- 期刊
Molecular Study on the Infantile Form of Pompe Disease in Chinese in Taiwan
台灣地區國人嬰兒型龐佩症（Pompe Disease）之分子生物學研究
摘要
肝醣貯積症第二型“龐佩症”是酸性α-D-解甘酶活性欠損所造成之遺傳疾病。爲了探討國人龐佩症之致前不見古人機制，過去吾人制備了抗α-D-解甘酶之單株抗體，利用放射免疫沈澱法吾人已知國人的龐佩症患者無酸性α-D-解甘酶活性，但酸性α-D解甘酶蛋白及其傳信RNA的合成能力及大小均同於正常人。因此在本文之研究，首先吾人以四位龐佩症患者（一位之父母是近新結婚，其他三位則否）爲對象，先抽取其培養皮膚腺芽細胞中之RNA，再以反轉錄酶轉錄成DNA，分四段以聚合酶鏈反應技術予以放大後定序，發現四位患者均有共同的變異點，在第14個外顯子5端之第1935個核革甘酸由胞核甘異變爲腺甘酸(C1935→A)，造成第645個胺基酸由天門冬酸異變爲麩氨基酸(Asp-645→Glu)，接著吾人將此突變點置入正常的酸性α-D-解甘酶cDNA中，並使其於COS-1細胞中表現，發現Asp-645→Glu之突變會造成酸性α-D-解甘酶活性明顯的喪失。其次爲了大量篩檢國人龐佩症患者是否大部份均在C1935→A發生點變異，吾人找到一限制酶Aat Ⅱ，當病人發生C1935→A之點變異就不會被Aat Ⅱ切開仍爲861 bp，正常人則會被Aat Ⅱ切成728及133 bp之兩段，吾人篩檢台灣由北至南25 位之不同家庭之龐佩症病人的培養表皮皮膚腺芽細胞，結果具有此C1935→A點變異之機率爲0.8。以上結果顯示C1935→A使Asp-645→Glu之突變是造成在台灣國人龐佩症最主要的原因。
並列摘要
Glycogen-storage disease type Ⅱ, Pompe disease, is caused by the deficiency of acid α-D-glucosidase in lysosome. Previously we found that acidα-D-glucosidase did exist in the skin fibroblasts and there was also no difference of mRNA in quantity and size of Chinese infantile type Pompe disease patients in Taiwan. However, functional assay of the acidα-D-glucosidase of these patients showed its enzyme function to be defective. In the present study, first we identified a substitution site in four Chinese infantile patients with Pompe disease which is a cytidine to adenosine (C1935→A) transversion at 5' end of exon 14 causing substitution of glutamic acid for aspartic acid at position 645 of the acidα-D-glucosidase. This substitution was introduced in wild-type cDNA and expressed in COS-1 cells. The Asp-645→Glu substitution resulted in significant reduction of acidα-D-glucosidase activity. Second, according to the screening data in 25 Chinese Pompe disease patients using digestion of RTPCR amplified specific fragment with Aat Ⅱ, the restriction fragment length analysis showed that patients presented the 861 bp band and the normal individuals presented the 728 bp and 133 bp polymorphic bands. We found that the frequency of mutant allele is 0.8 in infantile patients with Chinese Pompe disease and 0 in normal individuals. These results therefore indicate that Asp-645→Glu mutation results in infantile form of Pompe disease as the major cause in Chinese patients in Taiwan.