Molecular genetic analysis and trial of making mouse model of α-mannosidosis.

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α-Mannosidosis is an autosomal recessive lysosomal storage disorder caused by a deficiency of lysosomal α-mannosidase activity. This disease shows a wide range of clinical phenotypes, from a severe, infantile form (type I). which is fatal before at several years ago, to a less severe, fate-onset form (type II), which ultimately may involve hearing loss, coarse face, mental retardation, and hepatosplenomegaly. We previously reported the mutational analysis of six patients with α-mannosidosis including our late-onset sister cases who have the homozygous R760X mutations in exon 19. To investigate the correlation between the R760X mutation and the milder clinical manifestation of the patients. we introduced the 1660X, R760X and A865X mutations in α-mannosidase cDNA, transfected into HEK293 cells, and analyzed the mRNAs or expressed proteins of α-mannosidase.The results showed that steady state level of α-mannosidase mRNA of cultured lymphoblasts of the patient was dramatically decreased to … More ress than ten % of normal control. Moreover, abnormally spliced α-mannosidase mRNA of lacking the exon 19, which was not present in normal lymphoblasts, was also detected from patient's sample. When normal cDNA was transfected in HEK293 cells, α-mannosidase activity was elevated to fifty times to that of Mock transfection, whereas there aren't any increase of the activity when mutant cDNA was transfected. Western blot analysis revealed that α-mannosidase protein produced by overexpression of mutant α-mannosidase cDNA (R760X) in HEK293 cells showed mostly one big protein band (more than 100kDa in size), implying that post translation processing was not occurred correctly. Taken together. the patient with α-mannosidosis who has R760X mutation produces the unstable and aberrantly spliced α-mannosidase mRNA and unprocessed α-mannosidase protein result in completely lacking the activities of the enzyme. This also demonstrated that the patient who have not any activities of α-mannosidase might present the milder forms of the disease. Less