Molecular analysis of Fukuyama muscular dystrophy and functional analysis of the gene product fukutin.

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Fukuyama-type congenital muscular dystrophy (FCMD) is an autosomal recessive severe muscular dystrophy accompanied by brain malformation, prevalent in Japan. This research was performed for the purpose of making the antibodies specific for the FCMD gene product fukutin, analyzing localization and function of fukutin, and creating the mouse model of this disease. Then the following things were clarified.Although some antibodies were obtained which detect overexpressed fukutin in mammalian cells, these could not detect endogenous fukutin. Since it is supposed that fukutin is a glycosyltransferase from our recent researches and that very small quantity of endogenous fukutin exists in cells like many of known glycosyltransferases, it was thought that detection of the endogenous fukutin by the antibodies cannot be made. That is, it turned out that the analysis is difficult using the fukutin antibodies. Moreover, it was shown that fukutin exists in a Golgi body by the immunohistochemical ana … More lysis of mammalian cells overexpressing fukutin, and that is not contradictory to the possibility of being a glycosyltransferase.Mutational analysis was performed in the FCMD patients and some additional mutations were newly discovered.Identification of fukutin-binding proteins is tried by affinity column chromatography using recombinant fukutin and by mass spectrometric analysis. Although some proteins were obtained, we are checking whether these are the actual fukutin-binding proteins. Moreover, another analysis is performed using 2-dimensional electrophoresis, immunoprecipitation, and two-hybrid methods in order to identify the target protein of fukutin as a possible glycosyltransferase and the partner protein of the possible fukutin complex.To create the knock-in mice which carry the retrotransposon insertion in 3'-untranslated region of the fukutin gene, the knock-in vector was constructed and introduced to embryonic stem cells.Muscle-eye-brain disease (MEB) bears a striking resemblance to FCMD, We identified the gene responsible for MEB which encodes POMGnT1 glycosyltransferase. Less