RESUMO
Meckel syndrome (MKS) is an inherited autosomal recessive hepatorenal fibrocystic syndrome, caused by mutations in TMEM67, characterized by occipital encephalocoele, renal cysts, hepatic fibrosis, and polydactyly. Here we describe an ovine model of MKS, with kidney and liver abnormalities, without polydactyly or occipital encephalocoele. Homozygous missense p.(Ile681Asn; Ile687Ser) mutations identified in ovine TMEM67 were pathogenic in zebrafish phenotype rescue assays. Meckelin protein was expressed in affected and unaffected kidney epithelial cells by immunoblotting, and in primary cilia of lamb kidney cyst epithelial cells by immunofluorescence. In contrast to primary cilia of relatively consistent length and morphology in unaffected kidney cells, those of affected cyst-lining cells displayed a range of short and extremely long cilia, as well as abnormal morphologies, such as bulbous regions along the axoneme. Putative cilia fragments were also consistently located within the cyst luminal contents. The abnormal ciliary phenotype was further confirmed in cultured interstitial fibroblasts from affected kidneys. These primary cilia dysmorphologies and length control defects were significantly greater in affected cells compared to unaffected controls. In conclusion, we describe abnormalities involving primary cilia length and morphology in the first reported example of a large animal model of MKS, in which we have identified TMEM67 mutations.
Assuntos
Anormalidades Múltiplas/genética , Síndrome de Dandy-Walker/genética , Síndrome Hepatorrenal/genética , Proteínas de Membrana/genética , Mutação/genética , Cisto Pancreático/genética , Anormalidades Múltiplas/patologia , Substituição de Aminoácidos , Animais , Sequência de Bases , Cromossomos de Mamíferos/genética , Cílios/patologia , Síndrome de Dandy-Walker/patologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Loci Gênicos , Complexo de Golgi/metabolismo , Síndrome Hepatorrenal/patologia , Homozigoto , Rim/patologia , Proteínas de Membrana/química , Mutação de Sentido Incorreto/genética , Cisto Pancreático/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ovinos , Peixe-ZebraAssuntos
Canais Iônicos/genética , Proteínas de Membrana/fisiologia , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/fisiopatologia , Proteínas/fisiologia , Animais , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Doenças Renais Policísticas/metabolismo , Proteínas/química , Proteínas/genética , Canais de Cátion TRPPRESUMO
PAX2, a member of the PAX gene family of developmental transcription factors, is expressed at high levels in the developing eyes, ears, central nervous and urogenital systems, as well as in Wilms' tumor and renal cell carcinoma. Expression of PAX2 in the urogenital system is associated with proliferating cells of the ureteric bud and the differentiating nephrogenic mesenchyme. To date, little is known about the molecular mechanisms controlling the regulation of PAX2 expression. This report describes the cloning and characterization of the human PAX2 gene promoter and localization of the transcription start sites in fetal kidney and Wilms' tumor. We identified two transcription start sites in a Wilms' tumor sample, which were found to be different from that in fetal kidney. The activity of a deletion series of the PAX2 promoter was assessed in NIH-3T3, COS-7, 293, and Madin-Darby canine kidney cells. Although some differences were observed in the activity of each promoter construct, the profile of activity for the promoter fragment series was similar in each experiment, regardless of cell type. The WT1 tumor suppressor protein, which has previously been shown to repress murine Pax2 expression in vitro, was shown to also repress expression from the human PAX2 promoter.