RESUMO
Enamel and enameloid, the highly mineralized tooth-covering tissues in living vertebrates, are different in their matrix composition. Enamel, a unique product of ameloblasts, principally contains enamel matrix proteins (EMPs), while enameloid possesses collagen fibrils and probably receives contributions from both odontoblasts and ameloblasts. Here we focused on type I collagen (COL1A1) and amelogenin (AMEL) gene expression during enameloid and enamel formation throughout ontogeny in the caudate amphibian, Pleurodeles waltl. In this model, pre-metamorphic teeth possess enameloid and enamel, while post-metamorphic teeth possess enamel only. In first-generation teeth, qPCR and in situ hybridization (ISH) on sections revealed that ameloblasts weakly expressed AMEL during late-stage enameloid formation, while expression strongly increased during enamel deposition. Using ISH, we identified COL1A1 transcripts in ameloblasts and odontoblasts during enameloid formation. COL1A1 expression in ameloblasts gradually decreased and was no longer detected after metamorphosis. The transition from enameloid-rich to enamel-rich teeth could be related to a switch in ameloblast activity from COL1A1 to AMEL synthesis. P. waltl therefore appears to be an appropriate animal model for the study of the processes involved during enameloid-to-enamel transition, especially because similar events probably occurred in various lineages during vertebrate evolution.
Assuntos
Ameloblastos/metabolismo , Amelogênese/fisiologia , Colágeno Tipo I/análise , Ameloblastos/citologia , Amelogenina/análise , Animais , Diferenciação Celular/fisiologia , Cadeia alfa 1 do Colágeno Tipo I , Esmalte Dentário/citologia , Esmalte Dentário/metabolismo , Dentinogênese/fisiologia , Órgão do Esmalte/anatomia & histologia , Metamorfose Biológica/fisiologia , Microscopia Eletrônica de Transmissão , Modelos Animais , Odontoblastos/citologia , Odontoblastos/metabolismo , Odontogênese/fisiologia , Pleurodeles , Germe de Dente/anatomia & histologiaRESUMO
In the literature, the enamelin gene ENAM has been repeatedly designated as a possible candidate for caries susceptibility. Here, we checked whether ENAM variants could increase caries susceptibility. To this aim, we sequenced coding exons and exon-intron boundaries of ENAM in 250 children with a severe caries phenotype and in 149 caries-free patients from 9 French hospital groups. In total, 23 single-nucleotide polymorphisms (SNPs) were found, but none appeared to be responsible for a direct change of ENAM function. Six SNPs had a high minor allele frequency (MAF) and 6 others were identified for the first time. Statistical and evolutionary analyses showed that none of these SNPs was associated with caries susceptibility or caries protection when studied separately and challenged with environmental factors. However, haplotype interaction analysis showed that the presence, in a same variant, of 2 exonic SNPs (rs7671281 and rs3796704; MAF 0.12 and 0.10, respectively), both changing an amino acid in the protein region encoded by exon 10 (p.I648T and p.R763Q, respectively), increased caries susceptibility 2.66-fold independent of the environmental risk factors. These findings support ENAM as a gene candidate for caries susceptibility in the studied population.
Assuntos
Cárie Dentária/genética , Haplótipos/genética , Proteínas/genética , Substituição de Aminoácidos/genética , Arginina/genética , Criança , Índice CPO , Suscetibilidade à Cárie Dentária/genética , Éxons/genética , Proteínas da Matriz Extracelular , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Variação Genética/genética , Glutamina/genética , Humanos , Íntrons/genética , Isoleucina/genética , Desequilíbrio de Ligação/genética , Masculino , Mutação de Sentido Incorreto/genética , Polimorfismo de Nucleotídeo Único/genética , Treonina/genética , Adulto JovemRESUMO
In this article, we focus on hypomaturation autosomal-recessive-type amelogenesis imperfecta (type IIA2) and describe 2 new causal Matrix metalloproteinase 20 (MMP20) mutations validated in two unrelated families: a missense mutation p.T130I at the expected homozygous state, and a compound heterozygous mutation having the same mutation combined with a nucleotide deletion, leading to a premature stop codon (p.N120fz*2). We characterized the enamel structure of the latter case using scanning electron microscopy analysis and microanalysis (Energy-dispersive X-ray Spectroscopy, EDX) and confirmed the hypomaturation-type amelogenesis imperfecta as identified in the clinical diagnosis. The mineralized content was slightly decreased, with magnesium substituting for calcium in the crystal structure. The anomalies affected enamel with minimal inter-rod enamel present and apatite crystals perpendicular to the enamel prisms, suggesting a possible new role for MMP20 in enamel formation.
Assuntos
Amelogênese Imperfeita/enzimologia , Heterozigoto , Homozigoto , Metaloproteinase 20 da Matriz/genética , Mutação/genética , Amelogênese Imperfeita/genética , Apatitas/análise , Sequência de Bases/genética , Cálcio/análise , Pré-Escolar , Códon sem Sentido/genética , Cristalografia , Citosina , Esmalte Dentário/ultraestrutura , Microanálise por Sonda Eletrônica , Éxons/genética , Feminino , Genes Recessivos/genética , Humanos , Magnésio/análise , Microscopia Eletrônica de Varredura , Minerais/análise , Mutação de Sentido Incorreto/genética , Deleção de Sequência/genética , TiminaRESUMO
Genetic approaches have shown that several genes could modify caries susceptibility; AmelogeninX (AMELX) has been repeatedly designated. Here, we hypothesized that AMELX mutations resulting in discrete changes of enamel microstructure may be found in children with a severe caries phenotype. In parallel, possible AMELX mutations that could explain resistance to caries may be found in caries-free patients. In this study, coding exons of AMELX and exon-intron boundaries were sequenced in 399 individuals with extensive caries (250) or caries-free (149) individuals from nine French hospital groups. No mutation responsible for a direct change of amelogenin function was identified. Seven single-nucleotide polymorphisms (SNPs) were found, 3 presenting a high allele frequency, and 1 being detected for the first time. Three SNPs were located in coding regions, 2 of them being non-synonymous. Both evolutionary and statistical analyses showed that none of these SNPs was associated with caries susceptibility, suggesting that AMELX is not a gene candidate in our studied population.
Assuntos
Amelogenina/genética , Suscetibilidade à Cárie Dentária/genética , Cárie Dentária/genética , Adolescente , Adulto , Criança , Pré-Escolar , Índice CPO , Índice de Placa Dentária , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Adulto JovemRESUMO
Molecular evolutionary analysis is an efficient method to predict and/or validate amino acid substitutions that could lead to a genetic disease and to highlight residues and motifs that could play an important role in the protein structure and/or function. We have applied such analysis to amelotin (AMTN), a recently identified enamel protein in the rat, mouse, and humans. An in silico search for AMTN provided 42 new mammalian sequences that were added to the 3 published sequences with which we performed the analysis using a dataset representative of all lineages (circa 220 million years of evolution), including 2 enamel-less species, sloth and armadillo. During evolution, of the 209 residues of human AMTN, 17 were unchanged and 34 had conserved their chemical properties. Substituting these important residues could lead to amelogenesis imperfecta (AI). Also, AMTN possesses a well-conserved signal peptide, 2 conserved motifs whose function is certainly important but unknown, and a putative phosphorylation site (SXE). In addition, the sequences of the 2 enamel-less species display mutations revealing that AMTN underwent pseudogenization, which suggests that AMTN is an enamel-specific protein.