RESUMEN

Amelogenesis imperfecta (AI) is a heterogeneous group of genetic diseases characterised by dental enamel malformation. Pathogenic variants in at least 33 genes cause syndromic or non-syndromic AI. Recently variants in RELT, encoding an orphan receptor in the tumour necrosis factor (TNF) superfamily, were found to cause recessive AI, as part of a syndrome encompassing small stature and severe childhood infections. Here we describe four additional families with autosomal recessive hypomineralised AI due to previously unreported homozygous mutations in RELT. Three families carried a homozygous missense variant in the fourth exon (c.164C>T, p.(T55I)) and a fourth family carried a homozygous missense variant in the 11th exon (c.1264C>T, p.(R422W)). We found no evidence of additional syndromic symptoms in affected individuals. Analyses of tooth microstructure with computerised tomography and scanning electron microscopy suggest a role for RELT in ameloblasts' coordination and interaction with the enamel matrix. Microsatellite genotyping in families segregating the T55I variant reveals a shared founder haplotype. These findings extend the RELT pathogenic variant spectrum, reveal a founder mutation in the UK Pakistani population and provide detailed analysis of human teeth affected by this hypomineralised phenotype, but do not support a possible syndromic presentation in all those with RELT-variant associated AI.

Asunto(s)

Amelogénesis Imperfecta/genética , Predisposición Genética a la Enfermedad , Receptores del Factor de Necrosis Tumoral/genética , Desmineralización Dental/genética , Amelogénesis Imperfecta/diagnóstico por imagen , Amelogénesis Imperfecta/patología , Exones , Femenino , Homocigoto , Humanos , Masculino , Mutación Missense/genética , Linaje , Fenotipo , Desmineralización Dental/diagnóstico por imagen , Desmineralización Dental/patología , Factor de Necrosis Tumoral alfa/genética

RESUMEN

In this chapter we discuss the potential of preparative SDS-PAGE for use in purifying native developing enamel matrix proteins. We believe that the methodology has the potential to provide the relatively large-scale single-step purification of any enamel protein that can be resolved as a single band during analytical SDS-PAGE. Of course, a single band on analytical SDS-PAGE does not guarantee absolute purity as the band may be comprised of two or more proteins migrating at the same apparent molecular weight on the gel. Where absolute purity is required, the methodology can be used in conjunction with other techniques such as ion-exchange chromatography or reverse-phase chromatography. We do not see preparative SDS-PAGE replacing chromatographic methodologies but believe that it can provide another powerful tool to add to the battery of purification techniques already available to researchers in the field.

Asunto(s)

Amelogenina/aislamiento & purificación , Proteínas del Esmalte Dental/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida/métodos , Acrilamida/química , Animales , Electroforesis en Gel de Poliacrilamida/instrumentación , Diseño de Equipo , Geles/química , Porosidad , Porcinos

RESUMEN

X-ray micro CT has become a popular methodology for the nondestructive analysis of dental tissues and has been used extensively in the amelogenesis field. The aim of this chapter is to introduce ImageJ/Fiji to researchers new to CT scanning and the analysis of CT image data. The program can be applied to analyzing X-ray CT images of enamel but can be extrapolated to other tissues as well.

Asunto(s)

Esmalte Dental/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Microtomografía por Rayos X/métodos , Animales , Densidad Ósea , Esmalte Dental/química , Aprendizaje Automático , Mandíbula/química , Mandíbula/ultraestructura , Ratones , Minerales/química , Diente Molar/química , Diente Molar/ultraestructura

RESUMEN

During the secretory phase of their life-cycle, ameloblasts are highly specialized secretory cells whose role is to elaborate an extracellular matrix that ultimately confers both form and function to dental enamel, the most highly mineralized of all mammalian tissues. In common with many other "professional" secretory cells, ameloblasts employ the unfolded protein response (UPR) to help them cope with the large secretory cargo of extracellular matrix proteins transiting their ER (endoplasmic reticulum)/Golgi complex and so minimize ER stress. However, the UPR is a double-edged sword, and, in cases where ER stress is severe and prolonged, the UPR switches from pro-survival to pro-apoptotic mode. The purpose of this review is to consider the role of the ameloblast UPR in the biology and pathology of amelogenesis; specifically in respect of amelogenesis imperfecta (AI) and fluorosis. Some forms of AI appear to correspond to classic proteopathies, where pathological intra-cellular accumulations of protein tip the UPR toward apoptosis. Fluorosis also involves the UPR and, while not of itself a classic proteopathic disease, shares some common elements through the involvement of the UPR. The possibility of therapeutic intervention by pharmacological modulation of the UPR in AI and fluorosis is also discussed.

RESUMEN

Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with "tags" that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond.

RESUMEN

Amelogenesis imperfecta (AI) is the name given to a heterogeneous group of conditions characterized by inherited developmental enamel defects. AI enamel is abnormally thin, soft, fragile, pitted and/or badly discolored, with poor function and aesthetics, causing patients problems such as early tooth loss, severe embarrassment, eating difficulties, and pain. It was first described separately from diseases of dentine nearly 80 years ago, but the underlying genetic and mechanistic basis of the condition is only now coming to light. Mutations in the gene AMELX, encoding an extracellular matrix protein secreted by ameloblasts during enamel formation, were first identified as a cause of AI in 1991. Since then, mutations in at least eighteen genes have been shown to cause AI presenting in isolation of other health problems, with many more implicated in syndromic AI. Some of the encoded proteins have well documented roles in amelogenesis, acting as enamel matrix proteins or the proteases that degrade them, cell adhesion molecules or regulators of calcium homeostasis. However, for others, function is less clear and further research is needed to understand the pathways and processes essential for the development of healthy enamel. Here, we review the genes and mutations underlying AI presenting in isolation of other health problems, the proteins they encode and knowledge of their roles in amelogenesis, combining evidence from human phenotypes, inheritance patterns, mouse models, and in vitro studies. An LOVD resource (http://dna2.leeds.ac.uk/LOVD/) containing all published gene mutations for AI presenting in isolation of other health problems is described. We use this resource to identify trends in the genes and mutations reported to cause AI in the 270 families for which molecular diagnoses have been reported by 23rd May 2017. Finally we discuss the potential value of the translation of AI genetics to clinical care with improved patient pathways and speculate on the possibility of novel treatments and prevention strategies for AI.

RESUMEN

"Amelogenesis imperfecta" (AI) describes a group of genetic conditions that result in defects in tooth enamel formation. Mutations in many genes are known to cause AI, including the gene encoding the serine protease, kallikrein related peptidase 4 (KLK4), expressed during the maturation stage of amelogenesis. In this study we report the fourth KLK4 mutation to be identified in autosomal recessively-inherited hypomaturation type AI, c.632delT, p.(L211Rfs*37) (NM_004917.4, NP_004908.4). This homozygous variant was identified in five Pakistani AI families and is predicted to result in a transcript with a premature stop codon that escapes nonsense mediated decay. However, the protein may misfold, as three of six disulphide bonds would be disrupted, and may be degraded or non-functional as a result. Primary teeth were obtained from one affected individual. The enamel phenotype was characterized using high-resolution computerized X-ray tomography (CT), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and microhardness testing (MH). Enamel from the affected individual (referred to as KLK4 enamel) was hypomineralised in comparison with matched control enamel. Furthermore, KLK4 inner enamel was hypomineralised compared with KLK4 outer enamel. SEM showed a clear structural demarcation between KLK4 inner and outer enamel, although enamel structure was similar to control tissue overall. EDX showed that KLK4 inner enamel contained less calcium and phosphorus and more nitrogen than control inner enamel and KLK4 outer enamel. MH testing showed that KLK4 inner enamel was significantly softer than KLK4 outer enamel (p < 0.001). However, the hardness of control inner enamel was not significantly different to that of control outer enamel. Overall, these findings suggest that the KLK4 c.632delT mutation may be a common cause of autosomal recessive AI in the Pakistani population. The phenotype data obtained mirror findings in the Klk4-/- mouse and suggest that KLK4 is required for the hardening and mineralization of the inner enamel layer but is less essential for hardening and mineralization of the outer enamel layer.

RESUMEN

We identified two homozygous missense variants (c.428C>T, p.(T143M) and c.746C>T, p.(P249L)) in ACPT, the gene encoding acid phosphatase, testicular, which segregates with hypoplastic amelogenesis imperfecta in two unrelated families. ACPT is reported to play a role in odontoblast differentiation and mineralisation by supplying phosphate during dentine formation. Analysis by computerised tomography and scanning electron microscopy of a primary molar tooth from an individual homozygous for the c.746C>T variant revealed an enamel layer that was hypoplastic, but mineralised with prismatic architecture. These findings implicate variants in ACPT as a cause of early failure of amelogenesis during the secretory phase.

Asunto(s)

Fosfatasa Ácida/genética , Amelogénesis Imperfecta/genética , Mutación Missense , Fosfatasa Ácida/metabolismo , Ameloblastos/metabolismo , Amelogénesis Imperfecta/diagnóstico , Genes Recesivos , Homocigoto , Humanos , Diente Molar/diagnóstico por imagen , Linaje

RESUMEN

'Amelogenesis imperfecta' (AI) describes a group of inherited diseases of dental enamel that have major clinical impact. Here, we identify the aetiology driving AI in mice carrying a p.S55I mutation in enamelin; one of the most commonly mutated proteins underlying AI in humans. Our data indicate that the mutation inhibits the ameloblast secretory pathway leading to ER stress and an activated unfolded protein response (UPR). Initially, with the support of the UPR acting in pro-survival mode, Enamp.S55I heterozygous mice secreted structurally normal enamel. However, enamel secreted thereafter was structurally abnormal; presumably due to the UPR modulating ameloblast behaviour and function in an attempt to relieve ER stress. Homozygous mutant mice failed to produce enamel. We also identified a novel heterozygous ENAMp.L31R mutation causing AI in humans. We hypothesize that ER stress is the aetiological factor in this case of human AI as it shared the characteristic phenotype described above for the Enamp.S55I mouse. We previously demonstrated that AI in mice carrying the Amelxp.Y64H mutation is a proteinopathy. The current data indicate that AI in Enamp.S55I mice is also a proteinopathy, and based on comparative phenotypic analysis, we suggest that human AI resulting from the ENAMp.L31R mutation is another proteinopathic disease. Identifying a common aetiology for AI resulting from mutations in two different genes opens the way for developing pharmaceutical interventions designed to relieve ER stress or modulate the UPR during enamel development to ameliorate the clinical phenotype.

Asunto(s)

Amelogénesis Imperfecta/genética , Amelogénesis Imperfecta/metabolismo , Ameloblastos/metabolismo , Animales , Esmalte Dental/metabolismo , Proteínas del Esmalte Dental/genética , Proteínas del Esmalte Dental/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/fisiología , Estrés del Retículo Endoplásmico/genética , Estrés del Retículo Endoplásmico/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación Puntual , Estrés Fisiológico , Respuesta de Proteína Desplegada

RESUMEN

Amelogenesis imperfecta (AI) is a heterogeneous group of genetic conditions that result in defective dental enamel formation. Amelotin (AMTN) is a secreted protein thought to act as a promoter of matrix mineralization in the final stage of enamel development, and is strongly expressed, almost exclusively, in maturation stage ameloblasts. Amtn overexpression and Amtn knockout mouse models have defective enamel with no other associated phenotypes, highlighting AMTN as an excellent candidate gene for human AI. However, no AMTN mutations have yet been associated with human AI. Using whole exome sequencing, we identified an 8,678 bp heterozygous genomic deletion encompassing exons 3-6 of AMTN in a Costa Rican family segregating dominant hypomineralised AI. The deletion corresponds to an in-frame deletion of 92 amino acids, shortening the protein from 209 to 117 residues. Exfoliated primary teeth from an affected family member had enamel that was of a lower mineral density compared to control enamel and exhibited structural defects at least some of which appeared to be associated with organic material as evidenced using elemental analysis. This study demonstrates for the first time that AMTN mutations cause non-syndromic human AI and explores the human phenotype, comparing it with that of mice with disrupted Amtn function.

Asunto(s)

Amelogénesis Imperfecta/genética , Proteínas del Esmalte Dental/genética , Esmalte Dental/patología , Predisposición Genética a la Enfermedad , Amelogénesis Imperfecta/fisiopatología , Secuencia de Aminoácidos/genética , Animales , Esmalte Dental/crecimiento & desarrollo , Modelos Animales de Enfermedad , Exones/genética , Humanos , Ratones , Ratones Noqueados , Fenotipo , Eliminación de Secuencia/genética

RESUMEN

Heimler syndrome (HS) consists of recessively inherited sensorineural hearing loss, amelogenesis imperfecta (AI) and nail abnormalities, with or without visual defects. Recently HS was shown to result from hypomorphic mutations in PEX1 or PEX6, both previously implicated in Zellweger Syndrome Spectrum Disorders (ZSSD). ZSSD are a group of conditions consisting of craniofacial and neurological abnormalities, sensory defects and multi-organ dysfunction. The finding of HS-causing mutations in PEX1 and PEX6 shows that HS represents the mild end of the ZSSD spectrum, though these conditions were previously thought to be distinct nosological entities. Here, we present six further HS families, five with PEX6 variants and one with PEX1 variants, and show the patterns of Pex1, Pex14 and Pex6 immunoreactivity in the mouse retina. While Ratbi et al. found more HS-causing mutations in PEX1 than in PEX6, as is the case for ZSSD, in this cohort PEX6 variants predominate, suggesting both genes play a significant role in HS. The PEX6 variant c.1802G>A, p.(R601Q), reported previously in compound heterozygous state in one HS and three ZSSD cases, was found in compound heterozygous state in three HS families. Haplotype analysis suggests a common founder variant. All families segregated at least one missense variant, consistent with the hypothesis that HS results from genotypes including milder hypomorphic alleles. The clinical overlap of HS with the more common Usher syndrome and lack of peroxisomal abnormalities on plasma screening suggest that HS may be under-diagnosed. Recognition of AI is key to the accurate diagnosis of HS.

Asunto(s)

Adenosina Trifosfatasas/genética , Amelogénesis Imperfecta/genética , Mutación del Sistema de Lectura , Pérdida Auditiva Sensorineural/genética , Proteínas de la Membrana/genética , Mutación Missense , Uñas Malformadas/genética , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/metabolismo , Amelogénesis Imperfecta/diagnóstico , Animales , Exoma , Pérdida Auditiva Sensorineural/diagnóstico , Heterocigoto , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Uñas Malformadas/diagnóstico , Linaje , Fenotipo , Retina/metabolismo

RESUMEN

The enamel matrix derivative (EMD) contains hundreds of peptides in different levels of proteolytic processing that may provide a range of biological effects of importance in wound healing. The aim of the present study was to compare the effect of EMD and its fractions on the cytokine profiles from human gingival fibroblasts in vitro and in gingival crevicular fluid (GCF) in a randomized controlled split-mouth clinical study (n = 12). Levels of cytokines in cell culture medium and in GCF were measured by Luminex over a 2-week period. In the clinical study, levels of pro-inflammatory cytokines and chemokines were increased, whereas the levels of transforming growth factor-α (TGF-α) and platelet-derived growth factor-BB (PDGF-BB) were reduced. The in vitro study showed that EMD and its high and low molecular weight fractions reduced the secretion of pro-inflammatory cytokines and chemokines compared to untreated cells. EMD had an effect on levels of cytokines related to fibroplasia, angiogenesis, inflammation and chemotaxis both in vitro and in vivo, however, the anti-inflammatory effect induced by EMD observed in the in vitro study could not be confirmed clinically.

Asunto(s)

Periodontitis Crónica/tratamiento farmacológico , Citocinas/metabolismo , Proteínas del Esmalte Dental/farmacología , Periodoncio/efectos de los fármacos , Adulto , Anciano , Animales , Becaplermina , Células Cultivadas , Periodontitis Crónica/metabolismo , Periodontitis Crónica/cirugía , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Encía/citología , Líquido del Surco Gingival/efectos de los fármacos , Líquido del Surco Gingival/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Persona de Mediana Edad , Periodoncio/fisiopatología , Periodoncio/cirugía , Proteínas Proto-Oncogénicas c-sis/metabolismo , Regeneración/efectos de los fármacos , Factor de Crecimiento Transformador alfa/metabolismo , Cicatrización de Heridas/efectos de los fármacos

RESUMEN

Enamel matrix derivative is used to promote periodontal regeneration during the corrective phase of the treatment of periodontal defects. Our main goal was to analyze the bioactivity of different molecular weight fractions of enamel matrix derivative. Enamel matrix derivative, a complex mixture of proteins, was separated into 13 fractions using size-exclusion chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-electrospray ionization-tandem mass spectrometry. Human periodontal ligament fibroblasts were treated with either enamel matrix derivative or the different fractions. Proliferation and cytokine secretion to the cell culture medium were measured and compared to untreated cells. The liquid chromatography-electrospray ionization-tandem mass spectrometry analyses revealed that the most abundant peptides were amelogenin and leucine-rich amelogenin peptide related. The fractions containing proteins above 20 kDa induced an increase in vascular endothelial growth factor and interleukin-6 secretion, whereas lower molecular weight fractions enhanced proliferation and secretion of interleukin-8 and monocyte chemoattractant protein-1 and reduced interleukin-4 release. The various molecular components in the enamel matrix derivative formulation might contribute to reported effects on tissue regeneration through their influence on vascularization, the immune response, and chemotaxis.

RESUMEN

Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In situ hybridization and immunohistodetection were performed in mandibular tissues using AMELX KO mice as controls. We identified AMELX-producing (RNA-positive) cells lining the adjacent alveolar bone and AMBN and AMELX proteins in the microenvironment surrounding EMPs-producing cells. Western blotting of proteins extracted by non-dissociative means revealed that AMELX and AMBN are not exclusive to mineralized matrix; they are present to some degree in a solubilized state in mandibular bone and presumably have some capacity to diffuse. Our data support the notion that AMELX and AMBN may function as growth factor-like molecules solubilized in the aqueous microenvironment. In jaws, they might play some role in bone physiology through autocrine/paracrine pathways, particularly during development and stress-induced remodeling.

Asunto(s)

Amelogenina/fisiología , Proteínas del Esmalte Dental/fisiología , Mandíbula/metabolismo , Amelogenina/análisis , Amelogenina/deficiencia , Amelogenina/genética , Animales , Proteínas del Esmalte Dental/análisis , Proteínas del Esmalte Dental/genética , Difusión , Células Epiteliales/metabolismo , Proteínas del Ojo/análisis , Proteínas del Ojo/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Mandíbula/crecimiento & desarrollo , Mesodermo/metabolismo , Ratones , Ratones Noqueados , Proteínas Musculares/análisis , Proteínas Musculares/fisiología , Especificidad de Órganos , Ovario/crecimiento & desarrollo , Ovario/metabolismo , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Solubilidad , Testículo/crecimiento & desarrollo , Testículo/metabolismo , Lengua/crecimiento & desarrollo , Lengua/metabolismo , Vísceras/crecimiento & desarrollo , Vísceras/metabolismo

RESUMEN

Amelogenesis imperfecta (AI) describes a heterogeneous group of inherited dental enamel defects reflecting failure of normal amelogenesis. Ameloblastin (AMBN) is the second most abundant enamel matrix protein expressed during amelogenesis. The pivotal role of AMBN in amelogenesis has been confirmed experimentally using mouse models. However, no AMBN mutations have been associated with human AI. Using autozygosity mapping and exome sequencing, we identified genomic deletion of AMBN exon 6 in a second cousin consanguineous family with three of the six children having hypoplastic AI. The genomic deletion corresponds to an in-frame deletion of 79 amino acids, shortening the protein from 447 to 368 residues. Exfoliated primary teeth (unmatched to genotype) were available from family members. The most severely affected had thin, aprismatic enamel (similar to that reported in mice homozygous for Ambn lacking exons 5 and 6). Other teeth exhibited thicker but largely aprismatic enamel. One tooth had apparently normal enamel. It has been suggested that AMBN may function in bone development. No clinically obvious bone or other co-segregating health problems were identified in the family investigated. This study confirms for the first time that AMBN mutations cause non-syndromic human AI and that mouse models with disrupted Ambn function are valid.

Asunto(s)

Amelogénesis Imperfecta/patología , Proteínas del Esmalte Dental/genética , Eliminación de Secuencia , Diente/ultraestructura , Amelogénesis Imperfecta/genética , Secuencia de Aminoácidos , Animales , Exones , Femenino , Humanos , Masculino , Ratones , Linaje , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Diente/patología

RESUMEN

Inherited diseases caused by genetic mutations can arise due to loss of protein function. Alternatively, mutated proteins may mis-fold, impairing endoplasmic reticulum (ER) trafficking, causing ER stress and triggering the unfolded protein response (UPR). The UPR attempts to restore proteostasis but if unsuccessful drives affected cells towards apoptosis. Previously, we reported that in mice, the p.Tyr64His mutation in the enamel extracellular matrix (EEM) protein amelogenin disrupts the secretory pathway in the enamel-forming ameloblasts, resulting in eruption of malformed tooth enamel that phenocopies human amelogenesis imperfecta (AI). Defective amelogenin post-secretory self-assembly and processing within the developing EEM has been suggested to underlie the pathogenesis of X chromosome-linked AI. Here, we challenge this concept by showing that AI pathogenesis associated with the p.Tyr64His amelogenin mutation involves ameloblast apoptosis induced by ER stress. Furthermore, we show that 4-phenylbutyrate can rescue the enamel phenotype in affected female mice by promoting cell survival over apoptosis such that they are able to complete enamel formation despite the presence of the mutation, offering a potential therapeutic option for patients with this form of AI and emphasizing the importance of ER stress in the pathogenesis of this inherited conformational disease.

Asunto(s)

Amelogénesis Imperfecta/tratamiento farmacológico , Amelogénesis Imperfecta/metabolismo , Fenilbutiratos/uso terapéutico , Amelogénesis Imperfecta/genética , Amelogenina/genética , Animales , Western Blotting , Estrés del Retículo Endoplásmico/efectos de los fármacos , Masculino , Ratones , Ratones Mutantes , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Mutación

RESUMEN

We identified a family in which pitted hypomineralized amelogenesis imperfecta (AI) with premature enamel failure segregated in an autosomal recessive fashion. Whole-exome sequencing revealed a missense mutation (c.586C>A, p.P196T) in the I-domain of integrin-ß6 (ITGB6), which is consistently predicted to be pathogenic by all available programmes and is the only variant that segregates with the disease phenotype. Furthermore, a recent study revealed that mice lacking a functional allele of Itgb6 display a hypomaturation AI phenotype. Phenotypic characterization of affected human teeth in this study showed areas of abnormal prismatic organization, areas of low mineral density and severe abnormal surface pitting in the tooth's coronal portion. We suggest that the pathogenesis of this form of AI may be due to ineffective ligand binding of ITGB6 resulting in either compromised cell-matrix interaction or compromised ITGB6 activation of transforming growth factor-ß (TGF-ß) impacting indirectly on ameloblast-ameloblast interactions and proteolytic processing of extracellular matrix proteins via MMP20. This study adds to the list of genes mutated in AI and further highlights the importance of cell-matrix interactions during enamel formation.

Asunto(s)

Amelogénesis Imperfecta/genética , Amelogénesis Imperfecta/patología , Genes Recesivos , Cadenas beta de Integrinas/fisiología , Mutación Missense/genética , Amelogénesis Imperfecta/metabolismo , Secuencia de Aminoácidos , Animales , ADN/genética , Esmalte Dental/metabolismo , Esmalte Dental/patología , Exoma/genética , Humanos , Técnicas para Inmunoenzimas , Ratones , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Linaje , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Homología de Secuencia de Aminoácido , Microtomografía por Rayos X

RESUMEN

BACKGROUND:Studies have shown that dental pulp cells can differentiate into odontoblastic like cells under certain stimulations, which is regulated by several signal pathways network. OBJECTIVE:To summarize the research progress of stimulating molecules and underlying molecular mechanisms of dental pulp cells’ odontoblastic differentiation. METHODS:A computer-based online search of CNKI and Wanfang databases was undertaken for the related Chinese articles dated from January 1998 to July 2014 with the keywords of“dental pulp cells”and“odontoblastic differentiation”in Chinese. Meanwhile, PubMed database was searched for the related English articles dated from January 1998 to July 2014 with the same keywords in English. Those with unrelated research subjects, or repetitive studies were excluded. Final y, 63 articles were reviewed. RESULTS AND CONCLUSION:Various stimuli, including bone morphogenetic protein,β-glycerophosphate and ascorbic acid, can stimulate dental pulp cells differentiation into odontoblastic like cells under certain conditions regulated by several signal pathways network. But odontoblastic differentiation is a complex process, and further research into stimulating molecules and underlying molecular mechanisms of dental pulp cells’ odontoblastic differentiation is significantly important for clinical applications of dental regenerative therapy.