RESUMO
The FaceBase Consortium was established by the National Institute of Dental and Craniofacial Research in 2009 as a 'big data' resource for the craniofacial research community. Over the past decade, researchers have deposited hundreds of annotated and curated datasets on both normal and disordered craniofacial development in FaceBase, all freely available to the research community on the FaceBase Hub website. The Hub has developed numerous visualization and analysis tools designed to promote integration of multidisciplinary data while remaining dedicated to the FAIR principles of data management (findability, accessibility, interoperability and reusability) and providing a faceted search infrastructure for locating desired data efficiently. Summaries of the datasets generated by the FaceBase projects from 2014 to 2019 are provided here. FaceBase 3 now welcomes contributions of data on craniofacial and dental development in humans, model organisms and cell lines. Collectively, the FaceBase Consortium, along with other NIH-supported data resources, provide a continuously growing, dynamic and current resource for the scientific community while improving data reproducibility and fulfilling data sharing requirements.
Assuntos
Pesquisa em Odontologia/métodos , Ossos Faciais/fisiologia , Crânio/fisiologia , Animais , Bases de Dados Factuais , Humanos , Reprodutibilidade dos Testes , PesquisadoresRESUMO
Zellweger spectrum disorder (ZSD) results from biallelic mutations in PEX genes required for peroxisome biogenesis. PEX1-G843D is a common hypomorphic allele in the patient population that is associated with milder disease. In prior work using a PEX1-G843D/null patient fibroblast line expressing a green fluorescent protein (GFP) reporter with a peroxisome-targeting signal (GFP-PTS1), we demonstrated that treatments with the chemical chaperone betaine and flavonoid acacetin diacetate recovered peroxisome functions. To identify more effective compounds for preclinical investigation, we evaluated 54 flavonoids using this cell-based phenotype assay. Diosmetin showed the most promising combination of potency and efficacy (EC50 2.5 µM). All active 5',7'-dihydroxyflavones showed greater average efficacy than their corresponding flavonols, whereas the corresponding flavanones, isoflavones, and chalcones tested were inactive. Additional treatment with the proteostasis regulator bortezomib increased the percentage of import-rescued cells over treatment with flavonoids alone. Cotreatments of diosmetin and betaine showed the most robust additive effects, as confirmed by three independent functional assays in primary PEX1-G843D patient cells, but neither agent was active alone or in combination in patient cells homozygous for the PEX1 c.2097_2098insT null allele. Moreover, diosmetin treatment increased PEX1, PEX6, and PEX5 protein levels in PEX1-G843D patient cells, but none of these proteins increased in PEX1 null cells. We propose that diosmetin acts as a pharmacological chaperone that improves the stability, conformation, and functions of PEX1/PEX6 exportomer complexes required for peroxisome assembly. We suggest that diosmetin, in clinical use for chronic venous disease, and related flavonoids warrant further preclinical investigation for the treatment of PEX1-G843D-associated ZSD.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Alelos , Fibroblastos/metabolismo , Flavonoides/farmacologia , Proteínas de Membrana/genética , Peroxissomos/efeitos dos fármacos , Síndrome de Zellweger/patologia , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Trifosfato de Adenosina/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Quimioterapia Combinada , Flavonoides/uso terapêutico , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/farmacologia , Chaperonas Moleculares/uso terapêutico , Sinais de Orientação para Peroxissomos , Peroxissomos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Síndrome de Zellweger/tratamento farmacológicoRESUMO
BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is a genetic disorder leading to the accumulation of very long chain fatty acids (VLCFA) due to a mutation in the ABCD1 gene. ABCD1 mutations lead to a variety of phenotypes, including cerebral X-ALD and adrenomyeloneuropathy (AMN) in affected males and 80% of carrier females. There is no definite genotype-phenotype correlation with intrafamilial variability. Cerebral X-ALD typically presents in childhood, but can also present in juveniles and adults. The most affected tissues are the white matter of the brain and adrenal cortex. MRI demonstrates a characteristic imaging appearance in cerebral X-ALD that is used as a diagnostic tool. OBJECTIVES: We aim to correlate a mutation in the ABCD1 gene in a chimpanzee to the human disease X-ALD based on MRI features, neurologic symptoms, and plasma levels of VLCFA. METHODS: Diagnosis of X-ALD made using MRI, blood lipid profiling, and DNA sequencing. RESULTS: An 11-year-old chimpanzee showed remarkably similar features to juvenile onset cerebral X-ALD in humans including demyelination of frontal lobes and corpus callosum on MRI, elevated plasma levels of C24:0 and C26:0, and identification of the c.1661G>A ABCD1 variant. CONCLUSIONS: This case study presents the first reported case of a leukodystrophy in a great ape, and underscores the fidelity of MRI pattern recognition in this disorder across species.
Assuntos
Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/genética , Adrenoleucodistrofia/genética , Encéfalo/fisiopatologia , Pan troglodytes/genética , Adrenoleucodistrofia/diagnóstico por imagem , Adulto , Idade de Início , Animais , Encéfalo/anatomia & histologia , Encéfalo/diagnóstico por imagem , Coenzima A Ligases/sangue , Doenças Desmielinizantes , Feminino , Lobo Frontal/patologia , Estudos de Associação Genética , Humanos , Lipídeos/sangue , Imageamento por Ressonância Magnética , Masculino , Mutação , Fenótipo , Análise de Sequência de DNA/métodosRESUMO
BACKGROUND: Current clinical tools have limited accuracy in differentiating patients with localized prostate cancer who are at risk of recurrence from patients with indolent disease. We aimed to identify a gene expression signature that jointly with clinical variables could improve upon the prediction of clinical recurrence after RP for patients with stage T2 PCa. METHODS: The study population includes consented patients who underwent a radical retropubic prostatectomy (RP) and bilateral pelvic lymph node dissection at the University of Southern California in the PSA-era (1988-2008). We used a nested case-control study of 187 organ-confined patients (pT2N0M0): 154 with no recurrence ("controls") and 33 with clinical recurrence ("cases"). RNA was obtained from laser capture microdissected malignant glands representative of the overall Gleason score of each patient. Whole genome gene expression profiles (29,000 transcripts) were obtained using the Whole Genome DASL HT platform (Illumina, Inc). A gene expression signature of PCa clinical recurrence was identified using stability selection with elastic net regularized logistic regression. Three existing datasets generated with the Affymetrix Human Exon 1.0ST array were used for validation: Mayo Clinic (MC, n = 545), Memorial Sloan Kettering Cancer Center (SKCC, n = 150), and Erasmus Medical Center (EMC, n = 48). The areas under the ROC curve (AUCs) were obtained using repeated fivefold cross-validation. RESULTS: A 28-gene expression signature was identified that jointly with key clinical variables (age, Gleason score, pre-operative PSA level, and operation year) was predictive of clinical recurrence (AUC of clinical variables only was 0.67, AUC of clinical variables, and 28-gene signature was 0.99). The AUC of this gene signature fitted in each of the external datasets jointly with clinical variables was 0.75 (0.72-0.77) (MC), 0.90 (0.86-0.94) (MSKCC), and 0.82 (0.74-0.91) (EMC), whereas the AUC for clinical variables only in each dataset was 0.72 (0.70-0.74), 0.86 (0.82-0.91), and 0.76 (0.67-0.85), respectively. CONCLUSIONS: We report a novel gene-expression based classifier identified using agnostic approaches from whole genome expression profiles that can improve upon the accuracy of clinical indicators to stratify early stage localized patients at risk of clinical recurrence after RP. Prostate 76:1239-1256, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Recidiva Local de Neoplasia/diagnóstico , Recidiva Local de Neoplasia/genética , Prostatectomia , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Seguimentos , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/cirurgia , Valor Preditivo dos Testes , Prostatectomia/métodos , Neoplasias da Próstata/cirurgiaRESUMO
Peroxisome biogenesis disorders in the Zellweger spectrum (PBD-ZSD) are a heterogeneous group of genetic disorders caused by mutations in PEX genes responsible for normal peroxisome assembly and functions. As a result of impaired peroxisomal activities, individuals with PBD-ZSD can manifest a complex spectrum of clinical phenotypes that typically result in shortened life spans. The extreme variability in disease manifestation ranging from onset of profound neurologic symptoms in newborns to progressive degenerative disease in adults presents practical challenges in disease diagnosis and medical management. Recent advances in biochemical methods for newborn screening and genetic testing have provided unprecedented opportunities for identifying patients at the earliest possible time and defining the molecular bases for their diseases. Here, we provide an overview of current clinical approaches for the diagnosis of PBD-ZSD and provide broad guidelines for the treatment of disease in its wide variety of forms. Although we anticipate future progress in the development of more effective targeted interventions, the current guidelines are meant to provide a starting point for the management of these complex conditions in the context of personalized health care.
Assuntos
Mutação , Transtornos Peroxissômicos/diagnóstico , Transtornos Peroxissômicos/terapia , Síndrome de Zellweger/diagnóstico , Síndrome de Zellweger/terapia , Adulto , Testes Genéticos , Perda Auditiva Neurossensorial/etiologia , Perda Auditiva Neurossensorial/fisiopatologia , Humanos , Proteínas de Membrana/genética , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Peroxissomos/genética , Fenótipo , Guias de Prática Clínica como Assunto , Medicina de Precisão , Distrofias Retinianas/etiologia , Distrofias Retinianas/fisiopatologiaRESUMO
The tongue is a muscular organ and plays a crucial role in speech, deglutition and taste. Despite the important physiological functions of the tongue, little is known about the regulatory mechanisms of tongue muscle development. TGFß family members play important roles in regulating myogenesis, but the functional significance of Smad-dependent TGFß signaling in regulating tongue skeletal muscle development remains unclear. In this study, we have investigated Smad4-mediated TGFß signaling in the development of occipital somite-derived myogenic progenitors during tongue morphogenesis through tissue-specific inactivation of Smad4 (using Myf5-Cre;Smad4(flox/flox) mice). During the initiation of tongue development, cranial neural crest (CNC) cells occupy the tongue buds before myogenic progenitors migrate into the tongue primordium, suggesting that CNC cells play an instructive role in guiding tongue muscle development. Moreover, ablation of Smad4 results in defects in myogenic terminal differentiation and myoblast fusion. Despite compromised muscle differentiation, tendon formation appears unaffected in the tongue of Myf5-Cre;Smad4(flox/flox) mice, suggesting that the differentiation and maintenance of CNC-derived tendon cells are independent of Smad4-mediated signaling in myogenic cells in the tongue. Furthermore, loss of Smad4 results in a significant reduction in expression of several members of the FGF family, including Fgf6 and Fgfr4. Exogenous Fgf6 partially rescues the tongue myoblast fusion defect of Myf5-Cre;Smad4(flox/flox) mice. Taken together, our study demonstrates that a TGFß-Smad4-Fgf6 signaling cascade plays a crucial role in myogenic cell fate determination and lineage progression during tongue myogenesis.
Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Desenvolvimento Muscular/fisiologia , Transdução de Sinais/fisiologia , Proteína Smad4/metabolismo , Língua/embriologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Western Blotting , Células Cultivadas , Fator 6 de Crescimento de Fibroblastos/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Hibridização In Situ , Camundongos , Camundongos Mutantes , Microscopia Eletrônica de Varredura , Mioblastos/citologia , Crista Neural/embriologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Proteína Smad4/genética , Tendões/citologia , Língua/citologia , beta-GalactosidaseRESUMO
BACKGROUND: In the rodent incisor during amelogenesis, as ameloblast cells transition from secretory stage to maturation stage, their morphology and transcriptome profiles change dramatically. Prior whole genome transcriptome analysis has given a broad picture of the molecular activities dominating both stages of amelogenesis, but this type of analysis has not included miRNA transcript profiling. In this study, we set out to document which miRNAs and corresponding target genes change significantly as ameloblasts transition from secretory- to maturation-stage amelogenesis. RESULTS: Total RNA samples from both secretory- and maturation-stage rat enamel organs were subjected to genome-wide miRNA and mRNA transcript profiling. We identified 59 miRNAs that were differentially expressed at the maturation stage relative to the secretory stage of enamel development (False Discovery Rate (FDR)<0.05, fold change (FC)≥1.8). In parallel, transcriptome profiling experiments identified 1,729 mRNA transcripts that were differentially expressed in the maturation stage compared to the secretory stage (FDR<0.05, FC≥1.8). Based on bioinformatics analyses, 5.8% (629 total) of these differentially expressed genes (DEGS) were highlighted as being the potential targets of 59 miRNAs that were differentially expressed in the opposite direction, in the same tissue samples. Although the number of predicted target DEGs was not higher than baseline expectations generated by examination of stably expressed miRNAs, Gene Ontology (GO) analysis showed that these 629 DEGS were enriched for ion transport, pH regulation, calcium handling, endocytotic, and apoptotic activities. Seven differentially expressed miRNAs (miR-21, miR-31, miR-488, miR-153, miR-135b, miR-135a and miR298) in secretory- and/or maturation-stage enamel organs were confirmed by in situ hybridization. Further, we used luciferase reporter assays to provide evidence that two of these differentially expressed miRNAs, miR-153 and miR-31, are potential regulators for their predicated target mRNAs, Lamp1 (miR-153) and Tfrc (miR-31). CONCLUSIONS: In conclusion, these data indicate that miRNAs exhibit a dynamic expression pattern during the transition from secretory-stage to maturation-stage tooth enamel formation. Although they represent only one of numerous mechanisms influencing gene activities, miRNAs specific to the maturation stage could be involved in regulating several key processes of enamel maturation by influencing mRNA stability and translation.
Assuntos
Amelogênese/genética , Genoma , MicroRNAs/genética , Transcriptoma/genética , Animais , Análise por Conglomerados , Esmalte Dentário/crescimento & desenvolvimento , Esmalte Dentário/metabolismo , Regulação para Baixo/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Redes Reguladoras de Genes , Hibridização In Situ , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Masculino , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reprodutibilidade dos Testes , Regulação para Cima/genéticaRESUMO
Zellweger spectrum disorder (ZSD) is a disease continuum that results from inherited defects in PEX genes essential for normal peroxisome assembly. These autosomal recessive disorders impact brain development and also cause postnatal liver, adrenal, and kidney dysfunction, as well as loss of vision and hearing. The hypomorphic PEX1-G843D missense allele, observed in approximately 30% of ZSD patients, is associated with milder clinical and biochemical phenotypes, with some homozygous individuals surviving into early adulthood. Nonetheless, affected children with the PEX1-G843D allele have intellectual disability, failure to thrive, and significant sensory deficits. To enhance our ability to test candidate therapies that improve human PEX1-G843D function, we created the novel Pex1-G844D knock-in mouse model that represents the murine equivalent of the common human mutation. We show that Pex1-G844D homozygous mice recapitulate many classic features of mild ZSD cases, including growth retardation and fatty livers with cholestasis. In addition, electrophysiology, histology, and gene expression studies provide evidence that these animals develop a retinopathy similar to that observed in human patients, with evidence of cone photoreceptor cell death. Similar to skin fibroblasts obtained from ZSD patients with a PEX1-G843D allele, we demonstrate that murine cells homozygous for the Pex1-G844D allele respond to chaperone-like compounds, which normalizes peroxisomal ß-oxidation. Thus, the Pex1-G844D mouse provides a powerful model system for testing candidate therapies that address the most common genetic cause of ZSD. In addition, this murine model will enhance studies focused on mechanisms of pathogenesis.
Assuntos
Adenosina Trifosfatases/genética , Modelos Animais de Doenças , Mutação de Sentido Incorreto/genética , Síndrome de Zellweger/patologia , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/metabolismo , Animais , Animais Recém-Nascidos , Ácidos e Sais Biliares/metabolismo , Ácidos Graxos/sangue , Feminino , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Crescimento e Desenvolvimento , Audição , Heterozigoto , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Chaperonas Moleculares/metabolismo , Fenótipo , Retina/patologia , Retina/fisiopatologia , Comportamento Sexual Animal , Pele/patologia , Análise de Sobrevida , Visão Ocular , Síndrome de Zellweger/sangue , Síndrome de Zellweger/genética , Síndrome de Zellweger/fisiopatologiaRESUMO
Cleft palate represents one of the most common congenital birth defects. Transforming growth factor ß (TGFß) signaling plays crucial functions in regulating craniofacial development, and loss of TGFß receptor type II in cranial neural crest cells leads to craniofacial malformations, including cleft palate in mice (Tgfbr2(fl/fl);Wnt1-Cre mice). Here we have identified candidate target genes of TGFß signaling during palatal formation. These target genes were selected based on combining results from gene expression profiles of embryonic day 14.5 palates from Tgfbr2(fl/fl);Wnt1-Cre mice and previously identified cleft palate phenotypes in genetically engineered mouse models. We found that fibroblast growth factor 9 (Fgf9) and transcription factor pituitary homeobox 2 (Pitx2) expressions are significantly down-regulated in the palate of Tgfbr2(fl/fl);Wnt1-Cre mice, and Fgf9 and Pitx2 loss of function mutations result in cleft palate in mice. Pitx2 expression is down-regulated by siRNA knockdown of Fgf9, suggesting that Fgf9 is upstream of Pitx2. We detected decreased expression of both cyclins D1 and D3 in the palates of Tgfbr2(fl/fl);Wnt1-Cre mice, consistent with the defect in cell proliferation. Significantly, exogenous FGF9 restores expression of cyclins D1 and D3 in a Pitx2-dependent manner and rescues the cell proliferation defect in the palatal mesenchyme of Tgfbr2(fl/fl);Wnt1-Cre mice. Our study indicates that a TGFß-FGF9-PITX2 signaling cascade regulates cranial neural crest cell proliferation during palate formation.
Assuntos
Proliferação de Células , Fator 9 de Crescimento de Fibroblastos/metabolismo , Proteínas de Homeodomínio/metabolismo , Mesoderma/embriologia , Palato/embriologia , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Fissura Palatina/embriologia , Fissura Palatina/genética , Fator 9 de Crescimento de Fibroblastos/genética , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Transgênicos , Crista Neural/embriologia , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Proteína Homeobox PITX2RESUMO
Nonsyndromic orofacial clefts are common birth defects whose etiology is influenced by complex genetic and environmental factors and gene-environment interactions. Although these risk factors are not yet fully elucidated, it is known that alterations in transforming growth factor-beta (TGFß) signaling can cause craniofacial abnormalities, including cleft palate, in mammals. To elucidate the downstream targets of TGFß signaling in palatogenesis, we analyzed the gene expression profiles of Tgfbr2(fl/fl) ;Wnt1-Cre mouse embryos with cleft palate and other craniofacial deformities resulting from the targeted inactivation of the Tgfbr2 gene in their cranial neural crest (CNC) cells. Relative to controls, palatal tissues obtained from Tgfbr2(fl/fl) ;Wnt1-Cre mouse embryos at embryonic day 14.5 (E14.5) of gestation have a robust gene expression signature reflective of known defects in CNC-derived mesenchymal cell proliferation. Groups of differentially expressed genes (DEGs) were involved in diverse cellular processes and components associated with orofacial clefting, including the extracellular matrix, cholesterol metabolism, ciliogenesis, and multiple signaling pathways. A subset of the DEGs are known or suspected to be associated with an increased risk of orofacial clefting in humans and/or genetically engineered mice. Based on bioinformatics analyses, we highlight the functional relationships among differentially expressed transcriptional regulators of palatogenesis as well as transcriptional factors not previously associated with this process. We suggest that gene expression profiling studies of mice with TGFß signaling defects provide a valuable approach for identifying candidate mechanisms by which this pathway controls cell fate during palatogenesis and its role in the etiology of human craniofacial abnormalities.
Assuntos
Fissura Palatina/patologia , Perfilação da Expressão Gênica/métodos , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular , Proliferação de Células , Fissura Palatina/embriologia , Fissura Palatina/metabolismo , Biologia Computacional/métodos , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Inativação Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Motivos de Nucleotídeos , Palato/embriologia , Palato/metabolismo , Palato/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMO
BACKGROUND: Humans and rodents with impaired phytanic acid (PA) metabolism can accumulate toxic stores of PA that have deleterious effects on multiple organ systems. Ruminants and certain fish obtain PA from the microbial degradation of dietary chlorophyll and/or through chlorophyll-derived precursors. In contrast, humans cannot derive PA from chlorophyll and instead normally obtain it only from meat, dairy, and fish products. RESULTS: Captive apes and Old world monkeys had significantly higher red blood cell (RBC) PA levels relative to humans when all subjects were fed PA-deficient diets. Given the adverse health effects resulting from PA over accumulation, we investigated the molecular evolution of thirteen PA metabolism genes in apes, Old world monkeys, and New world monkeys. All non-human primate (NHP) orthologs are predicted to encode full-length proteins with the marmoset Phyh gene containing a rare, but functional, GA splice donor dinucleotide. Acox2, Scp2, and Pecr sequences had amino acid positions with accelerated substitution rates while Amacr had significant variation in evolutionary rates in apes relative to other primates. CONCLUSIONS: Unlike humans, diverse captive NHPs with PA-deficient diets rich in plant products have substantial RBC PA levels. The favored hypothesis is that NHPs can derive significant amounts of PA from the degradation of ingested chlorophyll through gut fermentation. If correct, this raises the possibility that RBC PA levels could serve as a biomarker for evaluating the digestive health of captive NHPs. Furthermore, the evolutionary rates of the several genes relevant to PA metabolism provide candidate genetic adaptations to NHP diets.
Assuntos
Callithrix/metabolismo , Clorofila/metabolismo , Dieta Vegetariana , Eritrócitos/metabolismo , Hominidae/metabolismo , Macaca mulatta/metabolismo , Papio anubis/metabolismo , Ácido Fitânico/metabolismo , Animais , Evolução Biológica , Callithrix/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Feminino , Expressão Gênica , Hominidae/genética , Humanos , Macaca mulatta/genética , Masculino , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Papio anubis/genética , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Especificidade da EspécieRESUMO
The gene repertoire regulating vertebrate biomineralization is poorly understood. Dental enamel, the most highly mineralized tissue in mammals, differs from other calcifying systems in that the formative cells (ameloblasts) lack remodeling activity and largely degrade and resorb the initial extracellular matrix. Enamel mineralization requires that ameloblasts undergo a profound functional switch from matrix-secreting to maturational (calcium transport, protein resorption) roles as mineralization progresses. During the maturation stage, extracellular pH decreases markedly, placing high demands on ameloblasts to regulate acidic environments present around the growing hydroxyapatite crystals. To identify the genetic events driving enamel mineralization, we conducted genome-wide transcript profiling of the developing enamel organ from rat incisors and highlight over 300 genes differentially expressed during maturation. Using multiple bioinformatics analyses, we identified groups of maturation-associated genes whose functions are linked to key mineralization processes including pH regulation, calcium handling, and matrix turnover. Subsequent qPCR and Western blot analyses revealed that a number of solute carrier (SLC) gene family members were up-regulated during maturation, including the novel protein Slc24a4 involved in calcium handling as well as other proteins of similar function (Stim1). By providing the first global overview of the cellular machinery required for enamel maturation, this study provide a strong foundation for improving basic understanding of biomineralization and its practical applications in healthcare.
Assuntos
Amelogênese/fisiologia , Esmalte Dentário/química , Esmalte Dentário/metabolismo , Perfilação da Expressão Gênica/métodos , Genoma , Calcificação de Dente/genética , Ameloblastos/metabolismo , Animais , Cálcio/metabolismo , Matriz Extracelular/metabolismo , Expressão Gênica , Humanos , Incisivo/anatomia & histologia , Incisivo/metabolismo , Ratos , Ratos WistarRESUMO
BACKGROUND: Zellweger syndrome (ZS) is a peroxisome biogenesis disorder due to mutations in any one of 13 PEX genes. Increased incidence of ZS has been suspected in French-Canadians of the Saguenay-Lac-St-Jean region (SLSJ) of Quebec, but this remains unsolved. METHODS: We identified 5 ZS patients from SLSJ diagnosed by peroxisome dysfunction between 1990-2010 and sequenced all coding exons of known PEX genes in one patient using Next Generation Sequencing (NGS) for diagnostic confirmation. RESULTS: A homozygous mutation (c.802_815del, p.[Val207_Gln294del, Val76_Gln294del]) in PEX6 was identified and then shown in 4 other patients. Parental heterozygosity was confirmed in all. Incidence of ZS was estimated to 1 in 12,191 live births, with a carrier frequency of 1 in 55. In addition, we present data suggesting that this mutation abolishes a SF2/ASF splice enhancer binding site, resulting in the use of two alternative cryptic donor splice sites and predicted to encode an internally deleted in-frame protein. CONCLUSION: We report increased incidence of ZS in French-Canadians of SLSJ caused by a PEX6 founder mutation. To our knowledge, this is the highest reported incidence of ZS worldwide. These findings have implications for carrier screening and support the utility of NGS for molecular confirmation of peroxisomal disorders.
Assuntos
Adenosina Trifosfatases/genética , Efeito Fundador , Mutação , População Branca/genética , Síndrome de Zellweger/epidemiologia , Síndrome de Zellweger/genética , ATPases Associadas a Diversas Atividades Celulares , Sequência de Bases , Feminino , França/etnologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Quebeque/epidemiologia , Síndrome de Zellweger/enzimologiaRESUMO
Untargeted metabolomics is a global molecular profiling technology that can be used to screen for inborn errors of metabolism (IEMs). Metabolite perturbations are evaluated based on current knowledge of specific metabolic pathway deficiencies, a manual diagnostic process that is qualitative, has limited scalability, and is not equipped to learn from accumulating clinical data. Our purpose was to improve upon manual diagnosis of IEMs in the clinic by developing novel computational methods for analyzing untargeted metabolomics data. We employed CTD, an automated computational diagnostic method that "connects the dots" between metabolite perturbations observed in individual metabolomics profiling data and modules identified in disease-specific metabolite co-perturbation networks learned from prior profiling data. We also extended CTD to calculate distances between any two individuals (CTDncd) and between an individual and a disease state (CTDdm), to provide additional network-quantified predictors for use in diagnosis. We show that across 539 plasma samples, CTD-based network-quantified measures can reproduce accurate diagnosis of 16 different IEMs, including adenylosuccinase deficiency, argininemia, argininosuccinic aciduria, aromatic L-amino acid decarboxylase deficiency, cerebral creatine deficiency syndrome type 2, citrullinemia, cobalamin biosynthesis defect, GABA-transaminase deficiency, glutaric acidemia type 1, maple syrup urine disease, methylmalonic aciduria, ornithine transcarbamylase deficiency, phenylketonuria, propionic acidemia, rhizomelic chondrodysplasia punctata, and the Zellweger spectrum disorders. Our approach can be used to supplement information from biochemical pathways and has the potential to significantly enhance the interpretation of variants of uncertain significance uncovered by exome sequencing. CTD, CTDdm, and CTDncd can serve as an essential toolset for biological interpretation of untargeted metabolomics data that overcomes limitations associated with manual diagnosis to assist diagnosticians in clinical decision-making. By automating and quantifying the interpretation of perturbation patterns, CTD can improve the speed and confidence by which clinical laboratory directors make diagnostic and treatment decisions, while automatically improving performance with new case data.
Assuntos
Doenças Metabólicas , Metabolômica , Diagnóstico por Computador , Humanos , Doenças Metabólicas/diagnóstico , Metabolômica/métodosRESUMO
Peroxisome biogenesis disorders (PBDs) are multisystemic autosomal recessive disorders resulting from mutations in PEX genes required for normal peroxisome assembly and metabolic activities. Here, we evaluated the potential effectiveness of aminoglycoside G418 (geneticin) and PTC124 (ataluren) nonsense suppression therapies for the treatment of PBD patients with disease-causing nonsense mutations. PBD patient skin fibroblasts producing stable PEX2 or PEX12 nonsense transcripts and Chinese hamster ovary (CHO) cells with a Pex2 nonsense allele all showed dramatic improvements in peroxisomal very long chain fatty acid catabolism and plasmalogen biosynthesis in response to G418 treatments. Cell imaging assays provided complementary confirmatory evidence of improved peroxisome assembly in G418-treated patient fibroblasts. In contrast, we observed no appreciable rescue of peroxisome lipid metabolism or assembly for any patient fibroblast or CHO cell culture treated with various doses of PTC124. Additionally, PTC124 did not show measurable nonsense suppression in immunoblot assays that directly evaluated the read-through of PEX7 nonsense alleles found in PBD patients with rhizomelic chondrodysplasia punctata type 1 (RCDP1). Overall, our results support the continued development of safe and effective nonsense suppressor therapies that could benefit a significant subset of individuals with PBDs. Furthermore, we suggest that the described cell culture assay systems could be useful for evaluating and screening for novel nonsense suppressor therapies.
Assuntos
Gentamicinas/uso terapêutico , Proteínas de Membrana/genética , Oxidiazóis/uso terapêutico , Alelos , Animais , Células CHO , Condrodisplasia Punctata Rizomélica/metabolismo , Condrodisplasia Punctata Rizomélica/terapia , Códon sem Sentido/efeitos dos fármacos , Cricetinae , Cricetulus , Feminino , Fibroblastos/metabolismo , Humanos , Metabolismo dos Lipídeos/genética , Fator 2 da Biogênese de Peroxissomos , Transtornos Peroxissômicos/tratamento farmacológico , Transtornos Peroxissômicos/genética , Receptor 2 de Sinal de Orientação para Peroxissomos , Plasmalogênios/genética , Plasmalogênios/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Enamel maturation is a dynamic process that involves high rates of mineral acquisition, associated fluctuations in extracellular pH, and resorption of extracellular enamel proteins. During maturation, ameloblasts change from having a tall, thin, and highly polarized organization, characteristic of the secretory stage, to having a low columnar and widened morphology in the maturation stage. To identify potential differences in gene expression throughout maturation, we obtained enamel organ epithelial cells derived from the early- and late-maturation stages of rat incisor and analyzed the global gene-expression profiles at each stage. Sixty-three candidate genes were identified as having potential roles in the maturation process. Quantitative PCR was used to confirm the results of this genome-wide analysis in a subset of genes. Transcripts enriched during late maturation (n = 38) included those associated with lysosomal activity, solute carrier transport, and calcium signaling. Also up-regulated were transcripts involved in cellular responses to oxidative stress, proton transport, cell death, and the immune system. Transcripts down-regulated during the late maturation stage (n =25) included those with functions related to cell adhesion, cell signaling, and T-cell activation. These results indicate that ameloblasts undergo widespread molecular changes during the maturation stage of amelogenesis and hence provide a basis for future functional investigations into the mechanistic basis of enamel mineralization.
Assuntos
Ameloblastos/citologia , Amelogênese/fisiologia , Órgão do Esmalte/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Calcificação de Dente/genética , Ameloblastos/metabolismo , Amelogênese/genética , Animais , Transporte Biológico/genética , Sinalização do Cálcio/genética , Adesão Celular/genética , Estudo de Associação Genômica Ampla , Concentração de Íons de Hidrogênio , Ativação Linfocitária/genética , Lisossomos/fisiologia , Ratos , Ratos Wistar , Transcriptoma/genética , Regulação para CimaRESUMO
BACKGROUND: Plasmalogens are ether phospholipids required for normal mammalian developmental, physiological, and cognitive functions. They have been proposed to act as membrane antioxidants and reservoirs of polyunsaturated fatty acids as well as influence intracellular signaling and membrane dynamics. Plasmalogens are particularly enriched in cells and tissues of the human nervous, immune, and cardiovascular systems. Humans with severely reduced plasmalogen levels have reduced life spans, abnormal neurological development, skeletal dysplasia, impaired respiration, and cataracts. Plasmalogen deficiency is also found in the brain tissue of individuals with Alzheimer disease. RESULTS: In a human and great ape cohort, we measured the red blood cell (RBC) levels of the most abundant types of plasmalogens. Total RBC plasmalogen levels were lower in humans than bonobos, chimpanzees, and gorillas, but higher than orangutans. There were especially pronounced cross-species differences in the levels of plasmalogens with a C16:0 moiety at the sn-1 position. Humans on Western or vegan diets had comparable total RBC plasmalogen levels, but the latter group showed moderately higher levels of plasmalogens with a C18:1 moiety at the sn-1 position. We did not find robust sex-specific differences in human or chimpanzee RBC plasmalogen levels or composition. Furthermore, human and great ape skin fibroblasts showed only modest differences in peroxisomal plasmalogen biosynthetic activity. Human and chimpanzee microarray data indicated that genes involved in plasmalogen biosynthesis show cross-species differential expression in multiple tissues. CONCLUSION: We propose that the observed differences in human and great ape RBC plasmalogens are primarily caused by their rates of biosynthesis and/or turnover. Gene expression data raise the possibility that other human and great ape cells and tissues differ in plasmalogen levels. Based on the phenotypes of humans and rodents with plasmalogen disorders, we propose that cross-species differences in tissue plasmalogen levels could influence organ functions and processes ranging from cognition to reproduction to aging.
Assuntos
Eritrócitos/metabolismo , Pan troglodytes/metabolismo , Plasmalogênios/metabolismo , Animais , Vias Biossintéticas , Células Cultivadas , Dieta Vegetariana , Feminino , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Gorilla gorilla , Humanos , Masculino , Pan paniscus , Peroxissomos/metabolismo , Fosfolipídeos/metabolismo , Filogenia , Plasmalogênios/biossíntese , Plasmalogênios/química , Pongo pygmaeusRESUMO
Patients with Zellweger spectrum disorder (ZSD) commonly present with vision loss due to mutations in PEX genes required for peroxisome assembly and function. Here, we evaluate PEX1 retinal gene augmentation therapy in a mouse model of mild ZSD bearing the murine equivalent (PEX1-p[Gly844Asp]) of the most common human mutation. Experimental adeno-associated virus 8.cytomegalovirus.human PEX1.hemagglutinin (AAV8.CMV.HsPEX1.HA) and control AAV8.CMV.EGFP vectors were administered by subretinal injection in contralateral eyes of early (5-week-old)- or later (9-week-old)-stage retinopathy cohorts. HsPEX1.HA protein was expressed in the retina with no gross histologic side effects. Peroxisomal metabolic functions, assessed by retinal C26:0 lysophosphatidylcholine (lyso-PC) levels, were partially normalized after therapeutic vector treatment. Full-field flash electroretinogram (ffERG) analyses at 8 weeks post-injection showed a 2-fold improved retinal response in the therapeutic relative to control vector-injected eyes. ffERG improved by 1.6- to 2.5-fold in the therapeutic vector-injected eyes when each cohort reached 25 weeks of age. At 32 weeks of age, the average ffERG response was double in the therapeutic relative to control vector-injected eyes in both cohorts. Optomotor reflex analyses trended toward improvement. These proof-of-concept studies represent the first application of gene augmentation therapy to treat peroxisome biogenesis disorders and support the potential for retinal gene delivery to improve vision in these patients.
RESUMO
Epigenetic remodeling is a hallmark of cancer, with the frequent acquisition of de novo DNA methylation in CpG islands. However, the functional relevance of de novo DNA methylation in cancer is less well defined. To begin to address this issue in B-cells, we used BeadArray assays to survey the methylation status of over 1,500 cancer-related CpG loci in two molecular subtypes of diffuse large B-cell lymphoma (ABC-DLBCL and GCB-DLBCL) and cognate normal B-cell populations. We identified 81 loci that showed frequent de novo DNA methylation in GCB-DLBCL and 67 loci that showed frequent de novo DNA methylation in ABC-DLBCL. These de novo methylated CpG loci included reported targets of polycomb repressive complexes (PRC) in stem cells. All candidate loci in GCB-DLBCL are proximal to genes that are poorly expressed or silent in purified normal germinal center (GC) B-cells. This is consistent with the hypothesis that de novo DNA methylation in cancer is more frequently involved in the maintenance rather than the initiation of gene silencing (de novo repression). This suggests that epigenetic switching occurs during tumorigenesis with de novo DNA methylation locking in gene silencing normally mediated by transcriptional repressors. Furthermore, we propose that similar to de novo genetic mutations, the majority of de novo DNA methylation events observed in tumors are passengers not causally involved in tumorigenesis.
Assuntos
Linfócitos B/patologia , Metilação de DNA/fisiologia , Linfoma Difuso de Grandes Células B/genética , Linhagem Celular Tumoral , Ilhas de CpG , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Linfoma Difuso de Grandes Células B/patologia , Células-Tronco Neoplásicas , Proteínas do Grupo Polycomb , Proteínas Repressoras/genéticaRESUMO
BACKGROUND: It has been proposed that anatomical differences in human and great ape guts arose in response to species-specific diets and energy demands. To investigate functional genomic consequences of these differences, we compared their physiological levels of phytanic acid, a branched chain fatty acid that can be derived from the microbial degradation of chlorophyll in ruminant guts. Humans who accumulate large stores of phytanic acid commonly develop cerebellar ataxia, peripheral polyneuropathy, and retinitis pigmentosa in addition to other medical conditions. Furthermore, phytanic acid is an activator of the PPAR-alpha transcription factor that influences the expression of genes relevant to lipid metabolism. RESULTS: Despite their trace dietary phytanic acid intake, all great ape species had elevated red blood cell (RBC) phytanic acid levels relative to humans on diverse diets. Unlike humans, chimpanzees showed sexual dimorphism in RBC phytanic acid levels, which were higher in males relative to females. Cultured skin fibroblasts from all species had a robust capacity to degrade phytanic acid. We provide indirect evidence that great apes, in contrast to humans, derive significant amounts of phytanic acid from the hindgut fermentation of plant materials. This would represent a novel reduction of metabolic activity in humans relative to the great apes. CONCLUSION: We identified differences in the physiological levels of phytanic acid in humans and great apes and propose this is causally related to their gut anatomies and microbiomes. Phytanic acid levels could contribute to cross-species and sex-specific differences in human and great ape transcriptomes, especially those related to lipid metabolism. Based on the medical conditions caused by phytanic acid accumulation, we suggest that differences in phytanic acid metabolism could influence the functions of human and great ape nervous, cardiovascular, and skeletal systems.