RESUMO
PURPOSE: ADAMTSL4 mutations cause autosomal recessive isolated ectopia lentis (IEL) and ectopia lentis et pupillae. Dominant FBN1 mutations cause IEL or syndromic ectopia lentis (Marfan syndrome and Weill-Marchesani syndrome). The authors sought to characterize recombinant ADAMTSL4 and the ocular distribution of ADAMTSL4 and to investigate whether ADAMTSL4 influences the biogenesis of fibrillin-1 microfibrils, which compose the zonule. METHODS: ADAMTSL4 was expressed by the transfection of HEK293F cells. Protein extracts and paraffin sections from human eyes were analyzed by Western blot analysis and by immunoperoxidase staining, respectively. Immunofluorescence was used to evaluate fibrillin-1 deposition in the ECM of fetal bovine nuchal ligament cells after culture in ADAMTSL4-conditioned medium or control medium. Confocal microscopy was performed to investigate ADAMTSL4 and fibrillin-1 colocalization in these cultures. RESULTS: Western blot analysis identified ADAMTSL4 as a glycoprotein in HEK293F cells and as a major band of 150 kDa in ocular tissues including ciliary body, sclera, cornea, and retina. Immunoperoxidase staining showed a broad ocular distribution of ADAMTSL4, associated with both cells and fibrillar ECM. When cultured in ADAMTSL4-containing medium, fetal bovine nuchal ligament cells showed accelerated fibrillin-1 deposition in ECM. ADAMTSL4 colocalized with fibrillin-1 microfibrils in the ECM of these cells. CONCLUSIONS: ADAMTSL4 is a secreted glycoprotein that is widely distributed in the human eye. Enhanced fibrillin-1 deposition in the presence of ADAMTSL4 and colocalization of ADAMTSL4 with fibrillin-1 in the ECM of cultured fibroblasts suggest a potential role for ADAMTSL4 in the formation or maintenance of the zonule.
Assuntos
Olho/metabolismo , Regulação da Expressão Gênica , Microfibrilas/genética , Proteínas dos Microfilamentos/metabolismo , RNA/genética , Trombospondinas/genética , Proteínas ADAMTS , Animais , Sítios de Ligação , Western Blotting , Bovinos , Células Cultivadas , Ectopia do Cristalino/genética , Ectopia do Cristalino/metabolismo , Ectopia do Cristalino/patologia , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular , Olho/patologia , Fibrilina-1 , Fibrilinas , Imunofluorescência , Humanos , Imuno-Histoquímica , Microfibrilas/metabolismo , Microscopia Confocal , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trombospondinas/biossíntese , Trombospondinas/metabolismoRESUMO
Accurate molecular diagnosis of genetic eye diseases has proven to be of great importance because of the prognostic and therapeutic value of an accurate ascertainment of the underlying genetic mutation. Efforts continue in diagnostic laboratories to develop strategies that allow the discovery of responsible gene/mutations in the individual patient using the least number of assays and economizing on the expenses and time involved in the process. Once the ophthalmologist has made the best possible clinical diagnosis, blood samples are obtained for genetic testing. In this paper we will review the basic laboratory methods utilized to identify the chromosomal or mutational etiology of genetic diseases that affect the eye.