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1.
Int J Mol Sci ; 23(4)2022 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-35216266

RESUMEN

BRIL (bone restricted ifitm-like; also known as IFITM5) is a transmembrane protein expressed in osteoblasts. Although its role in skeletal development and homeostasis is unknown, mutations in BRIL result in rare dominant forms of osteogenesis imperfecta. The pathogenic mechanism has been proposed to be a gain-of or neomorphic function. To understand the function of BRIL and its OI type V mutant (MALEP BRIL) and whether they could activate signaling pathways in osteoblasts, we performed a luciferase reporter assay screen based on the activity of 26 transcription factors. When overexpressed in MC3T3-E1 and MLO-A5 cells, the MALEP BRIL activated the reporters dependent on MEF2, NFATc, and NR4A significantly more. Additional co-transfection experiments with MEF2C and NFATc1 and a number of their modulators (HDAC4, calcineurin, RCAN, FK506) confirmed the additive or synergistic activation of the pathways by MALEP, and suggested a coordinated regulation involving calcineurin. Endogenous levels of Nr4a members, as well as Ptgs2, were upregulated by MALEP BRIL. Y2H and co-immunoprecipitation indicated that BRIL interacted with CAML, but its contribution as the most upstream stimulator of the Ca2+-calcineurin-MEF2/NFATc cascade was not confirmed convincingly. Altogether the data presented provide the first ever readout to monitor for BRIL activity and suggest a potential gain-of-function causative effect for MALEP BRIL in OI type V, leading to perturbed signaling events and gene expression.


Asunto(s)
Proteínas de la Membrana/genética , Mutación/genética , Factores de Transcripción NFATC/genética , Osteoblastos/metabolismo , Osteogénesis Imperfecta/genética , Activación Transcripcional/genética , Células 3T3 , Animales , Calcineurina/genética , Calcio/metabolismo , Línea Celular , Células HEK293 , Humanos , Ratones , Osteogénesis Imperfecta/metabolismo , Transducción de Señal/genética , Factores de Transcripción/genética
2.
Int J Mol Sci ; 22(10)2021 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-34069814

RESUMEN

Osteogenesis imperfecta (OI) is a bone fragility disorder that is usually caused by mutations affecting collagen type I. We compared the calvaria bone tissue transcriptome of male 10-week-old heterozygous Jrt (Col1a1 mutation) and homozygous oim mice (Col1a2 mutation) to their respective littermate results. We found that Jrt and oim mice shared 185 differentially expressed genes (upregulated: 106 genes; downregulated: 79 genes). A total of seven genes were upregulated by a factor of two or more in both mouse models (Cyp2e1, Slc13a5, Cgref1, Smpd3, Ifitm5, Cthrc1 and Rerg). One gene (Gypa, coding for a blood group antigen) was downregulated by a factor of two or more in both OI mouse models. Overrepresentation analyses revealed that genes involved in 'ossification' were significantly overrepresented among upregulated genes in both Jrt and oim mice, whereas hematopoietic genes were downregulated. Several genes involved in Wnt signaling and transforming growth factor beta signaling were upregulated in oim mice, but less so in Jrt mice. Thus, this study identified a set of genes that are dysregulated across various OI mouse models and are likely to play an important role in the pathophysiology of this disorder.


Asunto(s)
Osteogénesis Imperfecta/genética , Cráneo/metabolismo , Animales , Colágeno Tipo I/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Modelos Animales de Enfermedad , Fémur/metabolismo , Perfilación de la Expresión Génica/métodos , Heterocigoto , Homocigoto , Masculino , Ratones , Mutación , Osteogénesis , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/fisiopatología , Cráneo/fisiología , Esfingomielina Fosfodiesterasa/metabolismo , Simportadores/metabolismo , Transcriptoma/genética
3.
Eur J Oral Sci ; 127(4): 313-322, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31230388

RESUMEN

The junctional epithelium (JE) is a specialized portion of the gingiva that seals off the tooth-supporting tissues from the oral environment. This relationship is achieved via a unique adhesive extracellular matrix that is, in fact, a specialized basal lamina (sBL). Three unique proteins - amelotin (AMTN), odontogenic ameloblast-associated (ODAM), and secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1) - together with laminin-332 structure the supramolecular organization of this sBL and determine its adhesive capacity. Despite the constant challenge of the JE by the oral microbiome, little is known of the susceptibility of the sBL to bacterial degradation. Assays with trypsin-like proteases, as well as incubation with Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola, revealed that all constituents, except SCPPPQ1, were rapidly degraded. Porphyromonas gingivalis was also shown to alter the supramolecular network of reconstituted and native sBLs. These results provide evidence that proteolytic enzymes and selected gram-negative periodontopathogenic bacteria can attack this adhesive extracellular matrix, intimating that its degradation could contribute to progression of periodontal diseases.


Asunto(s)
Membrana Basal/microbiología , Inserción Epitelial/microbiología , Matriz Extracelular/patología , Encía , Diente , Amiloide , Proteínas de Unión al Calcio , Proteínas del Esmalte Dental , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Neoplasias , Fosfoproteínas , Porphyromonas gingivalis , Prevotella intermedia , Treponema denticola
4.
Am J Hum Genet ; 96(3): 425-31, 2015 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-25683117

RESUMEN

Cole-Carpenter syndrome is a severe bone fragility disorder that is characterized by frequent fractures, craniosynostosis, ocular proptosis, hydrocephalus, and distinctive facial features. To identify the cause of Cole-Carpenter syndrome in the two individuals whose clinical results were presented in the original description of this disorder, we performed whole-exome sequencing of genomic DNA samples from both individuals. The two unrelated individuals had the same heterozygous missense mutation in exon 9 of P4HB (NM_000918.3: c.1178A>G [p.Tyr393Cys]), the gene that encodes protein disulfide isomerase (PDI). In one individual, the P4HB mutation had arisen de novo, whereas in the other the mutation was transmitted from the clinically unaffected father who was a mosaic carrier of the variant. The mutation was located in the C-terminal disulfide isomerase domain of PDI, sterically close to the enzymatic center, and affected disulfide isomerase activity in vitro. Skin fibroblasts showed signs of increased endoplasmic reticulum stress, but despite the reported importance of PDI for collagen type I production, the rate of collagen type I secretion appeared normal. In conclusion, Cole-Carpenter syndrome is caused by a specific de novo mutation in P4HB that impairs the disulfide isomerase activity of PDI.


Asunto(s)
Craneosinostosis/genética , Anomalías del Ojo/genética , Heterocigoto , Hidrocefalia/genética , Mutación Missense , Osteogénesis Imperfecta/genética , Procolágeno-Prolina Dioxigenasa/genética , Proteína Disulfuro Isomerasas/genética , Preescolar , Femenino , Frecuencia de los Genes , Humanos , Lactante , Masculino , Linaje , Procolágeno-Prolina Dioxigenasa/metabolismo , Conformación Proteica , Proteína Disulfuro Isomerasas/metabolismo , Pliegue de Proteína , Análisis de Secuencia de ADN
5.
Am J Hum Genet ; 96(6): 979-85, 2015 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-26027498

RESUMEN

Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I and other proteins in the extracellular matrix. Using whole-exome sequencing to identify the molecular defect in two unrelated girls with severe bone fragility and a clinical diagnosis of osteogenesis imperfecta type IV, we identified two homozygous variants in SPARC (GenBank: NM_003118.3; c.497G>A [p.Arg166His] in individual 1; c.787G>A [p.Glu263Lys] in individual 2). Published modeling and site-directed mutagenesis studies had previously shown that the residues substituted by these mutations form an intramolecular salt bridge in SPARC and are essential for the binding of SPARC to collagen type I. The amount of SPARC secreted by skin fibroblasts was reduced in individual 1 but appeared normal in individual 2. The migration of collagen type I alpha chains produced by these fibroblasts was mildly delayed on SDS-PAGE gel, suggesting some overmodification of collagen during triple helical formation. Pulse-chase experiments showed that collagen type I secretion was mildly delayed in skin fibroblasts from both individuals. Analysis of an iliac bone sample from individual 2 showed that trabecular bone was hypermineralized on the material level. In conclusion, these observations show that homozygous mutations in SPARC can give rise to severe bone fragility in humans.


Asunto(s)
Modelos Moleculares , Mutación Missense/genética , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/patología , Osteonectina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Colágeno Tipo I/metabolismo , Electroforesis en Gel de Poliacrilamida , Exoma/genética , Femenino , Genes Recesivos/genética , Humanos , Immunoblotting , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Osteonectina/química , Osteonectina/metabolismo , Linaje , Conformación Proteica , Alineación de Secuencia , Análisis de Secuencia de ADN
6.
Hum Mol Genet ; 24(2): 516-24, 2015 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-25214535

RESUMEN

We had previously published the clinical characteristics of a bone fragility disorder in children that was characterized mainly by lower extremity fractures and a mineralization defect in bone tissue but not on the growth plate level. We have now performed whole-exome sequencing on four unrelated individuals with this phenotype. Three individuals were homozygous for a nucleotide change in BMP1, affecting the polyadenylation signal of the transcript that codes for the short isoform of BMP1 (BMP1-1) (c.*241T>C). In skin fibroblasts of these individuals, we found low levels of BMP1-1 transcript and protein. The fourth individual was compound heterozygous for the c.*241T>C variant in BMP1-1 and a variant in BMP1 exon 15 (c.2107G>C) that affected splicing in both BMP1-1 and the long isoform of BMP1 (BMP1-3). Both the homozygous 3'UTR variant and the compound heterozygous variants were associated with impaired procollagen type I C-propeptide cleavage, as the amount of free C-propeptide in the supernatant of skin fibroblasts was less than in controls. Peripheral quantitative computed tomography showed that all individuals had elevated volumetric cortical bone mineral density. Assessment of iliac bone samples by histomorphometry and quantitative backscattered electron imaging indicated that the onset of mineralization at bone formation sites was delayed, but that mineralized matrix was hypermineralized. These results show that isolated lack of BMP1-1 causes bone fragility in children.


Asunto(s)
Enfermedades Óseas/genética , Proteína Morfogenética Ósea 1/genética , Fracturas Óseas/genética , Regiones no Traducidas 3' , Enfermedades Óseas/metabolismo , Proteína Morfogenética Ósea 1/deficiencia , Niño , Preescolar , Colágeno Tipo I/metabolismo , Exones , Femenino , Fracturas Óseas/metabolismo , Humanos , Lactante , Masculino , Poliadenilación
7.
J Negat Results Biomed ; 16(1): 7, 2017 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-28412940

RESUMEN

BACKGROUND: In vitro studies suggest that the multiple functions of decorin are related to both its core protein and its dermatan sulfate chain. To determine the contribution of the dermatan sulfate chain to the functional properties of decorin in vivo, a mutant mouse whose decorin lacked a dermatan sulfate chain was generated. RESULTS: Homozygous mice expressing only the decorin core protein developed and grew in a similar manner to wild type mice. In both embryonic and postnatal mice, all connective tissues studied, including cartilage, skin and cornea, appeared to be normal upon histological examination, and their collagen fibrils were of normal diameter and organization. In addition, abdominal skin wounds healed in an identical manner in the mutant and wild type mice. CONCLUSIONS: The absence of a dermatan sulfate chain on decorin does not appear to overtly influence its functional properties in vivo.


Asunto(s)
Decorina/metabolismo , Dermatán Sulfato/metabolismo , Desarrollo Embrionario , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Secuencia de Bases , Cartílago/patología , Cartílago/ultraestructura , Decorina/química , Decorina/genética , Técnicas de Sustitución del Gen , Homocigoto , Ratones Endogámicos C57BL , Cicatrización de Heridas
8.
Am J Hum Genet ; 92(2): 252-8, 2013 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-23290074

RESUMEN

Metaphyseal dysplasia with maxillary hypoplasia and brachydactyly (MDMHB) is an autosomal-dominant bone dysplasia characterized by metaphyseal flaring of long bones, enlargement of the medial halves of the clavicles, maxillary hypoplasia, variable brachydactyly, and dystrophic teeth. We performed genome-wide SNP genotyping in five affected and four unaffected members of an extended family with MDMHB. Analysis for copy-number variations revealed that a 105 kb duplication within RUNX2 segregated with the MDMHB phenotype in a region with maximum linkage. Real-time PCR for copy-number variation in genomic DNA in eight samples, as well as sequence analysis of fibroblast cDNA from one subject with MDMHB confirmed that affected family members were heterozygous for the presence of an intragenic duplication encompassing exons 3 to 5 of RUNX2. These three exons code for the Q/A domain and the functionally essential DNA-binding runt domain of RUNX2. Transfection studies with murine Runx2 cDNA showed that cellular levels of mutated RUNX2 were markedly higher than those of wild-type RUNX2, suggesting that the RUNX2 duplication found in individuals with MDMHB leads to a gain of function. Until now, only loss-of-function mutations have been detected in RUNX2; the present report associates an apparent gain-of-function alteration of RUNX2 function with a distinct rare disease.


Asunto(s)
Braquidactilia/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Duplicación de Gen/genética , Osteocondrodisplasias/genética , Adolescente , Braquidactilia/diagnóstico por imagen , Cromosomas Humanos Par 6/genética , Exones/genética , Facies , Familia , Femenino , Dedos/anomalías , Dedos/diagnóstico por imagen , Genoma Humano/genética , Humanos , Masculino , Maxilar/anomalías , Maxilar/diagnóstico por imagen , Osteocondrodisplasias/diagnóstico por imagen , Linaje , Radiografía , Adulto Joven
9.
Calcif Tissue Int ; 98(6): 566-72, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26815784

RESUMEN

Osteogenesis imperfecta (OI) type VI is a recessively inherited form of OI that is caused by mutations in SERPINF1, the gene coding for pigment-epithelium derived factor (PEDF). Here, we report on two apparently unrelated children with OI type VI who had the same unusual homozygous variant in intron 6 of SERPINF1 (c.787-10C>G). This variant created a novel splice site that led to the in-frame addition of three amino acids to PEDF (p.Lys262_Ile263insLeuSerGln). Western blotting showed that skin fibroblasts with this mutation produced PEDF but failed to secrete it. Both children were treated with intravenous bisphosphonates, but the treatment of Individual 1 was switched to subcutaneous injections of denosumab (dose 1 mg per kg body weight, repeated every 3 months). An iliac bone sample obtained after 5 denosumab injections (and 3 months after the last injection) showed no change in the increased osteoid parameters that are typical of OI type VI, but the number of osteoclasts in trabecular bone was markedly increased. This suggests that the effect of denosumab on osteoclast suppression is of shorter duration in children with OI type VI than what has previously been reported on adults with osteoporosis.


Asunto(s)
Conservadores de la Densidad Ósea/uso terapéutico , Proteínas del Ojo/genética , Factores de Crecimiento Nervioso/genética , Osteogénesis Imperfecta/tratamiento farmacológico , Osteogénesis Imperfecta/genética , Serpinas/genética , Adolescente , Western Blotting , Canadá , Niño , Preescolar , Denosumab/uso terapéutico , Femenino , Humanos , Lactante , Masculino , Mutación
10.
Calcif Tissue Int ; 98(1): 76-84, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26478226

RESUMEN

Osteogenesis imperfecta (OI) type I is usually caused by COL1A1 stop or frameshift mutations, leading to COL1A1 haploinsufficiency. Here we report on 12 individuals from 5 families who had OI type I due to an unusual cause­heterozygous deletions of the entire COL1A1 gene. The deletions were initially detected by semiconductor-based sequencing of genomic DNA and confirmed by quantitative PCR. Array comparative genomic hybridization in DNA of the index patient in each family showed that deletion size varied from 18.5 kb to 2.23 Mb between families, encompassing between 1 and 47 genes (COL1A1 included). The skeletal phenotype of the affected individuals was similar to that of patients with haploinsufficiency caused by COL1A1 stop or frameshift mutations. However, one individual with a deletion that included also DLX3 and DLX4 had tooth discoloration and bone fragility. Three individuals from 2 families had deletions that included also CACNA1G, and these individuals had learning disabilities. These features are not usually observed in COL1A1 haploinsufficiency, but are in accordance with previously described individuals in whom deletions included the same genes. In summary, we found deletions of COL1A1 in 5 out of 161 families (3 %) with OI type I that were evaluated. Deletions encompassing not only COL1A1 but also neighboring genes can lead to contiguous gene syndromes that may include dental involvement and learning disability.


Asunto(s)
Colágeno Tipo I/genética , Eliminación de Gen , Osteogénesis Imperfecta/genética , Adolescente , Adulto , Niño , Preescolar , Cadena alfa 1 del Colágeno Tipo I , Hibridación Genómica Comparativa , Familia , Femenino , Humanos , Lactante , Masculino , Osteogénesis Imperfecta/epidemiología , Linaje , Polimorfismo Genético , Adulto Joven
11.
J Biol Chem ; 288(19): 13278-94, 2013 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-23530031

RESUMEN

BACKGROUND: BRIL is a bone-specific membrane protein that is involved in osteogenesis imperfecta type V. RESULTS: Bril transcription is activated by Sp1, Sp3, OSX, and GLI2 and by CpG demethylation. CONCLUSION: Regulation of Bril involves trans-acting factors integrating at conserved promoter elements and epigenetic modifications. SIGNIFICANCE: Identification of the mechanisms governing Bril transcription is important to understand its role in skeletal biology. Bril encodes a small membrane protein present in osteoblasts. In humans, a single recurrent mutation in the 5'-UTR of BRIL causes osteogenesis imperfecta type V. The exact function of BRIL and the mechanism by which it contributes to disease are still unknown. The goal of the current study was to characterize the mechanisms governing Bril transcription in humans, rats, and mice. In the three species, as detected by luciferase reporter assays in UMR106 cells, we found that most of the base-line regulatory activity was localized within ∼250 bp upstream of the coding ATG. Co-transfection experiments indicated that Sp1 and Sp3 were potent inducers of the promoter activity, through the binding of several GC-rich boxes. Osterix was a weak activator but acted cooperatively with Sp1 and GLI2 to synergistically induce the BRIL promoter. GLI2, a mediator of hedgehog signaling pathway, was also a potent activator of BRIL through a single GLI binding site. Correspondingly, agonists of the hedgehog pathway (purmorphamine and Indian hedgehog) in MC3T3 osteoblasts led to increased BRIL levels. The BRIL promoter activity was also found to be negatively modulated through two different mechanisms. First, the ZFP354C zinc finger protein repressed basal and Sp1-induced activity. Second, CpG methylation of the promoter region correlated with an inactive state and prevented Sp1 activation. The data provide the very first analyses of the cis- and trans-acting factors regulating Bril transcription. They revealed key roles for the Sp members and GLI2 that possibly cooperate to activate Bril when the promoter becomes demethylated.


Asunto(s)
Metilación de ADN , Silenciador del Gen , Proteínas de la Membrana/genética , Transcripción Genética , Activación Transcripcional , Células 3T3 , Animales , Secuencia de Bases , Huesos/metabolismo , Diferenciación Celular , Secuencia Conservada , Islas de CpG , Femenino , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/fisiología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas , Unión Proteica , Ratas , Proteínas Represoras/metabolismo , Transducción de Señal , Factor de Transcripción Sp1/fisiología , TATA Box , Proteína Gli2 con Dedos de Zinc
12.
J Cell Biochem ; 115(12): 2089-102, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25043819

RESUMEN

Phosphate is critical for mineralization and deficiencies in the regulation of free phosphate lead to disease. Inorganic polyphosphates (polyPs) may represent a physiological source of phosphate because they can be hydrolyzed by biological phosphatases. To investigate whether exogenous polyP could be utilized for mineral formation, mineralization was evaluated in two osteogenic cell lines, Saos-2 and MC3T3, expressing different levels of tissue non-specific alkaline phosphatase (tnALP). The role of tnALP was further explored by lentiviral-mediated overexpression in MC3T3 cells. When cells were cultured in the presence of three different phosphate sources, there was a strong mineralization response with ß-glycerophosphate (ßGP) and orthophosphate (Pi) but none of the cultures sustained mineralization in the presence of polyP (neither chain length 17-Pi nor 42-Pi). Even in the presence of mineralizing levels of phosphate, low concentrations of polyP (50 µM) were sufficient to inhibit mineral formation. Energy-dispersive X-ray spectroscopy confirmed the presence of apatite-like mineral deposits in MC3T3 cultures supplemented with ßGP, but not in those with polyP. While von Kossa staining was consistent with the presence or absence of mineral, an unusual Alizarin staining was obtained in polyP-treated MC3T3 cultures. This staining pattern combined with low Ca:P ratios suggests the persistence of Ca-polyP complexes, even with high residual ALP activity. In conclusion, under standard culture conditions, exogenous polyP does not promote mineral deposition. This is not due to a lack of active ALP, and unless conditions that favor significant processing of polyP are achieved, its mineral inhibitory capacity predominates.


Asunto(s)
Osteoblastos/fisiología , Polifosfatos/metabolismo , Fosfatasa Alcalina , Animales , Calcificación Fisiológica , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Medios de Cultivo , Humanos , Ratones , Osteogénesis , Medicina Regenerativa
13.
Cell Tissue Res ; 358(3): 843-55, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25193156

RESUMEN

Functional genomic screening of the rat enamel organ (EO) has led to the identification of a number of secreted proteins expressed during the maturation stage of amelogenesis, including amelotin (AMTN) and odontogenic ameloblast-associated (ODAM). In this study, we characterise the gene, protein and pattern of expression of a related protein called secretory calcium-binding phosphoprotein-proline-glutamine-rich 1 (SCPPPQ1). The Scpppq1 gene resides within the secretory calcium-binding phosphoprotein (Scpp) cluster. SCPPPQ1 is a highly conserved, 75-residue, secreted protein rich in proline, leucine, glutamine and phenylalanine. In silico data mining has revealed no correlation to any known sequences. Northern blotting of various rat tissues suggests that the expression of Scpppq1 is restricted to tooth and associated tissues. Immunohistochemical analyses show that the protein is expressed during the late maturation stage of amelogenesis and in the junctional epithelium where it localises to an atypical basal lamina at the cell-tooth interface. This discrete localisation suggests that SCPPPQ1, together with AMTN and ODAM, participates in structuring the basal lamina and in mediating attachment of epithelia cells to mineralised tooth surfaces.


Asunto(s)
Membrana Basal/metabolismo , Proteínas de Unión al Calcio/metabolismo , Fosfoproteínas/metabolismo , Diente/citología , Diente/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/metabolismo , Secuencia de Bases , Northern Blotting , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Línea Celular Tumoral , Clonación Molecular , ADN Complementario/genética , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Células HEK293 , Histidina , Humanos , Ratones , Diente Molar/citología , Diente Molar/crecimiento & desarrollo , Diente Molar/metabolismo , Datos de Secuencia Molecular , Oligopéptidos , Fosfoproteínas/química , Fosfoproteínas/genética , Ratas , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Diente/crecimiento & desarrollo , Diente/ultraestructura , Transfección
14.
Calcif Tissue Int ; 95(4): 323-31, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25086671

RESUMEN

Metabolic bone disorders in children frequently are heritable, but the expanding number of genes associated with these conditions makes it difficult to perform molecular diagnosis. In the present study, we therefore evaluated a semiconductor (SC)-based sequencing system for this purpose. A total of 65 DNA samples were analyzed comprising 24 samples from patients with 27 known pathogenic mutations, 6 samples from patients with prior negative Sanger sequencing, and 35 consecutive samples from patients with suspected heritable metabolic bone disorders who had not had prior molecular diagnosis. In the samples with known pathogenic mutations, 26 of 27 mutations were identified by SC sequencing. All single nucleotide variants were correctly identified, but a 7-nucleotide duplication in CYP27B1 was not detected. SC sequencing revealed two pathogenic mutations in the six samples where prior Sanger sequencing had failed to identify a mutation. Finally, pathogenic mutations were found in 27 samples of patients with unknown mutation status (15 in COL1A1, 9 in COL1A2, 1 in LEPRE1, 1 in LRP5, 1 in PHEX). Subsequent Sanger sequencing confirmed the mutations in all 27 samples. In conclusion, we found that SC sequencing is suitable for the diagnosis of heritable metabolic bone disorders in children.


Asunto(s)
Huesos/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Niño , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , ADN/genética , Predisposición Genética a la Enfermedad , Humanos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Glicoproteínas de Membrana/genética , Mutación , Endopeptidasa Neutra Reguladora de Fosfato PHEX/genética , Pediatría , Prolil Hidroxilasas , Proteoglicanos/genética , Semiconductores , Análisis de Secuencia de ADN/métodos
15.
J Med Genet ; 50(5): 345-8, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23434763

RESUMEN

BACKGROUND: Osteogenesis imperfecta (OI) is a heritable bone fragility disorder that is usually due to dominant mutations in COL1A1 or COL1A2. Rare recessive forms of OI, caused by mutations in genes involved in various aspects of bone formation, have been described as well. OBJECTIVE: To identify the cause of OI in eight children with severe bone fragility and a clinical diagnosis of OI type IV who had had negative results on COL1A1/COL1A2 Sanger sequencing. METHODS: Whole exome sequencing was performed in genomic DNA samples from all eight individuals. RESULTS: WNT1 mutations were found in four children from three families. WNT1 was the only gene where mutations were found in all of these four patients. Two siblings from a consanguineous family had a homozygous missense mutation affecting a highly conserved cysteine residue in WNT1 (c.428G>T (p.Cys143Phe)). One girl had a homozygous frameshift deletion (c.287_300del(p.Gln96Profs)). A girl from a third family was compound heterozygous for a frameshift insertion and a missense mutation affecting a conserved amino acid (c.946_949insAACA (p.Ser317Lysfs); c.1063G>T (p.Val355Phe)). All of these children had short stature, low bone density, and severe vertebral compression fractures in addition to multiple long bone fractures in the first years of life. The Wnt signalling pathway is one of the key regulators of osteoblast activity. CONCLUSIONS: Recessive inactivating mutations in WNT1 are a new cause of OI type IV.


Asunto(s)
Huesos/diagnóstico por imagen , Modelos Moleculares , Osteogénesis Imperfecta/genética , Conformación Proteica , Proteína Wnt1/genética , Secuencia de Aminoácidos , Secuencia de Bases , Femenino , Mutación del Sistema de Lectura/genética , Humanos , Datos de Secuencia Molecular , Mutación Missense/genética , Linaje , Radiografía , Análisis de Secuencia de ADN , Proteína Wnt1/química
16.
J Med Genet ; 50(1): 21-4, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23240094

RESUMEN

BACKGROUND: Osteogenesis imperfecta (OI) type V is an autosomal dominant bone fragility disorder that we had described a decade ago. Recent research has shown that OI type V is caused by a recurrent c.-14C>T mutation in IFITM5. In the present study, we assessed all patients diagnosed with OI type V at our institutions for the presence of the IFITM5 mutation. METHODS: IFITM5 exon 1 was analysed by Sanger sequencing in genomic DNA from 42 patients with OI type V (age: 2-67 years; 18 female). RESULTS: The c.-14C>T mutation of IFITM5 was detected in all individuals. Indicators of disease severity varied widely: Height z-scores (n=38) ranged from -8.7 to -0.1, median -3.5. Median final height was 147 cm in men (N=15) and 145 cm in women (N=10). Lumbar spine areal bone mineral density z-scores in the absence of bisphosphonate treatment (n=29) were between -7.7 and -0.7, median -5.3. Scoliosis was present in 57%, vertebral compression fractures in 90% of patients. CONCLUSIONS: Even though the disease-causing mutation is identical among patients with OI type V, the interindividual phenotypic variability is considerable.


Asunto(s)
Proteínas de la Membrana/genética , Mutación , Osteogénesis Imperfecta/genética , Fenotipo , Adolescente , Adulto , Anciano , Huesos/diagnóstico por imagen , Huesos/patología , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteogénesis Imperfecta/diagnóstico , Radiografía , Adulto Joven
17.
Periodontol 2000 ; 63(1): 59-66, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23931054

RESUMEN

Two novel proteins - odontogenic ameloblast-associated protein and amelotin - have recently been identified in maturation-stage ameloblasts and in the junctional epithelium. This article reviews the structure and function of the junctional epithelium, the pattern of expression of odontogenic ameloblast-associated and amelotin proteins and the potential involvement of these proteins in the formation and regeneration of the junctional epithelium.


Asunto(s)
Proteínas Portadoras/fisiología , Proteínas del Esmalte Dental/fisiología , Inserción Epitelial/anatomía & histología , Amiloide , Membrana Basal/anatomía & histología , Membrana Basal/fisiología , Inserción Epitelial/fisiología , Proteínas de la Matriz Extracelular/fisiología , Regulación de la Expresión Génica , Hemidesmosomas/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Neoplasias , Ligamento Periodontal/anatomía & histología , Ligamento Periodontal/fisiología , Regeneración/fisiología
18.
Clin Oral Investig ; 17(1): 131-7, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22373776

RESUMEN

OBJECTIVES: Clinicians occasionally face the challenge of moving a tooth through the maxillary sinus. The objective of this study was to evaluate tissue remodeling during tooth movement into the maxillary sinus, more specifically as regards to bone formation. MATERIALS AND METHODS: The maxillary first molar of 20 male mice was moved toward the palatal side by a nickel-titanium super elastic wire for 1 to 14 days, and the bone remodeling around the root was evaluated using histomorphometry and immunodetection of bone-restricted Ifitm-like (Bril) protein, a novel marker of active bone formation. RESULTS: When mechanical stress was applied to the tooth, the periodontal ligament on the palatal side was immediately compressed to approximately half of its original width by the tipping movement of the tooth. At the same time, osteoblasts deposited new bone on the wall of the maxillary sinus prior to bone resorption by osteoclasts on the periodontal side, as evidenced by the high level of expression of Bril at this site. As a result of these sequential processes, bone on the sinus side maintained a consistent thickness during the entire observation period. No root resorption was observed. CONCLUSIONS: Bone formation on the surface of the maxillary sinus was evoked by mechanotransduction of mechanical stress applied to a tooth over a 2-week period, and was induced ahead of bone resorption on the periodontal ligament side. CLINICAL RELEVANCE: Mechanical stress can be exploited to induce bone formation in the maxillary sinus so that teeth can be moved into the sinus without losing bone or causing root damage.


Asunto(s)
Seno Maxilar/anatomía & histología , Osteogénesis/fisiología , Técnicas de Movimiento Dental/métodos , Proceso Alveolar/anatomía & histología , Animales , Biomarcadores/análisis , Fenómenos Biomecánicos , Remodelación Ósea/fisiología , Resorción Ósea/fisiopatología , Aleaciones Dentales/química , Cemento Dental/anatomía & histología , Masculino , Mecanotransducción Celular/fisiología , Proteínas de la Membrana/análisis , Ratones , Modelos Animales , Diente Molar/anatomía & histología , Níquel/química , Osteoblastos/fisiología , Osteoclastos/fisiología , Hueso Paladar/anatomía & histología , Ligamento Periodontal/anatomía & histología , Estrés Mecánico , Factores de Tiempo , Titanio/química , Raíz del Diente/anatomía & histología
19.
J Bone Miner Res ; 38(8): 1125-1134, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37293821

RESUMEN

DNA sequencing is a reliable tool for identifying genetic variants in osteogenesis imperfecta (OI) but cannot always establish pathogenicity, particularly in variants altering splicing. RNA sequencing can provide functional evidence of the effect of a variant on the transcript but requires cells expressing the relevant genes. Here, we used urine-derived cells (UDC) to characterize genetic variants in patients with suspected or confirmed OI and provide evidence on the pathogenicity of variants of uncertain significance (VUS). Urine samples were obtained from 45 children and adolescents; UDC culture was successful in 40 of these participants (age range 4-20 years, 21 females), including 18 participants with OI or suspected OI who had a candidate variant or VUS on DNA sequencing. RNA was extracted from UDC and sequenced on an Illumina NextSeq550 device. Principal component analysis showed that the gene expression profiles of UDC and fibroblasts (based on Genotype Tissue Expression [GTEx] Consortium data) clustered close together and had less variability than those of whole blood cells. Transcript abundance was sufficient for analysis by RNA sequencing (defined as a median gene expression level of ≥10 transcripts per million) for 25 of the 32 bone fragility genes (78%) that were included in our diagnostic DNA sequencing panel. These results were similar to GTEx data for fibroblasts. Abnormal splicing was identified in 7 of the 8 participants with pathogenic or likely pathogenic variants in the splice region or deeper within the intron. Abnormal splicing was also observed in 2 VUS (COL1A1 c.2829+5G>A and COL1A2 c.693+6T>G), but no splice abnormality was observed in 3 other VUS. Abnormal deletions and duplications could also be observed in UDC transcripts. In conclusion, UDC are suitable for RNA transcript analysis in patients with suspected OI and can provide functional evidence for pathogenicity, in particular of variants affecting splicing. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).


Asunto(s)
Osteogénesis Imperfecta , Niño , Femenino , Adolescente , Humanos , Preescolar , Adulto Joven , Adulto , Osteogénesis Imperfecta/diagnóstico , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/patología , Cadena alfa 1 del Colágeno Tipo I , Mutación , Colágeno Tipo I/genética , Análisis de Secuencia de ARN
20.
J Cell Physiol ; 227(4): 1776-85, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21732355

RESUMEN

Transcellular bicarbonate transport is suspected to be an important pathway used by ameloblasts to regulate extracellular pH and support crystal growth during enamel maturation. Proteins that play a role in amelogenesis include members of the ABC transporters (SLC gene family and CFTR). A number of carbonic anhydrases (CAs) have also been identified. The defined functions of these genes are likely interlinked during enamel mineralization. The purpose of this study is to quantify relative mRNA levels of individual SLC, Cftr, and CAs in enamel cells obtained from secretory and maturation stages on rat incisors. We also present novel data on the enamel phenotypes for two animal models, a mutant porcine (CFTR-ΔF508) and the NBCe1-null mouse. Our data show that two SLCs (AE2 and NBCe1), Cftr, and Car2, Car3, Car6, and Car12 are all significantly up-regulated at the onset of the maturation stage of amelogenesis when compared to the secretory stage. The remaining SLCs and CA gene transcripts showed negligible expression or no significant change in expression from secretory to maturation stages. The enamel of CFTR-ΔF508 adult pigs was hypomineralized and showed abnormal crystal growth. NBCe1-null mice enamel was structurally defective and had a marked decrease in mineral content relative to wild-type. These data demonstrate the importance of many non-matrix proteins to amelogenesis and that the expression levels of multiple genes regulating extracellular pH are modulated during enamel maturation in response to an increased need for pH buffering during hydroxyapatite crystal growth.


Asunto(s)
Esmalte Dental/crecimiento & desarrollo , Esmalte Dental/metabolismo , Amelogénesis/genética , Amelogénesis/fisiología , Animales , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Antiportadores/genética , Antiportadores/metabolismo , Secuencia de Bases , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Cartilla de ADN/genética , Esmalte Dental/anomalías , Concentración de Iones de Hidrógeno , Transporte Iónico , Masculino , Ratones , Ratones Noqueados , Microscopía Electrónica de Rastreo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Proteínas SLC4A , Simportadores de Sodio-Bicarbonato/deficiencia , Simportadores de Sodio-Bicarbonato/genética , Simportadores de Sodio-Bicarbonato/metabolismo , Intercambiador de Sodio-Calcio/genética , Sus scrofa
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