radius in Fam83h-/- rabbits. This novel Fam83h-/- rabbit model may facilitate understanding the function of Fam83h and the pathogenic mechanism of the Fam83h mutation.

Kohlschütter-Tönz syndrome: Case report with novel feature and detailed review of features associated with ROGDI variants.

Kohlschütter-Tönz syndrome (KTS) is a rare, autosomal recessive syndrome characterized by a triad of epilepsy, amelogenesis imperfecta and severe global developmental delay. It was first described in a Swiss family in 1974 by Alfried Kohlschütter and Otmar Tönz. It is caused by pathogenic variants in the ROGDI gene. To the best of our knowledge, there are currently 43 patients with a confirmed ROGDI gene pathogenic variant reported. Here, we review in detail the clinical manifestations of KTS, provide an overview of all reported genetically confirmed patients, and document an additional case of KTS-a 6-year-old Latvian girl-with a confirmed ROGDI gene pathogenic variant. In contrast to previous reports, we detected idiopathic bilateral nephrocalcinosis in this newly identified KTS patient. Perampanel proved an effective treatment for our patient with prolonged super-refractory status epilepticus. In order to better characterize this rare syndrome and its clinical course, it is important to report any additional symptoms and also the effectiveness of used therapies. Future research should focus on elucidating the mechanisms by which the absence/insufficiency of ROGDI-encoded protein causes the clinical manifestations of KTS. This knowledge could shape possible ways of influencing the disease's natural history with more effective therapies.