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1.
Proc Natl Acad Sci U S A ; 119(31): e2116974119, 2022 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-35881792

RESUMEN

Ribosomal RNA (rRNA) transcription by RNA polymerase I (Pol I) is a critical rate-limiting step in ribosome biogenesis, which is essential for cell survival. Despite its global function, disruptions in ribosome biogenesis cause tissue-specific birth defects called ribosomopathies, which frequently affect craniofacial development. Here, we describe a cellular and molecular mechanism underlying the susceptibility of craniofacial development to disruptions in Pol I transcription. We show that Pol I subunits are highly expressed in the neuroepithelium and neural crest cells (NCCs), which generate most of the craniofacial skeleton. High expression of Pol I subunits sustains elevated rRNA transcription in NCC progenitors, which supports their high tissue-specific levels of protein translation, but also makes NCCs particularly sensitive to rRNA synthesis defects. Consistent with this model, NCC-specific deletion of Pol I subunits Polr1a, Polr1c, and associated factor Tcof1 in mice cell-autonomously diminishes rRNA synthesis, which leads to p53 protein accumulation, resulting in NCC apoptosis and craniofacial anomalies. Furthermore, compound mutations in Pol I subunits and associated factors specifically exacerbate the craniofacial anomalies characteristic of the ribosomopathies Treacher Collins syndrome and Acrofacial Dysostosis-Cincinnati type. Mechanistically, we demonstrate that diminished rRNA synthesis causes an imbalance between rRNA and ribosomal proteins. This leads to increased binding of ribosomal proteins Rpl5 and Rpl11 to Mdm2 and concomitantly diminished binding between Mdm2 and p53. Altogether, our results demonstrate a dynamic spatiotemporal requirement for rRNA transcription during mammalian cranial NCC development and corresponding tissue-specific threshold sensitivities to disruptions in rRNA transcription in the pathogenesis of congenital craniofacial disorders.


Asunto(s)
Anomalías Craneofaciales , ARN Polimerasa I , ARN Ribosómico , Proteínas Ribosómicas , Cráneo , Transcripción Genética , Animales , Anomalías Craneofaciales/genética , Disostosis Mandibulofacial/genética , Ratones , Cresta Neural/embriología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Polimerasa I/metabolismo , ARN Ribosómico/genética , Proteínas Ribosómicas/metabolismo , Cráneo/embriología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
2.
Oral Dis ; 28(5): 1306-1326, 2022 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-35226783

RESUMEN

Clefts of the lip and palate (CLP), the major causes of congenital facial malformation globally, result from failure of fusion of the facial processes during embryogenesis. With a prevalence of 1 in 500-2500 live births, CLP causes major morbidity throughout life as a result of problems with facial appearance, feeding, speaking, obstructive apnoea, hearing and social adjustment and requires complex, multi-disciplinary care at considerable cost to healthcare systems worldwide. Long-term outcomes for affected individuals include increased mortality compared with their unaffected siblings. The frequent occurrence and major healthcare burden imposed by CLP highlight the importance of dissecting the molecular mechanisms driving facial development. Identification of the genetic mutations underlying syndromic forms of CLP, where CLP occurs in association with non-cleft clinical features, allied to developmental studies using appropriate animal models is central to our understanding of the molecular events underlying development of the lip and palate and, ultimately, how these are disturbed in CLP.


Asunto(s)
Labio Leporino , Fisura del Paladar , Labio Leporino/complicaciones , Labio Leporino/genética , Fisura del Paladar/complicaciones , Fisura del Paladar/genética , Desarrollo Embrionario/genética , Cara , Humanos
3.
PLoS Genet ; 15(7): e1008253, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31291240

RESUMEN

Endoreplication is a cell cycle variant that entails cell growth and periodic genome duplication without cell division, and results in large, polyploid cells. Cells switch from mitotic cycles to endoreplication cycles during development, and also in response to conditional stimuli during wound healing, regeneration, aging, and cancer. In this study, we use integrated approaches in Drosophila to determine how mitotic cycles are remodeled into endoreplication cycles, and how similar this remodeling is between induced and developmental endoreplicating cells (iECs and devECs). Our evidence suggests that Cyclin A / CDK directly activates the Myb-MuvB (MMB) complex to induce transcription of a battery of genes required for mitosis, and that repression of CDK activity dampens this MMB mitotic transcriptome to promote endoreplication in both iECs and devECs. iECs and devECs differed, however, in that devECs had reduced expression of E2F1-dependent genes that function in S phase, whereas repression of the MMB transcriptome in iECs was sufficient to induce endoreplication without a reduction in S phase gene expression. Among the MMB regulated genes, knockdown of AurB protein and other subunits of the chromosomal passenger complex (CPC) induced endoreplication, as did knockdown of CPC-regulated cytokinetic, but not kinetochore, proteins. Together, our results indicate that the status of a CycA-Myb-MuvB-AurB network determines the decision to commit to mitosis or switch to endoreplication in both iECs and devECs, and suggest that regulation of different steps of this network may explain the known diversity of polyploid cycle types in development and disease.


Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Endorreduplicación , Animales , Aurora Quinasa B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina A/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Femenino , Perfilación de la Expresión Génica , Mitosis , Poliploidía , Proteínas Proto-Oncogénicas c-myb/metabolismo
4.
Hum Mutat ; 42(8): 1066-1078, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34004033

RESUMEN

Genome-wide association studies (GWAS) have generated unprecedented insights into the genetic etiology of orofacial clefting (OFC). The moderate effect sizes of associated noncoding risk variants and limited access to disease-relevant tissue represent considerable challenges for biological interpretation of genetic findings. As rare variants with stronger effect sizes are likely to also contribute to OFC, an alternative approach to delineate pathogenic mechanisms is to identify private mutations and/or an increased burden of rare variants in associated regions. This report describes a framework for targeted resequencing at selected noncoding risk loci contributing to nonsyndromic cleft lip with/without cleft palate (nsCL/P), the most frequent OFC subtype. Based on GWAS data, we selected three risk loci and identified candidate regulatory regions (CRRs) through the integration of credible SNP information, epigenetic data from relevant cells/tissues, and conservation scores. The CRRs (total 57 kb) were resequenced in a multiethnic study population (1061 patients; 1591 controls), using single-molecule molecular inversion probe technology. Combining evidence from in silico variant annotation, pedigree- and burden analyses, we identified 16 likely deleterious rare variants that represent new candidates for functional studies in nsCL/P. Our framework is scalable and represents a promising approach to the investigation of additional congenital malformations with multifactorial etiology.


Asunto(s)
Labio Leporino , Fisura del Paladar , Labio Leporino/genética , Fisura del Paladar/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Polimorfismo de Nucleótido Simple
5.
Semin Cell Dev Biol ; 91: 75-83, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-28803895

RESUMEN

Development of the secondary palate involves a complex series of embryonic events which, if disrupted, result in the common congenital anomaly cleft palate. The secondary palate forms from paired palatal shelves which grow initially vertically before elevating to a horizontal position above the tongue and fusing together in the midline via the medial edge epithelia. As the epithelia of the vertical palatal shelves are in contact with the mandibular and lingual epithelia, pathological fusions between the palate and the mandible and/or the tongue must be prevented. This function is mediated by the single cell layered periderm which forms in a distinct and reproducible pattern early in embryogenesis, exhibits highly polarised expression of adhesion complexes, and is shed from the outer surface as the epidermis acquires its barrier function. Disruption of periderm formation and/or function underlies a series of birth defects that exhibit multiple inter-epithelial adhesions including the autosomal dominant popliteal pterygium syndrome and the autosomal recessive cocoon syndrome and Bartsocas Papas syndrome. Genetic analyses of these conditions have shown that IRF6, IKKA, SFN, RIPK4 and GRHL3, all of which are under the transcriptional control of p63, play a key role in periderm formation. Despite these observations, the medial edge epithelia must rapidly acquire the capability to fuse if the palatal shelves are not to remain cleft. This process is driven by TGFß3-mediated, down-regulation of p63 in the medial edge epithelia which allows periderm migration out of the midline epithelial seam and reduces the proliferative potential of the midline epithelial seam thereby preventing cleft palate. Together, these findings indicate that periderm plays a transient but fundamental role during embryogenesis in preventing pathological adhesion between intimately apposed, adhesion-competent epithelia.


Asunto(s)
Fisura del Paladar/embriología , Epidermis/embriología , Epitelio/embriología , Hueso Paladar/embriología , Animales , Diferenciación Celular/genética , Fisura del Paladar/genética , Epidermis/metabolismo , Epitelio/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Hueso Paladar/citología , Hueso Paladar/metabolismo , Transducción de Señal/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética
6.
PLoS Genet ; 13(6): e1006828, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28604778

RESUMEN

Cleft palate is a common congenital disorder that affects up to 1 in 2500 live births and results in considerable morbidity to affected individuals and their families. The aetiology of cleft palate is complex with both genetic and environmental factors implicated. Mutations in the transcription factor p63 are one of the major individual causes of cleft palate; however, the gene regulatory networks in which p63 functions remain only partially characterized. Our findings demonstrate that p63 functions as an essential regulatory molecule in the spatio-temporal control of palatal epithelial cell fate to ensure appropriate fusion of the palatal shelves. Initially, p63 induces periderm formation and controls its subsequent maintenance to prevent premature adhesion between adhesion-competent, intra-oral epithelia. Subsequently, TGFß3-induced down-regulation of p63 in the medial edge epithelia of the palatal shelves is a pre-requisite for palatal fusion by facilitating periderm migration from, and reducing the proliferative potential of, the midline epithelial seam thereby preventing cleft palate.


Asunto(s)
Fisura del Paladar/genética , Redes Reguladoras de Genes/genética , Fosfoproteínas/genética , Transactivadores/genética , Factor de Crecimiento Transformador beta3/genética , Animales , Movimiento Celular/genética , Proliferación Celular/genética , Fisura del Paladar/fisiopatología , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Mutación , Fosfoproteínas/biosíntesis , Transducción de Señal/genética , Transactivadores/biosíntesis
7.
Hum Mol Genet ; 26(10): 1863-1876, 2017 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-28334996

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
8.
Hum Mol Genet ; 26(4): 829-842, 2017 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-28087736

RESUMEN

Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is among the most common human birth defects with multifactorial etiology. Here, we present results from a genome-wide imputation study of nsCL/P in which, after adding replication cohort data, four novel risk loci for nsCL/P are identified (at chromosomal regions 2p21, 14q22, 15q24 and 19p13). On a systematic level, we show that the association signals within this high-density dataset are enriched in functionally-relevant genomic regions that are active in both human neural crest cells (hNCC) and mouse embryonic craniofacial tissue. This enrichment is also detectable in hNCC regions primed for later activity. Using GCTA analyses, we suggest that 30% of the estimated variance in risk for nsCL/P in the European population can be attributed to common variants, with 25.5% contributed to by the 24 risk loci known to date. For each of these, we identify credible SNPs using a Bayesian refinement approach, with two loci harbouring only one probable causal variant. Finally, we demonstrate that there is no polygenic component of nsCL/P detectable that is shared with nonsyndromic cleft palate only (nsCPO). Our data suggest that, while common variants are strongly contributing to risk for nsCL/P, they do not seem to be involved in nsCPO which might be more often caused by rare deleterious variants. Our study generates novel insights into both nsCL/P and nsCPO etiology and provides a systematic framework for research into craniofacial development and malformation.


Asunto(s)
Cromosomas Humanos/genética , Labio Leporino/genética , Fisura del Paladar/genética , Bases de Datos Genéticas , Sitios Genéticos , Estudio de Asociación del Genoma Completo , Polimorfismo de Nucleótido Simple , Animales , Labio Leporino/metabolismo , Labio Leporino/patología , Fisura del Paladar/metabolismo , Fisura del Paladar/patología , Femenino , Humanos , Masculino , Ratones
9.
Dev Biol ; 415(2): 296-305, 2016 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-26772999

RESUMEN

The palate functions as the roof of the mouth in mammals, separating the oral and nasal cavities. Its complex embryonic development and assembly poses unique susceptibilities to intrinsic and extrinsic disruptions. Such disruptions may cause failure of the developing palatal shelves to fuse along the midline resulting in a cleft. In other cases the palate may fuse at an arch, resulting in a vaulted oral cavity, termed high-arched palate. There are many models available for studying the pathogenesis of cleft palate but a relative paucity for high-arched palate. One condition exhibiting either cleft palate or high-arched palate is Treacher Collins syndrome, a congenital disorder characterized by numerous craniofacial anomalies. We quantitatively analyzed palatal perturbations in the Tcof1(+/-) mouse model of Treacher Collins syndrome, which phenocopies the condition in humans. We discovered that 46% of Tcof1(+/-) mutant embryos and new born pups exhibit either soft clefts or full clefts. In addition, 17% of Tcof1(+/-) mutants were found to exhibit high-arched palate, defined as two sigma above the corresponding wild-type population mean for height and angular based arch measurements. Furthermore, palatal shelf length and shelf width were decreased in all Tcof1(+/-) mutant embryos and pups compared to controls. Interestingly, these phenotypes were subsequently ameliorated through genetic inhibition of p53. The results of our study therefore provide a simple, reproducible and quantitative method for investigating models of high-arched palate.


Asunto(s)
Desarrollo Maxilofacial/fisiología , Proteínas Nucleares/genética , Hueso Paladar/anomalías , Fosfoproteínas/genética , Animales , Fisura del Paladar/diagnóstico por imagen , Fisura del Paladar/embriología , Fisura del Paladar/genética , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Femenino , Técnicas de Inactivación de Genes , Genes p53 , Heterocigoto , Humanos , Imagenología Tridimensional , Péptidos y Proteínas de Señalización Intracelular , Masculino , Disostosis Mandibulofacial/diagnóstico por imagen , Disostosis Mandibulofacial/embriología , Disostosis Mandibulofacial/genética , Desarrollo Maxilofacial/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Microscopía Confocal , Proteínas Nucleares/fisiología , Hueso Paladar/diagnóstico por imagen , Hueso Paladar/embriología , Fenotipo , Fosfoproteínas/fisiología , Especificidad de la Especie
10.
Nat Rev Genet ; 12(3): 167-78, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21331089

RESUMEN

Clefts of the lip and/or palate (CLP) are common birth defects of complex aetiology. CLP can occur in isolation or as part of a broad range of chromosomal, Mendelian or teratogenic syndromes. Although there has been marked progress in identifying genetic and environmental triggers for syndromic CLP, the aetiology of the more common non-syndromic (isolated) forms remains poorly characterized. Recently, using a combination of epidemiology, careful phenotyping, genome-wide association studies and analysis of animal models, several distinct genetic and environmental risk factors have been identified and confirmed for non-syndromic CLP. These findings have advanced our understanding of developmental biology and created new opportunities for clinical translational research.


Asunto(s)
Labio Leporino/genética , Fisura del Paladar/genética , Animales , Labio Leporino/epidemiología , Labio Leporino/etiología , Fisura del Paladar/epidemiología , Fisura del Paladar/etiología , Ambiente , Femenino , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Ratones , Mutación
11.
Hum Mol Genet ; 23(9): 2468-80, 2014 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-24362885

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
12.
Hum Mol Genet ; 23(10): 2569-79, 2014 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-24363063

RESUMEN

Heterozygous loss-of-function (LOF) mutations in the gene encoding the DNA-binding protein, SATB2, result in micrognathia and cleft palate in both humans and mice. In three unrelated individuals, we show that translocation breakpoints (BPs) up to 896 kb 3' of SATB2 polyadenylation site cause a phenotype which is indistinguishable from that caused by SATB2 LOF mutations. This syndrome comprises long nose, small mouth, micrognathia, cleft palate, arachnodactyly and intellectual disability. These BPs map to a gene desert between PLCL1 and SATB2. We identified three putative cis-regulatory elements (CRE1-3) using a comparative genomic approach each of which would be placed in trans relative to SATB2 by all three BPs. CRE1-3 each bind p300 and mono-methylated H3K4 consistent with enhancer function. In silico analysis suggested that CRE1-3 contain one or more conserved SOX9-binding sites, and this binding was confirmed using chromatin immunoprecipitation on cells derived from mouse embryonic pharyngeal arch. Interphase bacterial artificial chromosome fluorescence in situ hybridization measurements in embryonic craniofacial tissues showed that the orthologous region in mice exhibits Satb2 expression-dependent chromatin decondensation consistent with Satb2 being a target gene of CRE1-3. To assess their in vivo function, we made multiple stable reporter transgenic lines for each enhancer in zebrafish. CRE2 was shown to drive SATB2-like expression in the embryonic craniofacial region. This expression could be eliminated by mutating the SOX9-binding site of CRE2. These observations suggest that SATB2 and SOX9 may be acting together via complex cis-regulation to coordinate the growth of the developing jaw.


Asunto(s)
Proteínas de Unión a la Región de Fijación a la Matriz/genética , Síndrome de Pierre Robin/diagnóstico , Factor de Transcripción SOX9/genética , Factores de Transcripción/genética , Adulto , Animales , Sitios de Unión , Células Cultivadas , Niño , Preescolar , Epistasis Genética , Femenino , Humanos , Lactante , Masculino , Ratones , Mutación , Síndrome de Pierre Robin/genética , Elementos Reguladores de la Transcripción , Adulto Joven , Pez Cebra
13.
Development ; 140(6): 1220-30, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23406900

RESUMEN

Cleft palate is one of the most common human birth defects and is associated with multiple genetic and environmental risk factors. Although mutations in the genes encoding transforming growth factor beta (TGFß) signaling molecules and interferon regulatory factor 6 (Irf6) have been identified as genetic risk factors for cleft palate, little is known about the relationship between TGFß signaling and IRF6 activity during palate formation. Here, we show that TGFß signaling regulates expression of Irf6 and the fate of the medial edge epithelium (MEE) during palatal fusion in mice. Haploinsufficiency of Irf6 in mice with basal epithelial-specific deletion of the TGFß signaling mediator Smad4 (Smad4(fl/fl);K14-Cre;Irf6(+/R84C)) results in compromised p21 expression and MEE persistence, similar to observations in Tgfbr2(fl/fl);K14-Cre mice, although the secondary palate of Irf6(+/R84C) and Smad4(fl/fl);K14-Cre mice form normally. Furthermore, Smad4(fl/fl);K14-Cre;Irf6(+/R84C) mice show extra digits that are consistent with abnormal toe and nail phenotypes in individuals with Van der Woude and popliteal pterygium syndromes, suggesting that the TGFß/SMAD4/IRF6 signaling cascade might be a well-conserved mechanism in regulating multiple organogenesis. Strikingly, overexpression of Irf6 rescued p21 expression and MEE degeneration in Tgfbr2(fl/fl);K14-Cre mice. Thus, IRF6 and SMAD4 synergistically regulate the fate of the MEE, and TGFß-mediated Irf6 activity is responsible for MEE degeneration during palatal fusion in mice.


Asunto(s)
Epistasis Genética , Factores Reguladores del Interferón/genética , Hueso Paladar/embriología , Proteína Smad4/genética , Factor de Crecimiento Transformador beta/farmacología , Animales , Animales Recién Nacidos , Fusión Celular , Células Cultivadas , Embrión de Mamíferos , Epistasis Genética/efectos de los fármacos , Epistasis Genética/fisiología , Femenino , Humanos , Factores Reguladores del Interferón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Organogénesis/efectos de los fármacos , Organogénesis/genética , Hueso Paladar/efectos de los fármacos , Hueso Paladar/metabolismo , Embarazo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína Smad4/metabolismo , Factor de Crecimiento Transformador beta/metabolismo
14.
J Virol ; 89(1): 262-74, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25320325

RESUMEN

UNLABELLED: Skin keratinocytes represent a primary entry site for herpes simplex virus 1 (HSV-1) in vivo. The cellular proteins nectin-1 and herpesvirus entry mediator (HVEM) act as efficient receptors for both serotypes of HSV and are sufficient for disease development mediated by HSV-2 in mice. How HSV-1 enters skin and whether both nectin-1 and HVEM are involved are not known. We addressed the impact of nectin-1 during entry of HSV-1 into murine epidermis and investigated the putative contribution of HVEM. Using ex vivo infection of murine epidermis, we showed that HSV-1 entered the basal keratinocytes of the epidermis very efficiently. In nectin-1-deficient epidermis, entry was strongly reduced. Almost no entry was observed, however, in nectin-1-deficient keratinocytes grown in culture. This observation correlated with the presence of HVEM on the keratinocyte surface in epidermis and with the lack of HVEM expression in nectin-1-deficient primary keratinocytes. Our results suggest that nectin-1 is the primary receptor in epidermis, while HVEM has a more limited role. For primary murine keratinocytes, on which nectin-1 acts as a single receptor, electron microscopy suggested that HSV-1 can enter both by direct fusion with the plasma membrane and via endocytic vesicles. Thus, we concluded that nectin-1 directs internalization into keratinocytes via alternative pathways. In summary, HSV-1 entry into epidermis was shown to strongly depend on the presence of nectin-1, but the restricted presence of HVEM can potentially replace nectin-1 as a receptor, illustrating the flexibility employed by HSV-1 to efficiently invade tissue in vivo. IMPORTANCE: Herpes simplex virus (HSV) can cause a range of diseases in humans, from uncomplicated mucocutaneous lesions to life-threatening infections. The skin is one target tissue of HSV, and the question of how the virus overcomes the protective skin barrier and penetrates into the tissue to reach its receptors is still open. Previous studies analyzing entry into cells grown in vitro revealed nectin-1 and HVEM as HSV receptors. To explore the contributions of nectin-1 and HVEM to entry into a natural target tissue, we established an ex vivo infection model. Using nectin-1- or HVEM-deficient mice, we demonstrated the distinct involvement of nectin-1 and HVEM for HSV-1 entry into epidermis and characterized the internalization pathways. Such advances in understanding the involvement of receptors in tissue are essential preconditions for unraveling HSV invasion of skin, which in turn will allow the development of antiviral reagents.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno , Queratinocitos/virología , Miembro 14 de Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Virales/metabolismo , Internalización del Virus , Animales , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Nectinas , Piel/virología
15.
Genet Med ; 18(11): 1158-1162, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-26963285

RESUMEN

PURPOSE: We aimed to identify a novel genetic cause of tooth agenesis (TA) and/or orofacial clefting (OFC) by combining whole-exome sequencing (WES) and targeted resequencing in a large cohort of TA and OFC patients. METHODS: WES was performed in two unrelated patients: one with severe TA and OFC and another with severe TA only. After deleterious mutations were identified in a gene encoding low-density lipoprotein receptor-related protein 6 (LRP6), all its exons were resequenced with molecular inversion probes in 67 patients with TA, 1,072 patients with OFC, and 706 controls. RESULTS: We identified a frameshift (c.4594delG, p.Cys1532fs) and a canonical splice-site mutation (c.3398-2A>C, p.?) in LRP6, respectively, in the patient with TA and OFC and in the patient with severe TA only. The targeted resequencing showed significant enrichment of unique LRP6 variants in TA patients but not in nonsyndromic OFC patients. Of the five variants in patients with TA, two affected the canonical splice site and three were missense variants; all variants segregated with the dominant phenotype, and in one case the missense mutation occurred de novo. CONCLUSION: Mutations in LRP6 cause TA in humans.Genet Med 18 11, 1158-1162.


Asunto(s)
Anodoncia/genética , Exoma/genética , Predisposición Genética a la Enfermedad , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Adolescente , Anodoncia/patología , Niño , Femenino , Mutación del Sistema de Lectura/genética , Humanos , Masculino , Mutación Missense/genética , Linaje , Análisis de Secuencia de ADN , Vía de Señalización Wnt/genética
16.
EMBO J ; 30(22): 4571-85, 2011 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-21909072

RESUMEN

While the pro-differentiation and tumour suppressive functions of Notch signalling in keratinocytes are well established, the underlying mechanisms remain poorly understood. We report here that interferon regulatory factor 6 (IRF6), an IRF family member with an essential role in epidermal development, is induced in differentiation through a Notch-dependent mechanism and is a primary Notch target in keratinocytes and keratinocyte-derived SCC cells. Increased IRF6 expression contributes to the impact of Notch activation on growth/differentiation-related genes, while it is not required for induction of 'canonical' Notch targets like p21(WAF1/Cip1), Hes1 and Hey1. Down-modulation of IRF6 counteracts differentiation of primary human keratinocytes in vitro and in vivo, promoting ras-induced tumour formation. The clinical relevance of these findings is illustrated by the strikingly opposite pattern of expression of Notch1 and IRF6 versus epidermal growth factor receptor in a cohort of clinical SCCs, as a function of their grade of differentiation. Thus, IRF6 is a primary Notch target in keratinocytes, which contributes to the role of this pathway in differentiation and tumour suppression.


Asunto(s)
Factores Reguladores del Interferón/metabolismo , Queratinocitos/fisiología , Receptor Notch1/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Proteínas de Unión al ADN/metabolismo , Receptores ErbB/biosíntesis , Receptores ErbB/genética , Genes Supresores de Tumor , Proteínas de Homeodominio/metabolismo , Humanos , Factores Reguladores del Interferón/biosíntesis , Factores Reguladores del Interferón/genética , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteína Oncogénica p21(ras)/metabolismo , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño , Receptor Notch1/genética , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Factor de Transcripción HES-1
17.
Am J Hum Genet ; 90(1): 69-75, 2012 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-22197488

RESUMEN

Pterygium syndromes are complex congenital disorders that encompass several distinct clinical conditions characterized by multiple skin webs affecting the flexural surfaces often accompanied by craniofacial anomalies. In severe forms, such as in the autosomal-recessive Bartsocas-Papas syndrome, early lethality is common, complicating the identification of causative mutations. Using exome sequencing in a consanguineous family, we identified the homozygous mutation c.1127C>A in exon 7 of RIPK4 that resulted in the introduction of the nonsense mutation p.Ser376X into the encoded ankyrin repeat-containing kinase, a protein that is essential for keratinocyte differentiation. Subsequently, we identified a second mutation in exon 2 of RIPK4 (c.242T>A) that resulted in the missense variant p.Ile81Asn in the kinase domain of the protein. We have further demonstrated that RIPK4 is a direct transcriptional target of the protein p63, a master regulator of stratified epithelial development, which acts as a nodal point in the cascade of molecular events that prevent pterygium syndromes.


Asunto(s)
Labio Leporino/genética , Fisura del Paladar/genética , Exoma , Proteínas Serina-Treonina Quinasas/genética , Pterigion/congénito , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Niño , Labio Leporino/diagnóstico , Fisura del Paladar/diagnóstico , Consanguinidad , Anomalías Craneofaciales/genética , Exones , Genes Recesivos , Sitios Genéticos , Humanos , Queratinocitos/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Mutación , Fosfoproteínas/metabolismo , Pterigion/diagnóstico , Pterigion/genética , Índice de Severidad de la Enfermedad , Anomalías Cutáneas , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo
18.
Am J Hum Genet ; 91(3): 565-71, 2012 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-22901946

RESUMEN

Autozygosity mapping and clonal sequencing of an Omani family identified mutations in the uncharacterized gene, C4orf26, as a cause of recessive hypomineralized amelogenesis imperfecta (AI), a disease in which the formation of tooth enamel fails. Screening of a panel of 57 autosomal-recessive AI-affected families identified eight further families with loss-of-function mutations in C4orf26. C4orf26 encodes a putative extracellular matrix acidic phosphoprotein expressed in the enamel organ. A mineral nucleation assay showed that the protein's phosphorylated C terminus has the capacity to promote nucleation of hydroxyapatite, suggesting a possible function in enamel mineralization during amelogenesis.


Asunto(s)
Amelogénesis Imperfecta/genética , Proteínas del Tejido Nervioso/genética , Amelogénesis/genética , Esmalte Dental/metabolismo , Durapatita/metabolismo , Femenino , Humanos , Masculino , Mutación , Linaje
19.
Am J Med Genet A ; 167A(3): 545-52, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25691407

RESUMEN

The popliteal pterygia syndromes are a distinct subset of the hundreds of Mendelian orofacial clefting syndromes. Popliteal pterygia syndromes have considerable variability in severity and in the associated phenotypic features but are all characterized by cutaneous webbing across one or more major joints, cleft lip and/or palate, syndactyly, and genital malformations. Heterozygous mutations in IRF6 cause popliteal pterygium syndrome (PPS) while homozygous mutations in RIPK4 or CHUK (IKKA) cause the more severe Bartsocas-Papas syndrome (BPS) and Cocoon syndrome, respectively. In this study, we report mutations in six pedigrees with children affected with PPS or BPS. Using a combination of Sanger and exome sequencing, we report the first case of an autosomal recessive popliteal pterygium syndrome caused by homozygous mutation of IRF6 and the first case of uniparental disomy of chromosome 21 leading to a recessive disorder. We also demonstrate that mutations in RIPK4 can cause features with a range of severity along the PPS-BPS spectrum and that mutations in IKKA can cause a range of features along the BPS-Cocoon spectrum. Our findings have clinical implications for genetic counseling of families with pterygia syndromes and further implicate IRF6, RIPK4, and CHUK (IKKA) in potentially interconnected pathways governing epidermal and craniofacial development.


Asunto(s)
Labio Leporino/diagnóstico , Labio Leporino/genética , Fisura del Paladar/diagnóstico , Fisura del Paladar/genética , Anomalías del Ojo/diagnóstico , Anomalías del Ojo/genética , Dedos/anomalías , Estudios de Asociación Genética , Articulación de la Rodilla/anomalías , Deformidades Congénitas de las Extremidades Inferiores/diagnóstico , Deformidades Congénitas de las Extremidades Inferiores/genética , Fenotipo , Sindactilia/diagnóstico , Sindactilia/genética , Anomalías Urogenitales/diagnóstico , Anomalías Urogenitales/genética , Hibridación Genómica Comparativa , Análisis Mutacional de ADN , Exoma , Femenino , Genes Recesivos , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Quinasa I-kappa B/genética , Lactante , Recién Nacido , Factores Reguladores del Interferón/genética , Rodilla/anomalías , Masculino , Mutación , Linaje , Proteínas Serina-Treonina Quinasas/genética
20.
PLoS Genet ; 8(3): e1002566, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22479190

RESUMEN

The cerebral cortex is a specialized region of the brain that processes cognitive, motor, somatosensory, auditory, and visual functions. Its characteristic architecture and size is dependent upon the number of neurons generated during embryogenesis and has been postulated to be governed by symmetric versus asymmetric cell divisions, which mediate the balance between progenitor cell maintenance and neuron differentiation, respectively. The mechanistic importance of spindle orientation remains controversial, hence there is considerable interest in understanding how neural progenitor cell mitosis is controlled during neurogenesis. We discovered that Treacle, which is encoded by the Tcof1 gene, is a novel centrosome- and kinetochore-associated protein that is critical for spindle fidelity and mitotic progression. Tcof1/Treacle loss-of-function disrupts spindle orientation and cell cycle progression, which perturbs the maintenance, proliferation, and localization of neural progenitors during cortical neurogenesis. Consistent with this, Tcof1(+/-) mice exhibit reduced brain size as a consequence of defects in neural progenitor maintenance. We determined that Treacle elicits its effect via a direct interaction with Polo-like kinase1 (Plk1), and furthermore we discovered novel in vivo roles for Plk1 in governing mitotic progression and spindle orientation in the developing mammalian cortex. Increased asymmetric cell division, however, did not promote increased neuronal differentiation. Collectively our research has therefore identified Treacle and Plk1 as novel in vivo regulators of spindle fidelity, mitotic progression, and proliferation in the maintenance and localization of neural progenitor cells. Together, Treacle and Plk1 are critically required for proper cortical neurogenesis, which has important implications in the regulation of mammalian brain size and the pathogenesis of congenital neurodevelopmental disorders such as microcephaly.


Asunto(s)
Proteínas de Ciclo Celular , Corteza Cerebral/crecimiento & desarrollo , Mitosis/genética , Neurogénesis/genética , Neuronas/citología , Proteínas Nucleares , Fosfoproteínas , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas , Animales , Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Centrosoma/metabolismo , Corteza Cerebral/anomalías , Regulación del Desarrollo de la Expresión Génica , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Puntos de Control de la Fase M del Ciclo Celular/genética , Disostosis Mandibulofacial/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Mutantes , Neuronas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Células Madre/citología , Células Madre/metabolismo , Quinasa Tipo Polo 1
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