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1.
Med Sci Monit ; 25: 6980-6989, 2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31527569

RESUMO

BACKGROUND The pathogenesis of idiopathic congenital clubfoot (CCF) is unknown. Although some familial patients have Pitx1 mutations, and the Pitx1+/- genotype causes a clubfoot-like phenotype in mice, the mechanism of Pitx1-induced CCF is unknown. MATERIAL AND METHODS We used tibialis anterior tendon samples to detect the expression of Pitx1 in idiopathic and neurogenic clubfoot patients. After obtaining Sprague-Dawley (SD) rat Achilles tendon cells, the expression of Pitx1 was knocked down by SiRNA. After 48 h of culture, mass spectrometry was used to quantitatively analyze proteins. Then, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to assess the downstream pathway of PITX1. The relationship between Pitx1 and the promoter region of deacetylase 1 (Sirtuin-1 and Sirt1) was examined by luciferase and ChIP assays. RESULTS We found that Pitx1 expression in the tendon samples of idiopathic CCF patients was downregulated. Mass spectrometry analysis revealed that the inhibition of Pitx1 induced the downregulation of Sirt1 expression in tendon cells. Luciferase and ChIP assays confirmed that Pitx1 binds to the promoter region of SIRT1 and promotes Sirt1 gene transcription. Further results showed that, after the inhibition of Pitx1 in tendon cells, CRABP2 acetylation increased, the nuclear import of CRABP2 was enhanced, and the expression of RARß2 increased. After the inhibition of Pitx1, RARß2 expression was further increased by RA treatment in tendon cells. In the presence of retinoic acid, the expression of Pitx1 was inhibited in tendon cells. CONCLUSIONS Pitx1 binds to the promoter region of SIRT1 and promotes the transcription of SIRT1. Positive feedback occurs between RA signaling and Pitx1.


Assuntos
Pé Torto Equinovaro/metabolismo , Pé Torto Equinovaro/patologia , Fatores de Transcrição Box Pareados/metabolismo , Transdução de Sinais , Tendões/patologia , Tretinoína/metabolismo , Animais , Animais Recém-Nascidos , Transporte Biológico , Criança , Pré-Escolar , Retroalimentação Fisiológica , Humanos , Masculino , Regiões Promotoras Genéticas/genética , Ratos Sprague-Dawley , Sirtuína 1/genética , Sirtuína 1/metabolismo
2.
J Orthop Res ; 37(3): 769-778, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30615219

RESUMO

Idiopathic pes equinovarus (clubfoot) is a congenital deformity of the feet and lower legs. Clubfoot belongs to a group of fibro-proliferative disorders but its origin remains unknown. Our study aimed to achieve the first complex proteomic comparison of clubfoot contracted tissue of the foot (medial side; n = 16), with non-contracted tissue (lateral side; n = 13). We used label-free mass spectrometry quantification and immunohistochemistry. Seven proteins were observed to be significantly upregulated in the medial side (asporin, collagen type III, V, and VI, versican, tenascin-C, and transforming growth factor beta induced protein) and four in the lateral side (collagen types XII and XIV, fibromodulin, and cartilage intermediate layer protein 2) of the clubfoot. Comparison of control samples from cadavers brought only two different protein concentrations (collagen types I and VI). We also revealed pathological calcification and intracellular positivity of transforming growth factor beta only in the contracted tissue of clubfoot. Most of the 11 differently expressed proteins are strongly related to the extracellular matrix architecture and we assume that they may play specific roles in the pathogenesis of this deformity. These proteins seem to be promising targets for future investigations and treatment of this disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.


Assuntos
Pé Torto Equinovaro/etiologia , Proteínas da Matriz Extracelular/metabolismo , Calcinose , Criança , Pré-Escolar , Pé Torto Equinovaro/metabolismo , Feminino , Humanos , Masculino , Espectrometria de Massas , Estudos Prospectivos , Proteoma , Fator de Crescimento Transformador beta/metabolismo
3.
Int J Mol Sci ; 19(8)2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30071673

RESUMO

Lymphedema is characterized by chronic swelling of any body part caused by malfunctioning or obstruction in the lymphatic system. Primary lymphedema is often considered genetic in origin. VEGFC, which is a gene encoding the ligand for the vascular endothelial growth factor receptor 3 (VEGFR3/FLT4) and important for lymph vessel development during lymphangiogenesis, has been associated with a specific subtype of primary lymphedema. Through Sanger sequencing of a proband with bilateral congenital pedal edema resembling Milroy disease, we identified a novel mutation (NM_005429.2; c.361+5G>A) in VEGFC. The mutation induced skipping of exon 2 of VEGFC resulting in a frameshift and the introduction of a premature stop codon (p.Ala50ValfsTer18). The mutation leads to a loss of the entire VEGF-homology domain and the C-terminus. Expression of this Vegfc variant in the zebrafish floorplate showed that the splice-site variant significantly reduces the biological activity of the protein. Our findings confirm that the splice-site variant, c.361+5G>A, causes the primary lymphedema phenotype in the proband. We examine the mutations and clinical phenotypes of the previously reported cases to review the current knowledge in this area.


Assuntos
Artrogripose/genética , Fissura Palatina/genética , Pé Torto Equinovaro/genética , Mutação da Fase de Leitura , Deformidades Congênitas da Mão/genética , Processamento de RNA/genética , Fator C de Crescimento do Endotélio Vascular/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Artrogripose/metabolismo , Artrogripose/patologia , Pré-Escolar , Fissura Palatina/metabolismo , Fissura Palatina/patologia , Pé Torto Equinovaro/metabolismo , Pé Torto Equinovaro/patologia , Feminino , Deformidades Congênitas da Mão/metabolismo , Deformidades Congênitas da Mão/patologia , Humanos , Lactente , Recém-Nascido , Masculino , Domínios Proteicos , Fator C de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
4.
Physiol Rep ; 6(14): e13728, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30030908

RESUMO

Active reabsorption of magnesium (Mg2+ ) in the distal convoluted tubule (DCT) of the kidney is crucial for maintaining Mg2+ homeostasis. Impaired activity of the Na+ -Cl- -cotransporter (NCC) has been associated with hypermagnesiuria and hypomagnesemia, while increased activity of NCC, as observed in patients with Gordon syndrome, is not associated with alterations in Mg2+ balance. To further elucidate the possible interrelationship between NCC activity and renal Mg2+ handling, plasma Mg2+ levels and urinary excretion of sodium (Na+ ) and Mg2+ were measured in a mouse model of Gordon syndrome. In this model, DCT1-specific expression of a constitutively active mutant form of the NCC-phosphorylating kinase, SPAK (CA-SPAK), increases NCC activity and hydrochlorothiazide (HCTZ)-sensitive Na+ reabsorption. These mice were normomagnesemic and HCTZ administration comparably reduced plasma Mg2+ levels in CA-SPAK mice and control littermates. As inferred by the initial response to HCTZ, CA-SPAK mice exhibited greater NCC-dependent Na+ reabsorption together with decreased Mg2+ reabsorption, compared to controls. Following prolonged HCTZ administration (4 days), CA-SPAK mice exhibited higher urinary Mg2+ excretion, while urinary Na+ excretion decreased to levels observed in control animals. Surprisingly, CA-SPAK mice had unaltered renal expression of Trpm6, encoding the Mg2+ -permeable channel TRPM6, or other magnesiotropic genes. In conclusion, CA-SPAK mice exhibit normomagnesemia, despite increased NCC activity and Na+ reabsorption. Thus, Mg2+ reabsorption is not coupled to increased thiazide-sensitive Na+ reabsorption, suggesting a similar process explains normomagnesemia in Gordon syndrome. Further research is required to unravel the molecular underpinnings of this phenomenon and the more pronounced Mg2+ excretion after prolonged HCTZ administration.


Assuntos
Artrogripose/metabolismo , Fissura Palatina/metabolismo , Pé Torto Equinovaro/metabolismo , Deformidades Congênitas da Mão/metabolismo , Magnésio/metabolismo , Reabsorção Renal , Sódio/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Feminino , Hidroclorotiazida/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Inibidores de Simportadores de Cloreto de Sódio/farmacologia , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo
5.
J Cell Biochem ; 119(5): 3809-3818, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29274279

RESUMO

RBM10 is an RNA binding motif (RBM) protein expressed in most, if not all, human and animal cells. Interest in RBM10 is rapidly increasing and its clinical importance is highlighted by its identification as the causative agent of TARP syndrome, a developmental condition that significantly impacts affected children. RBM10's cellular functions are beginning to be explored, with initial studies demonstrating a tumor suppressor role. Very recently, however, contradictory results have emerged, suggesting a tumor promoter role for RBM10. In this review, we describe the current state of knowledge on RBM10, and address this dichotomy in RBM10 function. Furthermore, we discuss what may be regulating RBM10 function, particularly the importance of RBM10 alternative splicing, and the relationship between RBM10 and its paralogue, RBM5. As RBM10-related work is gaining momentum, it is critical that the various aspects of RBM10 molecular biology revealed by recent studies be considered moving forward. It is only if these recent advances in RBM10 structure and function are considered that a clearer insight into RBM10 function, and the disease states with which RBM10 mutation is associated, will be gained.


Assuntos
Processamento Alternativo , Pé Torto Equinovaro , Cardiopatias Congênitas , Mutação , Síndrome de Pierre Robin , Proteínas de Ligação a RNA , Animais , Pé Torto Equinovaro/genética , Pé Torto Equinovaro/metabolismo , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Humanos , Síndrome de Pierre Robin/genética , Síndrome de Pierre Robin/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Relação Estrutura-Atividade
6.
Neuron ; 93(5): 989-991, 2017 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-28279361

RESUMO

In this issue of Neuron, Ben-Yaacov et al. (2017) dissect the interaction between AMPA receptors and auxiliary (TARP) subunits, revealing essential roles for the receptor transmembrane and cytoplasmic domains, as well as for the TARP extracellular EX2 loop.


Assuntos
Canais de Cálcio/metabolismo , Pé Torto Equinovaro/metabolismo , Cardiopatias Congênitas/metabolismo , Neurônios/metabolismo , Síndrome de Pierre Robin/metabolismo , Subunidades Proteicas/metabolismo , Receptores de AMPA/metabolismo , Animais , Pé Torto Equinovaro/genética , Cardiopatias Congênitas/genética , Humanos , Proteínas de Membrana/metabolismo , Síndrome de Pierre Robin/genética
7.
Hum Mol Genet ; 26(12): 2177-2191, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28334780

RESUMO

Biallelic loss-of-function mutations in the RNA-binding protein EIF4A3 cause Richieri-Costa-Pereira syndrome (RCPS), an autosomal recessive condition mainly characterized by craniofacial and limb malformations. However, the pathogenic cellular mechanisms responsible for this syndrome are entirely unknown. Here, we used two complementary approaches, patient-derived induced pluripotent stem cells (iPSCs) and conditional Eif4a3 mouse models, to demonstrate that defective neural crest cell (NCC) development explains RCPS craniofacial abnormalities. RCPS iNCCs have decreased migratory capacity, a distinct phenotype relative to other craniofacial disorders. Eif4a3 haploinsufficient embryos presented altered mandibular process fusion and micrognathia, thus recapitulating the most penetrant phenotypes of the syndrome. These defects were evident in either ubiquitous or NCC-specific Eif4a3 haploinsufficient animals, demonstrating an autonomous requirement of Eif4a3 in NCCs. Notably, RCPS NCC-derived mesenchymal stem-like cells (nMSCs) showed premature bone differentiation, a phenotype paralleled by premature clavicle ossification in Eif4a3 haploinsufficient embryos. Likewise, nMSCs presented compromised in vitro chondrogenesis, and Meckel's cartilage was underdeveloped in vivo. These findings indicate novel and essential requirements of EIF4A3 for NCC migration and osteochondrogenic differentiation during craniofacial development. Altogether, complementary use of iPSCs and mouse models pinpoint unique cellular mechanisms by which EIF4A3 mutation causes RCPS, and provide a paradigm to study craniofacial disorders.


Assuntos
Pé Torto Equinovaro/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Deformidades Congênitas da Mão/genética , Síndrome de Pierre Robin/genética , Animais , Osso e Ossos/metabolismo , Região Branquial/metabolismo , Diferenciação Celular/genética , Movimento Celular , Condrogênese/genética , Pé Torto Equinovaro/metabolismo , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/metabolismo , Modelos Animais de Doenças , Deformidades Congênitas da Mão/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Crista Neural/crescimento & desenvolvimento , Crista Neural/metabolismo , Osteogênese/genética , Síndrome de Pierre Robin/metabolismo
8.
Clin Orthop Relat Res ; 474(7): 1726-35, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27020427

RESUMO

BACKGROUND: Isolated nonsyndromic clubfoot is a common birth defect affecting 135,000 newborns worldwide each year. Although treatment has improved, substantial long-term morbidity persists. Genetic causes have been implicated in family-based studies but the genetic changes have eluded identification. Previously, using a candidate gene approach in our family-based dataset, we identified associations between clubfoot and four single nucleotide polymorphisms (SNPs) located in potential regulatory regions of genes involved in muscle development and patterning (HOXA9) and muscle function (TPM1 and TPM2) were identified. QUESTIONS/PURPOSES: Four SNPs, rs3801776/HOXA9, rs4075583/TPM1, rs2025126/TPM2, and rs2145925/TPM2, located in potential regulatory regions, were evaluated to determine whether they altered promoter activity. METHODS: Electrophoretic mobility shift assays were performed on these four SNPs to identify allele-specific DNA-protein interactions. SNPs showing differential banding patterns were assessed for effect on promoter activity by luciferase assay. Undifferentiated (for HOXA9) and differentiated (for TPM1 and TPM2) mouse cells were used in functional assays as a proxy for the in vivo developmental stage. RESULTS: Functional analyses showed that the ancestral alleles of rs3801776/HOXA9, rs4075583/TPM1, and rs2025126/TPM2 and the alternate allele of rs2145925/TPM2 created allele-specific nuclear protein interactions and caused higher promoter activity. Interestingly, while rs4075583/TPM1 showed an allele-specific nuclear protein interaction, an effect on promoter activity was observed only when rs4075583/TPM1 was expressed in the 1.7kb haplotype construct. CONCLUSION: Our results show that associated promoter variants in HOXA9, TPM1, and TPM2, alter promoter expression suggesting that they have a functional role. Moreover and importantly, we show that alterations in promoter activity may be observed only in the context of the genomic architecture. Therefore, future studies focusing on proteins binding to these regulatory SNPs may provide important key insights into gene regulation in clubfoot. CLINICAL RELEVANCE: Identifying the genetic risk signature for clubfoot is important to provide accurate genetic counseling for at-risk families, for development of prevention programs and new treatments.


Assuntos
Pé Torto Equinovaro/genética , Proteínas de Homeodomínio/genética , Polimorfismo de Nucleotídeo Único , Tropomiosina/genética , Animais , Sítios de Ligação , Linhagem Celular , Pé Torto Equinovaro/diagnóstico , Pé Torto Equinovaro/metabolismo , Bases de Dados Genéticas , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica , Frequência do Gene , Genes Reporter , Estudos de Associação Genética , Marcadores Genéticos , Predisposição Genética para Doença , Haplótipos , Proteínas de Homeodomínio/metabolismo , Humanos , Luciferases/biossíntese , Luciferases/genética , Camundongos , Fenótipo , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Risco , Transfecção , Tropomiosina/metabolismo
9.
Clin Genet ; 88(5): 405-15, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25865758

RESUMO

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNA transcripts. Mutations in EFTUD2, encoding a component of the major spliceosome, have recently been identified as the cause of mandibulofacial dysostosis, Guion-Almeida type (MFDGA), characterized by mandibulofacial dysostosis, microcephaly, external ear malformations and intellectual disability. Mutations in several other genes involved in spliceosomal function or linked aspects of mRNA processing have also recently been identified in human disorders with specific craniofacial malformations: SF3B4 in Nager syndrome, an acrofacial dysostosis (AFD); SNRPB in cerebrocostomandibular syndrome, characterized by Robin sequence and rib defects; EIF4A3 in the AFD Richieri-Costa-Pereira syndrome, characterized by Robin sequence, median mandibular cleft and limb defects; and TXNL4A in Burn-McKeown syndrome, involving specific craniofacial dysmorphisms. Here, we review phenotypic and molecular aspects of these syndromes. Given the apparent sensitivity of craniofacial development to defects in mRNA processing, it is possible that mutations in other proteins involved in spliceosomal function will emerge in the future as causative for related human disorders.


Assuntos
Atresia das Cóanas/metabolismo , Pé Torto Equinovaro/metabolismo , Surdez/congênito , Deformidades Congênitas da Mão/metabolismo , Cardiopatias Congênitas/metabolismo , Deficiência Intelectual/metabolismo , Disostose Mandibulofacial/metabolismo , Micrognatismo/metabolismo , Mutação , Síndrome de Pierre Robin/metabolismo , Costelas/anormalidades , Spliceossomos/metabolismo , Atresia das Cóanas/genética , Pé Torto Equinovaro/genética , RNA Helicases DEAD-box/genética , Surdez/genética , Surdez/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Facies , Feminino , Deformidades Congênitas da Mão/genética , Cardiopatias Congênitas/genética , Humanos , Deficiência Intelectual/genética , Masculino , Disostose Mandibulofacial/genética , Micrognatismo/genética , Fatores de Alongamento de Peptídeos/genética , Síndrome de Pierre Robin/genética , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U5/genética , Costelas/metabolismo , Spliceossomos/genética
10.
Mol Cell Biochem ; 401(1-2): 133-9, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25472880

RESUMO

Idiopathic pes equinovarus is a congenital deformity of the foot and lower leg defined as a fixation of the foot in adduction, supination, and varus. Although the pathogenesis of clubfoot remains unclear, it has been suggested that fibroblasts and growth factors are involved. To directly analyze the protein composition of the extracellular matrix in contracted tissue of patients with clubfoot. A total of 13 infants with idiopathic clubfoot treated with the Ponseti method were included in the present study. Tissue samples were obtained from patients undergoing surgery for relapsed clubfeet. Contracted tissues were obtained from the medial aspect of the talonavicular joint. Protein was extracted after digestion and delipidation using zip-tip C18. Individual collagenous fractions were detected using a chemiluminescent assay. Amino acid analysis of tissue samples revealed a predominance of collagens, namely collagen types I, III, and VI. The high content of glycine and h-proline suggests a predominance of collagens I and III. A total of 19 extracellular matrix proteins were identified. The major result of the present study was the observation that the extracellular matrix in clubfoot is composed of an additional 16 proteins, including collagens V, VI, and XII, as well as the previously described collagen types I and III and transforming growth factor ß. The characterization of the general protein composition of the extracellular matrix in various regions of clubfoot may help in understanding the pathogenesis of this anomaly and, thus, contribute to the development of more efficacious therapeutic approaches.


Assuntos
Pé Torto Equinovaro/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteômica/métodos , Aminoácidos/análise , Pé Torto Equinovaro/patologia , Pé Torto Equinovaro/terapia , Colágeno/metabolismo , Feminino , Humanos , Lactente , Masculino , Fator de Crescimento Transformador beta/metabolismo
11.
Int J Clin Exp Pathol ; 7(2): 677-84, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24551289

RESUMO

OBJECTIVE: To investigate the apoptotic gene expression of placenta in an all-trans-retinoic acid (ATRA) induced fetus congenital clubfoot pregnant rat model. METHODS: Sprague-Dawley (SD) rats were divided randomly into ATRA-exposed group and control group. On day 10 of pregnancy, a dose of 120 mg/kg ATRA dissolved in mineral oil was given intragastrically to the rats in the ATRA-exposed group and equivalent volume of mineral oil was given intragastrically to the control rats. Fetuses were delivered on day 20 of pregnancy, the placenta was collected for the pathological and biochemical analysis. RESULTS: Clubfoot-like deformity fetuses were observed in the ATRA-exposed group and none with deformity was found in the control group. The pro-apoptosis in placenta of ATRA-exposed group was measured by flow cytometry. Moreover, compared with the control group, lower expression of Bcl-2 and higher expression of BAX were found in the ATRA-exposed group in both mRNA and protein level. Immunohistochemical labeling of Bcl-2 in the control group was more intense while BAX labeling in the ATRA-exposed group was more intense. Additionally, the caspase-3 activity was also significantly increased in the ATRA-exposed group than control group. CONCLUSION: In our research, we found a pro-apoptosis in placenta in the ATRA-exposed pregnant rats, indicating a possible association between placental apoptosis and congenital clubfoot.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Pé Torto Equinovaro/metabolismo , Placenta/metabolismo , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Caspase 3/metabolismo , Pé Torto Equinovaro/induzido quimicamente , Pé Torto Equinovaro/genética , Pé Torto Equinovaro/patologia , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Imuno-Histoquímica , Placenta/patologia , Gravidez , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Tretinoína , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
12.
Mol Med Rep ; 7(3): 821-5, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23254326

RESUMO

The collagen, type IX, alpha 1 (COL9A1) gene was previously identified as a candidate gene for idiopathic congenital talipes equinovarus (ICTEV), a congenital lower limb deformity in humans. In the present study, increased expression levels of COL9A1 were identified in the abductor hallucis muscle of ICTEV patients compared with those in control samples. The COL9A1 gene is regulated by SRY (sex­determining region Y)­box 9 (SOX9). Immunofluorescence analysis of SOX9 and COL9A1 proteins identified colocalization to the sarcolemma, endomysium and muscle membrane in muscle samples of ICTEV. No mutations in the exons and promoters of SOX9 were detected in blood samples of 84 ICTEV patients by denaturing gradient gel electrophoresis. mRNA and protein expression levels of SOX9 were detected by real­time polymerase chain reaction and western blot analysis, respectively and were found to be significantly higher in ICTEV muscle samples compared with those in control samples. Based on present observations, we hypothesize that overexpression of the SOX9 gene may play a role in the genetic etiology of ICTEV.


Assuntos
Pé Torto Equinovaro/metabolismo , Fatores de Transcrição SOX9/metabolismo , Criança , Pré-Escolar , Pé Torto Equinovaro/patologia , Colágeno Tipo IX/genética , Colágeno Tipo IX/metabolismo , Feminino , Humanos , Masculino , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOX9/genética , Sarcolema/metabolismo
13.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 29(3): 260-5, 2012 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-22678783

RESUMO

OBJECTIVE: To investigate the relationship between GLI3 gene and pathogenesis of idiopathic congenital talipes equinovarus (ICTEV). METHOD: Potential mutations in the coding region of GLI3 were detected among 84 patients with ICTEV by denaturing gradient electrophoresis. Expression of GLI3 in the ICTEV patients' disease tissues was assessed by reverse transcription PCR. Following generation of rat model for ICTEV, mRNA and protein levels of GLI3 were evaluated by real-time PCR and immunohistochemistry and Western blotting. RESULTS: No mutation was found in exons 1 - 8 and 13 of GLI3 gene among the 84 ICTEV patients. No expression of GLI3 gene was detected in the flexor hallucis longus of ICTEV patients or normal controls. Expression of Gli3, in terms of both mRNA and protein, was stronger in the hindlimb of ICTEV rat embryos compared with normal controls. CONCLUSION: Mutation in the coding region of GLI3 may not be responsible for the occurrence of ICTEV. However, there may still be connection between abnormal expression of the gene and pathogenesis of ICTEV.


Assuntos
Pé Torto Equinovaro/genética , Fatores de Transcrição Kruppel-Like/genética , Proteínas do Tecido Nervoso/genética , Animais , Pé Torto Equinovaro/metabolismo , Pé Torto Equinovaro/patologia , Expressão Gênica , Predisposição Genética para Doença , Humanos , Fatores de Transcrição Kruppel-Like/biossíntese , Mutação , Proteínas do Tecido Nervoso/biossíntese , Ratos , Ratos Wistar , Proteína Gli3 com Dedos de Zinco
14.
Scand J Clin Lab Invest ; 71(7): 576-82, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21834619

RESUMO

BACKGROUND: We aimed to investigate serum prolidase activity and to find out its association with oxidative-antioxidative status in patients with idiopathic clubfoot and during the course of the disease. MATERIAL AND METHODS: Oxidative status parameters, including total free sulfhydryl groups (-SH), total antioxidant capacity (TAC), total oxidant status (TOS), and oxidative stress index (OSI), as well as serum prolidase activity were assessed at the beginning of the treatment in patients with idiopathic clubfoot (n = 38), at the end of 3 months during the treatment of the disease and in healthy controls (n = 40). All patients were managed with the Ponseti method and severity of the foot deformity was evaluated according to the Pirani Severity Score. RESULTS: Serum prolidase activity, TOS and OSI values of the patients at the beginning of the treatment were found to be significantly higher but -SH and TAC values were found to be significantly lower as compared to controls. In the treatment process, a significant decrease in serum prolidase activity, TOS and OSI values and Pirani Severity Score of the patients was observed, however a significant increase in -SH and TAC values of the patients was observed at the end of 3 months during the treatment of the disease as compared to the beginning of the treatment. CONCLUSION: Elevated levels of serum prolidase activity, TOS and OSI, and decreased levels of -SH and TAC may be associated with idiopathic clubfoot, and that these parameters may be useful adjunctive tools for follow-up in patients with idiopathic clubfoot.


Assuntos
Antioxidantes/metabolismo , Pé Torto Equinovaro/metabolismo , Colágeno/metabolismo , Dipeptidases/metabolismo , Antioxidantes/análise , Estudos de Casos e Controles , Moldes Cirúrgicos , Pé Torto Equinovaro/patologia , Pé Torto Equinovaro/fisiopatologia , Pé Torto Equinovaro/terapia , Dipeptidases/análise , Feminino , Seguimentos , Humanos , Lactente , Recém-Nascido , Masculino , Oxirredução , Estresse Oxidativo , Índice de Gravidade de Doença , Compostos de Sulfidrila/análise , Compostos de Sulfidrila/metabolismo , Turquia
15.
Hum Mol Genet ; 20(20): 3943-52, 2011 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-21775501

RESUMO

Clubfoot affects 1 in 1000 live births, although little is known about its genetic or developmental basis. We recently identified a missense mutation in the PITX1 bicoid homeodomain transcription factor in a family with a spectrum of lower extremity abnormalities, including clubfoot. Because the E130K mutation reduced PITX1 activity, we hypothesized that PITX1 haploinsufficiency could also cause clubfoot. Using copy number analysis, we identified a 241 kb chromosome 5q31 microdeletion involving PITX1 in a patient with isolated familial clubfoot. The PITX1 deletion segregated with autosomal dominant clubfoot over three generations. To study the role of PITX1 haploinsufficiency in clubfoot pathogenesis, we began to breed Pitx1 knockout mice. Although Pitx1(+/-) mice were previously reported to be normal, clubfoot was observed in 20 of 225 Pitx1(+/-) mice, resulting in an 8.9% penetrance. Clubfoot was unilateral in 16 of the 20 affected Pitx1(+/-) mice, with the right and left limbs equally affected, in contrast to right-sided predominant hindlimb abnormalities previously noted with complete loss of Pitx1. Peroneal artery hypoplasia occurred in the clubfoot limb and corresponded spatially with small lateral muscle compartments. Tibial and fibular bone volumes were also reduced. Skeletal muscle gene expression was significantly reduced in Pitx1(-/-) E12.5 hindlimb buds compared with the wild-type, suggesting that muscle hypoplasia was due to abnormal early muscle development and not disuse atrophy. Our morphological data suggest that PITX1 haploinsufficiency may cause a developmental field defect preferentially affecting the lateral lower leg, a theory that accounts for similar findings in human clubfoot.


Assuntos
Pé Torto Equinovaro/genética , Haploinsuficiência , Fatores de Transcrição Box Pareados/genética , Fenótipo , Animais , Deleção Cromossômica , Cromossomos Humanos Par 5 , Pé Torto Equinovaro/diagnóstico , Pé Torto Equinovaro/metabolismo , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Estudo de Associação Genômica Ampla , Humanos , Ossos da Perna/patologia , Imagem por Ressonância Magnética , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Atrofia Muscular/genética , Fatores de Transcrição Box Pareados/metabolismo , Linhagem
17.
BMC Musculoskelet Disord ; 10: 142, 2009 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-19925654

RESUMO

BACKGROUND: Idiopathic congenital talipes equinovarus (ICTEV) is a congenital limb deformity. Based on extended transmission disequilibrium testing, Gli-Kruppel family member 3 (Gli3) has been identified as a candidate gene for ICTEV. Here, we verify the role of Gli3 in ICTEV development. METHODS: Using the rat ICTEV model, we analyzed the differences in Gli3 expression levels between model rats and normal control rats. We used luciferase reporter gene assays and ChIP/EMSA assays to analyze the regulatory elements of Gli3. RESULTS: Gli3 showed higher expression levels in ICTEV model rats compared to controls (P < 0.05). We identified repressor and activator regions in the rat Gli3 promoter. The Gli3 promoter also contains two putative Hoxd13 binding sites. Using EMSA, the Hoxd13 binding site 2 was found to directly interact with Hoxd13 in vitro. ChIP assays of the Hoxd13-Gli3 promoter complex from a developing limb confirmed that endogenous Hoxd13 interacts with this region in vivo. CONCLUSION: Our findings suggest that HoxD13 directly interacts with the promoter of Gli3. The increase of Gli3 expression in ICTEV model animal might result from the low expression of HoxD13.


Assuntos
Pé Torto Equinovaro/metabolismo , Membro Posterior/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Cadáver , Células Cultivadas , Criança , Pré-Escolar , Imunoprecipitação da Cromatina , Pé Torto Equinovaro/induzido quimicamente , Pé Torto Equinovaro/genética , Modelos Animais de Doenças , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Membro Posterior/anormalidades , Proteínas de Homeodomínio/genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Masculino , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Gravidez , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fatores de Transcrição/genética , Transfecção , Tretinoína , Proteína Gli3 com Dedos de Zinco
18.
Clin Orthop Relat Res ; 467(5): 1180-5, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19169765

RESUMO

The contracted tissues from clubfeet resemble tissues from other fibroproliferative disorders such as palmar fibromatosis. Beta-catenin-mediated signaling is a crucial pathway controlling the fibroproliferative response in many fibroproliferative disorders. To determine if beta-catenin signaling plays a role in clubfoot, contracted and less contracted tissues from clubfeet were studied using Western analysis to determine the protein level of beta-catenin. Primary cell cultures were established from these tissues, and they were treated with either lithium to increase beta-catenin or Dickkopf-1 to inhibit beta-catenin. RNA was extracted from the cells and analyzed to determine how beta-catenin regulates expression of Type III collagen, an extracellular matrix protein upregulated in contracted clubfoot tissue. There was a more than twofold increase in beta-catenin protein in the contracted tissues. Treatment with either lithium or Dickkopf-1 showed Type III collagen RNA expression positively correlated with the protein level of beta-catenin. These data support the concept that beta-catenin-mediated signaling plays an important role regulating contracture in clubfeet. Because pharmacologic agents are under development to block this signaling pathway, such drugs could be used in cases of severe stiffness to improve range of motion or to decrease the need for radical surgical approaches.


Assuntos
Pé Torto Equinovaro/metabolismo , Células do Tecido Conjuntivo/metabolismo , Contratura , Transdução de Sinais , beta Catenina/metabolismo , Proliferação de Células , Células Cultivadas , Pé Torto Equinovaro/patologia , Pé Torto Equinovaro/fisiopatologia , Colágeno Tipo III/metabolismo , Células do Tecido Conjuntivo/efeitos dos fármacos , Células do Tecido Conjuntivo/patologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Cloreto de Lítio/farmacologia , Fosforilação , RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima
19.
Yi Chuan ; 31(12): 1214-20, 2009 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-20042388

RESUMO

To investigate the role of gene Gli3 in idiopathic congenital talipes equinovarus (ICTEV), we constructed the Gli3 luciferase reporter gene expression vectors to analyze the promoter activity of the rat gene Gli3. The regulatory element in the promoter region of the rat Gli3 was predicted using P-Match software and further verified by ChIP experiment. Meanwhile, the correlation between the rat En1 and ICTEV was evaluated by RT-PCR, immunohistochemistry, and Western blotting analyses. The result from P-Match software prediction showed that only one of the three possible En1 binding sites in Gli3 promoter region was interacted directly with En1 in vivo, which was confirmed by ChIP analysis. The results from RT-PCR, immunohistochemistry and Western blotting analyses suggested that En1 was down-regulated in ICTEV model rats compared to the controls. Our results indicated that En1 might be the negative regulatory element in the upstream of Gli3. The low expression level of EN1 in ICTEV could contribute to the up-regulation of GLI3, which led to the genesis of ICTEV.


Assuntos
Pé Torto Equinovaro/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Animais , Sequência de Bases , Pé Torto Equinovaro/embriologia , Pé Torto Equinovaro/genética , Pé Torto Equinovaro/patologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Dados de Sequência Molecular , Ligação Proteica , Ratos , Ratos Wistar , Proteína Gli3 com Dedos de Zinco
20.
Clin Orthop Relat Res ; 462: 27-31, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17417092

RESUMO

The genetic etiology of idiopathic clubfoot is unknown. There have been cases reported in which both clubfoot and vertical talus appears in the same family; therefore, the genes responsible for vertical talus are reasonable candidates for idiopathic clubfoot. A mutation in HOXD10 was previously identified in a family with isolated congenital vertical talus. To determine whether HOXD10 is involved in the etiology of idiopathic clubfoot, HOXD10 coding and 5' and 3' untranslated regions were resequenced in 190 patients (177 with clubfoot, 10 with sporadic vertical talus, and 3 with both clubfoot and vertical talus), and 160 ethnically matched control subjects. Rare nonsynonymous HOXD10 amino acid substitutions (Leu154Val, Asn202Lys, and Thr175Ala), likely benign variants, were all detected once in patients and control subjects. Nucleotide substitutions were also identified in HOXD10 intronic and 3' untranslated regions, but were not more frequent in cases compared to controls. To investigate the possibility that unsequenced regulatory regions play a role in this disorder, we performed linkage analysis with markers on chromosome 2q near HOXD10 in one large family. We found no evidence of linkage near the HOXD gene cluster on chromosome 2q, suggesting genes other than HOXD10 are responsible for idiopathic clubfoot.


Assuntos
Pé Torto Equinovaro/genética , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Mutação , Tálus/anormalidades , Fatores de Transcrição/genética , Criança , Cromossomos Humanos Par 2 , Pé Torto Equinovaro/diagnóstico , Pé Torto Equinovaro/metabolismo , Análise Mutacional de DNA , Feminino , Ligação Genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Linhagem , Polimorfismo de Nucleotídeo Único/genética , Fatores de Transcrição/metabolismo
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