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

α-甘露糖苷中毒小鼠模型的分子遗传学分析及制作试验。

基本信息

批准号：
11670630
负责人：
WAKAMATSU Nobuaki
金额：
$ 2.24万
依托单位：
Aichi Human Service Center
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

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

α-甘露糖尿病是由缺乏的α-甘露糖苷酶活性引起的常染色体隐性溶酶体储存障碍。，最终可能涉及听力损失，粗面和肝肾上腺素的形式（II型）。为了研究R760X突变与FOO患者的温和表现之间的相关性。培养的含有PA的含有的lypho的α-甘露糖苷酶mRNA急剧降低到……比正常对照的十％，此外，剪接的α-甘露糖苷酶mRNA缺乏外显子19在正常的淋巴细胞中，也从患者的样本中解散了α-甘露糖苷酶的活性，升高到五十倍的TOCK转染，在HEK293中，cDNA没有任何增加的cDNA。细胞大多显示一个大蛋白质带（大于100kDa），即未正确发生翻译后处理。蛋白质会导致酶的活性。