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

α-甘露糖苷病是一种常染色体隐性溶酶体贮积症，由溶酶体α-甘露糖苷酶活性缺乏引起。这种疾病表现出广泛的临床表型，从严重的婴儿型（I型）到较轻的命运型（II型），后者在几年前是致命的，最终可能包括听力损失、面部粗糙、智力迟钝和肝脾肿大。我们之前报道了6例α-甘露甘露病患者的突变分析，包括我们在19号外显子有纯合R760X突变的晚发姐妹病例。探讨R760X基因突变与患者轻度临床表现的相关性。我们引入α-甘露糖苷酶cDNA的1660X、R760X和A865X突变，转染HEK293细胞，分析α-甘露糖苷酶的mrna或表达蛋白。结果表明，患者培养的淋巴细胞α-甘露糖苷酶mRNA的稳态水平显著下降，为正常对照的10%以上。此外，在患者样本中还检测到缺乏19外显子的α-甘露糖苷酶mRNA的异常剪接，而正常淋巴母细胞中不存在这种情况。转染正常cDNA后，HEK293细胞α-甘露糖苷酶活性提高到模拟转染的50倍，转染突变cDNA后α-甘露糖苷酶活性无明显提高。Western blot分析显示，过表达突变体α-甘露糖苷酶cDNA （R760X）后产生的α-甘露糖苷酶蛋白在HEK293细胞中多为一条大蛋白带（大小大于100kDa），说明翻译后加工未正确进行。综上所述。R760X突变的α-甘露糖糖苷病患者产生的α-甘露糖糖苷酶mRNA不稳定且剪接异常，α-甘露糖糖苷酶蛋白未加工导致该酶完全缺乏活性。这也表明没有α-甘露糖苷酶活性的患者可能表现出较轻的疾病形式。少

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