RESUMO
Kinesin-2 enables ciliary assembly and maintenance as an anterograde intraflagellar transport (IFT) motor. Molecular motor activity is driven by a heterotrimeric complex comprised of KIF3A and KIF3B or KIF3C plus one non-motor subunit, KIFAP3. Using exome sequencing, we identified heterozygous KIF3B variants in two unrelated families with hallmark ciliopathy phenotypes. In the first family, the proband presents with hepatic fibrosis, retinitis pigmentosa, and postaxial polydactyly; he harbors a de novo c.748G>C (p.Glu250Gln) variant affecting the kinesin motor domain encoded by KIF3B. The second family is a six-generation pedigree affected predominantly by retinitis pigmentosa. Affected individuals carry a heterozygous c.1568T>C (p.Leu523Pro) KIF3B variant segregating in an autosomal-dominant pattern. We observed a significant increase in primary cilia length in vitro in the context of either of the two mutations while variant KIF3B proteins retained stability indistinguishable from wild type. Furthermore, we tested the effects of KIF3B mutant mRNA expression in the developing zebrafish retina. In the presence of either missense variant, rhodopsin was sequestered to the photoreceptor rod inner segment layer with a concomitant increase in photoreceptor cilia length. Notably, impaired rhodopsin trafficking is also characteristic of recessive KIF3B models as exemplified by an early-onset, autosomal-recessive, progressive retinal degeneration in Bengal cats; we identified a c.1000G>A (p.Ala334Thr) KIF3B variant by genome-wide association study and whole-genome sequencing. Together, our genetic, cell-based, and in vivo modeling data delineate an autosomal-dominant syndromic retinal ciliopathy in humans and suggest that multiple KIF3B pathomechanisms can impair kinesin-driven ciliary transport in the photoreceptor.
Assuntos
Ciliopatias/genética , Ciliopatias/patologia , Genes Dominantes/genética , Cinesinas/genética , Mutação , Retina/patologia , Sequência de Aminoácidos , Animais , Gatos , Pré-Escolar , Cílios/patologia , Feminino , Estudo de Associação Genômica Ampla , Heterozigoto , Humanos , Cinesinas/química , Cinesinas/metabolismo , Larva , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Células Fotorreceptoras/metabolismo , Retina/citologia , Retina/crescimento & desenvolvimento , Retina/metabolismo , Rodopsina/metabolismo , Adulto Jovem , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Fibroblast growth factor receptor 3 (FGFR3) gain-of-function mutations cause dwarfisms, including achondroplasia (ACH) and thanatophoric dysplasia (TD). The constitutive activation of FGFR3 disrupts the normal process of skeletal growth. Bone-growth anomalies have been identified in skeletal ciliopathies, in which primary cilia (PC) function is disrupted. In human ACH and TD, the impact of FGFR3 mutations on PC in growth plate cartilage remains unknown. Here we showed that in chondrocytes from human (ACH, TD) and mouse Fgfr3Y367C/+ cartilage, the constitutively active FGFR3 perturbed PC length and the sorting and trafficking of intraflagellar transport (IFT) 20 to the PC. We demonstrated that inhibiting FGFR3 with FGFR inhibitor, PD173074, rescued both PC length and IFT20 trafficking. We also studied the impact of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) pathway. Interestingly, mTOR inhibition also rescued PC length and IFT20 trafficking. Together, we provide evidence that the growth plate defects ascribed to FGFR3-related dwarfisms are potentially due to loss of PC function, and these dwarfisms may represent a novel type of skeletal disorders with defective ciliogenesis.
Assuntos
Acondroplasia/metabolismo , Proteínas de Transporte/metabolismo , Condrócitos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Acondroplasia/genética , Acondroplasia/patologia , Animais , Desenvolvimento Ósseo/genética , Proteínas de Transporte/genética , Cartilagem/metabolismo , Cartilagem/patologia , Diferenciação Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Condrócitos/patologia , Cílios/genética , Cílios/metabolismo , Modelos Animais de Doenças , Feminino , Lâmina de Crescimento/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Pirimidinas/farmacologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Transdução de SinaisRESUMO
Plants, including most food and feed plants, produce a broad range of bioactive chemical compounds. Among these compounds, polyphenols are reported to provide beneficial effects as anti-carcinogenic, anti-atherogenic, anti-inflammatory, immune modulating, anti-microbial, vasodilatory and analgesic. Cocoa (Theobroma cacao), a major, economically important, international crop, has been related to several nutritional benefits, which have been associated with the phenolic fraction. The main subclass of flavonoids found in cocoa is flavanols, particularly (epi)catechins monomers, and their oligomers, also known as procyanidins. In this study, these compounds were isolated by different methodologies as solid phase extraction (SPE), semi-preparative high-performance liquid chromatography (HPLC) and membrane technologies to obtain different polyphenolic profiles by HPLC coupled to electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and to test their cytotoxicity. Finally, different polyphenolic profiles were collected, where the combination of both semi-preparative HPLC and SPE technologies provided the most purified fractions. Filtration with membranes and SPE provide extracts with different composition depending on the pore size of membranes and on the solvent, respectively. In addition, the results of toxicity assay indicated low levels in all fractions.
Assuntos
Cacau/química , Flavonoides/isolamento & purificação , Compostos Fitoquímicos/isolamento & purificação , Extração em Fase Sólida/métodos , Cromatografia Líquida de Alta Pressão , Flavonoides/toxicidade , Inocuidade dos Alimentos , Análise de Perigos e Pontos Críticos de Controle , Humanos , Compostos Fitoquímicos/toxicidade , Polifenóis/isolamento & purificação , Polifenóis/toxicidade , Proantocianidinas/isolamento & purificação , Proantocianidinas/toxicidade , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Activating FGFR3 mutations in human result in achondroplasia (ACH), the most frequent form of dwarfism, where cartilages are severely disturbed causing long bones, cranial base and vertebrae defects. Because mandibular development and growth rely on cartilages that guide or directly participate to the ossification process, we investigated the impact of FGFR3 mutations on mandibular shape, size and position. By using CT scan imaging of ACH children and by analyzing Fgfr3Y367C/+ mice, a model of ACH, we show that FGFR3 gain-of-function mutations lead to structural anomalies of primary (Meckel's) and secondary (condylar) cartilages of the mandible, resulting in mandibular hypoplasia and dysmorphogenesis. These defects are likely related to a defective chondrocyte proliferation and differentiation and pan-FGFR tyrosine kinase inhibitor NVP-BGJ398 corrects Meckel's and condylar cartilages defects ex vivo. Moreover, we show that low dose of NVP-BGJ398 improves in vivo condyle growth and corrects dysmorphologies in Fgfr3Y367C/+ mice, suggesting that postnatal treatment with NVP-BGJ398 mice might offer a new therapeutic strategy to improve mandible anomalies in ACH and others FGFR3-related disorders.
Assuntos
Acondroplasia/genética , Cartilagem/anormalidades , Mandíbula/anormalidades , Côndilo Mandibular/anormalidades , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Acondroplasia/diagnóstico por imagem , Acondroplasia/tratamento farmacológico , Acondroplasia/fisiopatologia , Animais , Cartilagem/crescimento & desenvolvimento , Cartilagem/fisiopatologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Condrócitos/metabolismo , Condrócitos/patologia , Modelos Animais de Doenças , Humanos , Mandíbula/crescimento & desenvolvimento , Mandíbula/fisiopatologia , Côndilo Mandibular/crescimento & desenvolvimento , Côndilo Mandibular/fisiopatologia , Camundongos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Compostos de Fenilureia/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/administração & dosagemRESUMO
Autosomal dominant mutations in fibroblast growth factor receptor 3 (FGFR3) cause achondroplasia (Ach), the most common form of dwarfism in humans, and related chondrodysplasia syndromes that include hypochondroplasia (Hch), severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN), and thanatophoric dysplasia (TD). FGFR3 is expressed in chondrocytes and mature osteoblasts where it functions to regulate bone growth. Analysis of the mutations in FGFR3 revealed increased signaling through a combination of mechanisms that include stabilization of the receptor, enhanced dimerization, and enhanced tyrosine kinase activity. Paradoxically, increased FGFR3 signaling profoundly suppresses proliferation and maturation of growth plate chondrocytes resulting in decreased growth plate size, reduced trabecular bone volume, and resulting decreased bone elongation. In this review, we discuss the molecular mechanisms that regulate growth plate chondrocytes, the pathogenesis of Ach, and therapeutic approaches that are being evaluated to improve endochondral bone growth in people with Ach and related conditions. Developmental Dynamics 246:291-309, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Acondroplasia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/fisiologia , Acondroplasia/etiologia , Acondroplasia/patologia , Acondroplasia/terapia , Animais , Condrócitos/metabolismo , Lâmina de Crescimento/citologia , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/ultraestrutura , Humanos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/fisiologiaRESUMO
Impairment of the tightly regulated ossification process leads to a wide range of skeletal dysplasias and deciphering their molecular bases has contributed to the understanding of this complex process. Here, we report a homozygous mutation in the mitochondria-associated granulocyte macrophage colony stimulating factor-signaling gene (MAGMAS) in a novel and severe spondylodysplastic dysplasia. MAGMAS, also referred to as PAM16 (presequence translocase-associated motor 16), is a mitochondria-associated protein involved in preprotein translocation into the matrix. We show that MAGMAS is specifically expressed in trabecular bone and cartilage at early developmental stages and that the mutation leads to an instability of the protein. We further demonstrate that the mutation described here confers to yeast strains a temperature-sensitive phenotype, impairs the import of mitochondrial matrix pre-proteins and induces cell death. The finding of deleterious MAGMAS mutations in an early lethal skeletal dysplasia supports a key role for this mitochondrial protein in the ossification process.
Assuntos
Doenças do Desenvolvimento Ósseo/genética , Proteínas Mitocondriais/fisiologia , Sequência de Aminoácidos , Animais , Doenças do Desenvolvimento Ósseo/diagnóstico por imagem , Exoma , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Linhagem , RNA Mensageiro/genética , Radiografia , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
FGFR3 gain-of-function mutations lead to both chondrodysplasias and craniosynostoses. Achondroplasia (ACH), the most frequent dwarfism, is due to an FGFR3-activating mutation which results in impaired endochondral ossification. The effects of the mutation on membranous ossification are unknown. Fgfr3(Y367C/+) mice mimicking ACH and craniofacial analysis of patients with ACH and FGFR3-related craniosynostoses provide an opportunity to address this issue. Studying the calvaria and skull base, we observed abnormal cartilage and premature fusion of the synchondroses leading to modifications of foramen magnum shape and size in Fgfr3(Y367C/+) mice, ACH and FGFR3-related craniosynostoses patients. Partial premature fusion of the coronal sutures and non-ossified gaps in frontal bones were also present in Fgfr3(Y367C/+) mice and ACH patients. Our data provide strong support that not only endochondral ossification but also membranous ossification is severely affected in ACH. Demonstration of the impact of FGFR3 mutations on craniofacial development should initiate novel pharmacological and surgical therapeutic approaches.
Assuntos
Acondroplasia/enzimologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Acondroplasia/genética , Acondroplasia/patologia , Animais , Condrócitos/citologia , Condrócitos/enzimologia , Feminino , Humanos , Lactente , Masculino , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Ossificação Heterotópica , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Crânio/anatomia & histologia , Crânio/embriologia , Crânio/enzimologia , Crânio/patologiaRESUMO
Achondroplasia (ACH), the most common form of dwarfism, is an inherited autosomal-dominant chondrodysplasia caused by a gain-of-function mutation in fibroblast-growth-factor-receptor 3 (FGFR3). C-type natriuretic peptide (CNP) antagonizes FGFR3 downstream signaling by inhibiting the pathway of mitogen-activated protein kinase (MAPK). Here, we report the pharmacological activity of a 39 amino acid CNP analog (BMN 111) with an extended plasma half-life due to its resistance to neutral-endopeptidase (NEP) digestion. In ACH human growth-plate chondrocytes, we demonstrated a decrease in the phosphorylation of extracellular-signal-regulated kinases 1 and 2, confirming that this CNP analog inhibits fibroblast-growth-factor-mediated MAPK activation. Concomitantly, we analyzed the phenotype of Fgfr3(Y367C/+) mice and showed the presence of ACH-related clinical features in this mouse model. We found that in Fgfr3(Y367C/+) mice, treatment with this CNP analog led to a significant recovery of bone growth. We observed an increase in the axial and appendicular skeleton lengths, and improvements in dwarfism-related clinical features included flattening of the skull, reduced crossbite, straightening of the tibias and femurs, and correction of the growth-plate defect. Thus, our results provide the proof of concept that BMN 111, a NEP-resistant CNP analog, might benefit individuals with ACH and hypochondroplasia.
Assuntos
Acondroplasia/tratamento farmacológico , Peptídeo Natriurético Tipo C/análogos & derivados , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Acondroplasia/diagnóstico , Acondroplasia/genética , Animais , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Modelos Animais de Doenças , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/patologia , Humanos , Camundongos , Mutação , Peptídeo Natriurético Tipo C/química , Peptídeo Natriurético Tipo C/fisiologia , Peptídeo Natriurético Tipo C/uso terapêutico , Tamanho do Órgão/efeitos dos fármacos , Radiografia , Crânio/diagnóstico por imagem , Crânio/efeitos dos fármacos , Crânio/patologia , Resultado do TratamentoRESUMO
Tyrosine kinases are a wide family of targets with strong pharmacological relevance. These proteins undergo large-scale conformational motions able to inactivate them. By the end of one of these structural processes, a new cavity is opened allowing the access to a specific type of inhibitors, called type II. The kinase domain of fibroblast growth factor receptor 3 (FGFR3) falls into this family of kinases. We describe here, for the first time, its inactivation process through target molecular dynamics. The transient cavity, at the crossroad between the DFGout and Cα helix out inactivation is herein explored. Molecular docking calculations of known ligands demonstrated that type II inhibitors are able to interact with this metastable transient conformation of FGFR3 kinase. Besides, supplemental computations were conducted and clearly show that type II inhibitors drive the kinase inactivation process through specific stabilization with the DFG triad. This induced-fit effect of type II ligands toward FGFR3 might be extrapolated to other kinase systems and provides meaningful structural information for future drug developments.
Assuntos
Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/química , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , Inibidores de Proteínas Quinases/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Reprodutibilidade dos TestesRESUMO
The fibroblast growth factor receptor 3 (FGFR3) plays a critical role in the regulation of endochondral ossification. Fgfr3 gain-of-function mutations cause achondroplasia, the most common form of dwarfism, and a spectrum of chondrodysplasias. Despite a significant number of studies on the role of FGFR3 in cartilage, to date, none has investigated the influence of Fgfr3-mediated effects of the growth plate on bone formation. We studied three mouse models, each expressing Fgfr3 mutation either ubiquitously (CMV-Fgfr3(Y367C/+)), in chondrocytes (Col II-Fgfr3(Y367C/+)) or in mature osteoblasts (Col I-Fgfr3(Y367C/+)). Interestingly, we demonstrated that dwarfism with a significant defect in bone formation during growth was only observed in mouse models expressing mutant Fgfr3 in the cartilage. We observed a dramatic reduction in cartilage matrix mineralization and a strong defect of primary spongiosa. Anomalies of primary spongiosa were associated with an increase in osteoclast recruitment and a defect of osteoblasts at the mineralization front. A significant decrease in bone volume, trabecular thickness and number was also observed in the trabecular bone. Interestingly, no anomalies in proliferation and differentiation of primary osteoblasts from CMV-Fgfr3(Y367C/+) mice were observed. Based on these data, we excluded a potential function of Fgfr3 directly on osteoblasts at 3 weeks of age and we obtained evidence that the disorganization of the growth plate is responsible for the anomalies of the trabecular bone during bone formation. Herein, we propose that impaired FGFR3 signaling pathways may affect trabecular bone formation via a paracrine mechanism during growth. These results redefine our understanding of endochondral ossification in FGFR3-related chondrodysplasias.
Assuntos
Mutação , Comunicação Parácrina/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Animais , Proliferação de Células , Condrócitos/metabolismo , Lâmina de Crescimento/metabolismo , Camundongos , Camundongos Transgênicos , Osteoblastos/metabolismo , Osteogênese/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
Activating germline fibroblast growth factor receptor 3 (FGFR3) mutations cause achondroplasia (ACH), the most common form of human dwarfism and a spectrum of skeletal dysplasias. FGFR3 is a tyrosine kinase receptor and constitutive FGFR3 activation impairs endochondral ossification and triggers severe disorganization of the cartilage with shortening of long bones. To decipher the role of FGFR3 in endochondral ossification, we analyzed the impact of a novel tyrosine kinase inhibitor (TKI), A31, on both human and mouse mutant FGFR3-expressing cells and on the skeleton of Fgfr3(Y367C/+) dwarf mice. We found that A31 inhibited constitutive FGFR3 phosphorylation and restored the size of embryonic dwarf femurs using an ex vivo culture system. The increase in length of the treated mutant femurs was 2.6 times more than for the wild-type. Premature cell cycle exit and defective chondrocyte differentiation were observed in the Fgfr3(Y367C/+) growth plate. A31 restored normal expression of cell cycle regulators (proliferating cell nuclear antigen, KI67, cyclin D1 and p57) and allowed pre-hypertrophic chondrocytes to properly differentiate into hypertrophic chondocytes. Our data reveal a specific role for FGFR3 in the cell cycle and chondrocyte differentiation and support the development of TKIs for the treatment of FGFR3-related chondrodysplasias.
Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Modelos Animais , Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fêmur/efeitos dos fármacos , Fêmur/embriologia , Lâmina de Crescimento/efeitos dos fármacos , Técnicas In Vitro , Camundongos , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacologia , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/biossíntese , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genéticaRESUMO
Achondroplasia (ACH), the most common form of disproportionate short stature, is caused by gain-of-function point mutations in fibroblast growth factor receptor 3 (FGFR3). Abnormally elevated activation of FGFR3 modulates chondrocyte proliferation and differentiation via multiple signaling pathways, such as the MAPK pathway. Using a mouse model mimicking ACH (Fgfr3Y367C/+), we have previously shown that daily treatment with infigratinib (BGJ398), a selective and orally bioavailable FGFR1-3 inhibitor, at a dose of 2 mg/kg, significantly increased bone growth. In this study, we investigated the activity of infigratinib administered at substantially lower doses (0.2 and 0.5 mg/kg, given once daily) and using an intermittent dosing regimen (1 mg/kg every 3 days). Following a 15-day treatment period, these low dosages were sufficient to observe significant improvement of clinical hallmarks of ACH such as growth of the axial and appendicular skeleton and skull development. Immunohistological labeling demonstrated the positive impact of infigratinib on chondrocyte differentiation in the cartilage growth plate and the cartilage end plate of the vertebrae. Macroscopic and microcomputed analyses showed enlargement of the foramen magnum area at the skull base, thus improving foramen magnum stenosis, a well-recognized complication in ACH. No changes in FGF23 or phosphorus levels were observed, indicating that the treatment did not modify phosphate homeostasis. This proof-of-concept study demonstrates that infigratinib administered at low doses has the potential to be a safe and effective therapeutic option for children with ACH.
Assuntos
Acondroplasia , Modelos Animais de Doenças , Lâmina de Crescimento , Pirimidinas , Animais , Acondroplasia/tratamento farmacológico , Acondroplasia/patologia , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/patologia , Lâmina de Crescimento/metabolismo , Pirimidinas/farmacologia , Pirimidinas/administração & dosagem , Camundongos , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/administração & dosagem , Desenvolvimento Ósseo/efeitos dos fármacos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Relação Dose-Resposta a Droga , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Condrócitos/metabolismoRESUMO
By using a combination of array comparative genomic hybridization and a candidate gene approach, we identified nuclear factor I/X (NFIX) deletions or nonsense mutation in three sporadic cases of a Sotos-like overgrowth syndrome with advanced bone age, macrocephaly, developmental delay, scoliosis, and unusual facies. Unlike the aforementioned human syndrome, Nfix-deficient mice are unable to gain weight and die in the first 3 postnatal weeks, while they also present with a spinal deformation and decreased bone mineralization. These features prompted us to consider NFIX as a candidate gene for Marshall-Smith syndrome (MSS), a severe malformation syndrome characterized by failure to thrive, respiratory insufficiency, accelerated osseous maturation, kyphoscoliosis, osteopenia, and unusual facies. Distinct frameshift and splice NFIX mutations that escaped nonsense-mediated mRNA decay (NMD) were identified in nine MSS subjects. NFIX belongs to the Nuclear factor one (NFI) family of transcription factors, but its specific function is presently unknown. We demonstrate that NFIX is normally expressed prenatally during human brain development and skeletogenesis. These findings demonstrate that allelic NFIX mutations trigger distinct phenotypes, depending specifically on their impact on NMD.
Assuntos
Anormalidades Múltiplas/genética , Alelos , Códon sem Sentido/genética , Mutação/genética , Fatores de Transcrição NFI/genética , Estabilidade de RNA/genética , Adolescente , Adulto , Sequência de Bases , Criança , Cromossomos Humanos Par 19/genética , Hibridização Genômica Comparativa , Análise Mutacional de DNA , Feminino , Regulação da Expressão Gênica , Testes Genéticos , Humanos , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Fatores de Transcrição NFI/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SíndromeRESUMO
BACKGROUND: Achondroplasia is the most frequent FGFR3-related chondrodysplasia, leading to rhizomelic dwarfism, craniofacial anomalies, stenosis of the foramen magnum, and sleep apnea. Craniofacial growth and its correlation with obstructive sleep apnea syndrome has not been assessed in achondroplasia. In this study, we provide a multimodal analysis of craniofacial growth and anatomo-functional correlations between craniofacial features and the severity of obstructive sleep apnea syndrome. METHODS: A multimodal study was performed based on a paediatric cohort of 15 achondroplasia patients (mean age, 7.8 ± 3.3 years), including clinical and sleep study data, 2D cephalometrics, and 3D geometric morphometry analyses, based on CT-scans (mean age at CT-scan: patients, 4.9 ± 4.9 years; controls, 3.7 ± 4.2 years). RESULTS: Craniofacial phenotype was characterized by maxillo-zygomatic retrusion, deep nasal root, and prominent forehead. 2D cephalometric studies showed constant maxillo-mandibular retrusion, with excessive vertical dimensions of the lower third of the face, and modifications of cranial base angles. All patients with available CT-scan had premature fusion of skull base synchondroses. 3D morphometric analyses showed more severe craniofacial phenotypes associated with increasing patient age, predominantly regarding the midface-with increased maxillary retrusion in older patients-and the skull base-with closure of the spheno-occipital angle. At the mandibular level, both the corpus and ramus showed shape modifications with age, with shortened anteroposterior mandibular length, as well as ramus and condylar region lengths. We report a significant correlation between the severity of maxillo-mandibular retrusion and obstructive sleep apnea syndrome (p < 0.01). CONCLUSIONS: Our study shows more severe craniofacial phenotypes at older ages, with increased maxillomandibular retrusion, and demonstrates a significant anatomo-functional correlation between the severity of midface and mandible craniofacial features and obstructive sleep apnea syndrome.
Assuntos
Acondroplasia , Retrognatismo , Síndromes da Apneia do Sono , Apneia Obstrutiva do Sono , Humanos , Cefalometria , Acondroplasia/genéticaRESUMO
Hypochondroplasia (HCH) is a mild dwarfism caused by missense mutations in fibroblast growth factor receptor 3 (FGFR3), with the majority of cases resulting from a heterozygous p.Asn540Lys gain-of-function mutation. Here, we report the generation and characterization of the first mouse model (Fgfr3Asn534Lys/+) of HCH to our knowledge. Fgfr3Asn534Lys/+ mice exhibited progressive dwarfism and impairment of the synchondroses of the cranial base, resulting in defective formation of the foramen magnum. The appendicular and axial skeletons were both severely affected and we demonstrated an important role of FGFR3 in regulation of cortical and trabecular bone structure. Trabecular bone mineral density (BMD) of long bones and vertebral bodies was decreased, but cortical BMD increased with age in both tibiae and femurs. These results demonstrate that bones in Fgfr3Asn534Lys/+ mice, due to FGFR3 activation, exhibit some characteristics of osteoporosis. The present findings emphasize the detrimental effect of gain-of-function mutations in the Fgfr3 gene on long bone modeling during both developmental and aging processes, with potential implications for the management of elderly patients with hypochondroplasia and osteoporosis.
Assuntos
Nanismo , Osteoporose , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos , Animais , Camundongos , Calcificação Fisiológica , Nanismo/genética , Mutação com Ganho de Função , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genéticaRESUMO
Achondroplasia, the most common chondrodysplasia in humans, is caused by one of two gain of function mutations localized in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) leading to constitutive activation of FGFR3 and subsequent growth plate cartilage and bone defects. Phenotypic features of achondroplasia include macrocephaly with frontal bossing, midface hypoplasia, disproportionate shortening of the extremities, brachydactyly with trident configuration of the hand, and bowed legs. The condition is defined primarily on postnatal effects on bone and cartilage, and embryonic development of tissues in affected individuals is not well studied. Using the Fgfr3Y367C/+ mouse model of achondroplasia, we investigated the developing chondrocranium and Meckel's cartilage (MC) at embryonic days (E)14.5 and E16.5. Sparse hand annotations of chondrocranial and MC cartilages visualized in phosphotungstic acid enhanced three-dimensional (3D) micro-computed tomography (microCT) images were used to train our automatic deep learning-based 3D segmentation model and produce 3D isosurfaces of the chondrocranium and MC. Using 3D coordinates of landmarks measured on the 3D isosurfaces, we quantified differences in the chondrocranium and MC of Fgfr3Y367C/+ mice relative to those of their unaffected littermates. Statistically significant differences in morphology and growth of the chondrocranium and MC were found, indicating direct effects of this Fgfr3 mutation on embryonic cranial and pharyngeal cartilages, which in turn can secondarily affect cranial dermal bone development. Our results support the suggestion that early therapeutic intervention during cartilage formation may lessen the effects of this condition.
RESUMO
Central nervous system anomalies in Pfeiffer syndrome (PS) due to mutations in the FGFR2 gene are poorly understood, even though PS is often associated with serious cognitive impairment. The aim of this study is to describe the neuropathological phenotype in PS. We present four severe fetal cases of sporadic PS with FGFR2 mutations who underwent termination followed by fetopathological and neuropathological examination. We studied the expression pattern of Fgfr2 in the mouse brain using radioactive fluorescence in situ hybridization. PS is associated with brain deformations due to the abnormal skull shape, but FGFR2 mutations also induce specific brain developmental anomalies: megalencephaly, midline disorders, amygdala, and hippocampus malformations, and ventricular wall alterations. The expression pattern of Fgfr2 in mice matches the distribution of malformations in humans. The brain anomalies in PS result from the combination of mechanical deformations and intrinsic developmental disorders due to FGFR2 hyperactivity. Several similarities are noted between these anomalies and the brain lesions observed in other syndromes due to mutations in FGF-receptor genes. The specific involvement of the hippocampus and the amygdala should encourage the precise cognitive screening of patients with mild forms of PS.
Assuntos
Encéfalo/anormalidades , Craniossinostoses/genética , Craniossinostoses/patologia , Mutação , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Feto Abortado , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Craniossinostoses/diagnóstico , Estudos de Associação Genética , Humanos , Camundongos , Fenótipo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
Objective: Faciocraniosynostoses (FCS) are malformations affecting the development of the bones of the skull and face, due to the premature closure of one or more craniofacial sutures, mostly secondary to activating Fibroblast Growth Factor Receptor (FGFR) 1-3 mutations. Gain-of-function FGFR3 mutations are also responsible for various conditions referred to as osteochondrodysplasia (OCD), characterized by structural and functional abnormalities of growth plate cartilages. We hypothesized that patients with FGFR-related faciocraniosynostoses may present extra-cranial growth anomalies. Study design: We retrospectively collected height and weight data from a cohort of 70 patients. Included patients were admitted for FGFR-related FCS between 2000 and 2021 at the Craniofacial Unit of Necker - Enfants Malades University Hospital in Paris, France. Results: We showed that FGFR-related faciocraniosynostoses had significantly reduced heights and weights relative to controls, and that two specific time periods (1-3 years and > 8 years of age) were associated with lower height and weight values. Four patients had received growth hormone treatment but remained below normal values for growth in height and weight. Conclusions: Patients with FGFR-related faciocraniosynostoses have clinically significant extra-cranial anomalies which are not currently investigated and managed in usual protocols; these patients could benefit from a systematic pre-pubertal endocrine assessment. More generally, our results extend the scope of extracranial anomalies in FGFR-related faciocraniosynostoses and support the hypothesis that all conditions with activating FGFR mutations affect both membranous ossification and long bones.
RESUMO
Crouzon syndrome with acanthosis nigricans (CAN, a rare type of craniosynostosis characterized by premature suture fusion and neurological impairments) has been linked to a gain-of-function mutation (p.Ala391Glu) in fibroblast growth factor receptor 3 (FGFR3). To characterize the CAN mutation's impact on the skull and on brain functions, we developed the first mouse model (Fgfr3A385E/+) of this syndrome. Surprisingly, Fgfr3A385E/+ mice did not exhibit craniosynostosis but did show severe memory impairments, a structurally abnormal hippocampus, low activity-dependent synaptic plasticity, and overactivation of MAPK/ERK and Akt signaling pathways in the hippocampus. Systemic or brain-specific pharmacological inhibition of FGFR3 overactivation by BGJ398 injections rescued the memory impairments observed in Fgfr3A385E/+ mice. The present study is the first to have demonstrated cognitive impairments associated with brain FGFR3 overactivation, independently of skull abnormalities. Our results provide a better understanding of FGFR3's functional role and the impact of its gain-of-function mutation on brain functions. The modulation of FGFR3 signaling might be of value for treating the neurological disorders associated with craniosynostosis.
Assuntos
Acantose Nigricans , Disostose Craniofacial , Craniossinostoses , Acantose Nigricans/complicações , Acantose Nigricans/genética , Animais , Encéfalo , Disostose Craniofacial/complicações , Disostose Craniofacial/genética , Craniossinostoses/genética , Modelos Animais de Doenças , Transtornos da Memória/genética , Camundongos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genéticaRESUMO
A gain-of-function mutation in the fibroblast growth factor receptor 3 gene (FGFR3) results in achondroplasia (ACH), the most frequent form of dwarfism. Constitutive activation of FGFR3 impairs bone formation and elongation and many signal transduction pathways. Identification of new and relevant compounds targeting the FGFR3 signaling pathway is of broad importance for the treatment of ACH, and natural plant compounds are prime drug candidate sources. Here, we found that the phenolic compound (-)-epicatechin, isolated from Theobroma cacao, effectively inhibited FGFR3's downstream signaling pathways. Transcriptomic analysis in an Fgfr3 mouse model showed that ciliary mRNA expression was modified and influenced significantly by the Indian hedgehog and PKA pathways. (-)-Epicatechin is able to rescue mRNA expression impairments that control both the structural organization of the primary cilium and ciliogenesis-related genes. In femurs isolated from a mouse model (Fgfr3Y367C/+) of ACH, we showed that (-)-epicatechin eliminated bone growth impairment during 6 days of ex vivo culture. In vivo, we confirmed that daily subcutaneous injections of (-)-epicatechin to Fgfr3Y367C/+ mice increased bone elongation and rescued the primary cilium defects observed in chondrocytes. This modification to the primary cilia promoted the typical columnar arrangement of flat proliferative chondrocytes and thus enhanced bone elongation. The results of the present proof-of-principle study support (-)-epicatechin as a potential drug for the treatment of ACH.