RESUMO
Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by loss of apical microvilli and formation of cytoplasmic inclusions lined by microvilli in enterocytes. MVID is caused by mutations in the MYO5B gene, coding for the myosin Vb motor protein. Although myosin Vb is implicated in the organization of intracellular transport and cell surface polarity in epithelial cells, its precise role in the pathogenesis of MVID is unknown. We performed correlative immunohistochemistry analyses of sections from duodenal biopsies of a MVID patient, compound heterozygous for two novel MYO5B mutations, predicting loss of function of myosin Vb in duodenal enterocytes together with a stable MYO5B CaCo2 RNAi cell system. Our findings show that myosin Vb-deficient enterocytes display disruption of cell polarity as reflected by mislocalized apical and basolateral transporter proteins, altered distribution of certain endosomal/lysosomal constituents including Rab GTPases. Together, this severe disturbance of epithelial cell function could shed light on the pathology and symptoms of MVID.
Assuntos
Polaridade Celular , Síndromes de Malabsorção/metabolismo , Microvilosidades/patologia , Mucolipidoses/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Linhagem Celular Tumoral , Enterócitos/metabolismo , Enterócitos/patologia , Heterozigoto , Humanos , Recém-Nascido , Síndromes de Malabsorção/diagnóstico , Síndromes de Malabsorção/genética , Masculino , Microvilosidades/genética , Microvilosidades/metabolismo , Mucolipidoses/diagnóstico , Mucolipidoses/genética , Mutação , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Transporte ProteicoRESUMO
The sulfated polysaccharide dermatan sulfate (DS) forms proteoglycans with a number of distinct core proteins. Iduronic acid-containing domains in DS have a key role in mediating the functions of DS proteoglycans. Two tissue-specific DS epimerases, encoded by DSE and DSEL, and a GalNAc-4-O-sulfotransferase encoded by CHST14 are necessary for the formation of these domains. CHST14 mutations were previously identified for patients with the musculocontractural type of Ehlers-Danlos syndrome (MCEDS). We now identified a homozygous DSE missense mutation (c.803C>T, p.S268L) by the positional candidate approach in a male child with MCEDS, who was born to consanguineous parents. Heterologous expression of mutant full-length and soluble recombinant DSE proteins showed a loss of activity towards partially desulfated DS. Patient-derived fibroblasts also showed a significant reduction in epimerase activity. The amount of DS disaccharides was markedly decreased in the conditioned medium and the cell fraction from cultured fibroblasts of the patient when compared with a healthy control subject, whereas no apparent difference was observed in the chondroitin sulfate (CS) chains from the conditioned media. However, the total amount of CS disaccharides in the cell fraction from the patient was increased â¼1.5-fold, indicating an increased synthesis or a reduced conversion of CS chains in the cell fraction. Stable transfection of patient fibroblasts with a DSE expression vector increased the amount of secreted DS disaccharides. DSE deficiency represents a specific defect of DS biosynthesis. We demonstrate locus heterogeneity in MCEDS and provide evidence for the importance of DS in human development and extracellular matrix maintenance.
Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Síndrome de Ehlers-Danlos/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Sulfotransferases/genética , Células Cultivadas , Pré-Escolar , Consanguinidade , Proteínas de Ligação a DNA/deficiência , Decorina/metabolismo , Dermatan Sulfato/biossíntese , Dissacarídeos/metabolismo , Síndrome de Ehlers-Danlos/metabolismo , Matriz Extracelular/metabolismo , Heterogeneidade Genética , Humanos , Masculino , Mutação de Sentido Incorreto , Proteínas de Neoplasias/deficiência , Sulfotransferases/metabolismoRESUMO
BACKGROUND AND AIM: To describe the clinical and genetic characteristics of a mother and her son presenting with two distinct and rare forms of retinal degeneration. METHODS: Investigations in both patients comprised spectral domain optical coherence tomography (SD-OCT), fundus autofluorescence imaging, non-contact biometry, ultrasonography, electroretinography (ERG) and analysis of the mutational status of the KCNV2 and MFRP genes in genomic DNA. RESULTS: The clinical course and typical ERG pattern indicated a 'cone dystrophy with supernormal rod electroretinogram' in the proband, and SD-OCT demonstrated a subfoveal optical gap with loss of the inner segment/outer segment junction line. The proband was homozygous for a c.782C>A (p.Ala261Asp) mutation in KCNV2. Her son's axial length was shortened with refractive errors of +16.75 dioptres in the right and +14.0 dioptres in the left eye; ERG evidenced a rod-cone dystrophy, OCT showed central macular thickening with cystoid changes and ultrasonography revealed optic disc drusen. MFRP analysis disclosed a 1 bp deletion (c.498delC) that predicts a truncated protein. CONCLUSIONS: Two distinct ocular phenotypes with pathogenic mutations in two different genes segregated in this family. The coexistence of two independent autosomal recessive disorders should be considered even when dealing with diseases that bear low carrier frequencies in the general population.