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研究生: 陳易申
CHEN I SHEN
論文名稱: 臺灣兩位第一型黏多醣儲積症患者的突變篩檢與記述
Humanα-L-iduronidase gene : mutation screening and characterization in two Taiwainese MPS I patients
指導教授: 李桂楨
Lee, Guey-Jen
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2000
畢業學年度: 88
語文別: 中文
論文頁數: 58
中文關鍵詞: MPS I
英文關鍵詞: 第一型黏多醣儲積症
論文種類: 學術論文
相關次數: 點閱:118下載:1
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  • 本論文的目的在探討臺灣兩位第一型黏多醣儲積症 (MPS I) 患者的分子致因。利用聚合酵素鍊反應 (PCR) 放大患者包含 IDUA 基因各表現子 (exon) 及鄰近介入子 (intron) 的片段,進行單股核酸構形多型性 (SSCP) 分析及 DNA 定序分析。結果發現患者 1413 除了在表現子1發生遺傳自母親的 134del12 突變 (秦,1998) 外,尚有一遺傳自父親的 Q584X 突變,即第 1838 個核甘酸發生 C→T 的轉換,造成第 584 個胺基酸由麩醯胺酸 Gln 轉變成終止密碼。患者 174 在表現子 2 上發生 A79V 突變,即第 324 個核甘酸發生 C→T 的轉換,造成第 79 個胺基酸由丙胺酸 Ala 轉變成結頁胺酸 Val;在表現子 14 上則發生 R619G 突變,即第 1943 個核甘酸發生 C →G 的顛換,造成第 619 個胺基酸由精胺酸 Arg 轉變成甘胺酸 Gly。R619G 突變已於先前另一 MPS I 患者 766 的突變分析中被發現且記述 (秦,1998;湯,1998)。進一步構築含 134del12、Q584X、A79V 變異的 IDUA cDNA 重組質體,經 lipofection 的方式轉移至 COS-7 細胞中表現,發現 134del12 重組質體轉移細胞的 IDUA 酵素活性及 mRNA 表現量,和野生型的 IDUA 基因並無明顯差異,但在西方吸漬法卻檢測到大量的 IDUA 前趨蛋白;含 Q584X 變異的 IDUA 酵素活性僅為野生型的 0.4%,mRNA 表現量顯著下降,且未檢測到 IDUA 蛋白質;含 A79V 變異的 IDUA 酵素活性僅為野生型的 0.0%,而 mRNA 表現量雖和野生型無明顯差異,但蛋白質表現量顯著下降。

    The purpose of this study is to investigate the molecular lesion of two Taiwanese MPS I patients. The coding sequences and exon-intron borders of the IDUA gene were amplified by polymerase chain reaction (PCR) and subjected to single-strand conformation polymorphism (SSCP) and DNA sequencing analyses. The results demonstrated that patient 1413 has heterozygous mutations; the maternal allele has 134del12 (秦,1998) and the paternal allele has Q584X (C-to-T transition in codon 584). Patient 174 has A79V (C-to-T transition in codon 79) in exon 2 and R619G (C-to-G transversion in codon 619) in exon 14. The R619G mutation was identified and characterized previously during mutation sreening in MPS I patient 766 (秦,1998;湯,1998). Expression of 134del12 showed no diffrence in IDUA mRNA level and enzyme activity compared to those of wild type IDUA cDNA upon transfection into COS-7 cells. By western blot analysis, a lot of IDUA precusor protein was detected. In transfected COS-7 cells, Q584X showed traced amounts of α-L-iduronidase activity (0.4% of normal activity). IDUA mRNA level was reduced and no IDUA protein was detected in Q584X transfected COS-7 cells. Expression of A79V showed no appreciable α-L-iduronidase activity (0.0%). Although A79V did not cause apparent reduction in IDUA mRNA level, decreased amount of IDUA protein was observed.

    目錄………………………………………………………………………I 中文摘要……………………………………………………………….IV 英文摘要………………………………………………………………..V 圖次表………………………………………………………………….VI 壹、緒論…………………………………………………………………1 貳、研究材料與方法…………………………………………………..7 一、 MPS I 患者血液樣品…………………………………………...7 二、 基因組 DNA 的萃取…………………………………………....7 三、 聚合酵素鏈反應 (PCR)………………………………………...7 四、 單股核酸構形多型性 (SSCP) 分析…………………………...8 (一) 樣品處理………………………………………………….......8 (二) 電泳………………………………………………………….....8 (三) 銀染呈色…………………………………………………….....9 五、 DNA 定序…………………………………………………….....9 六、 Q584X 突變、R619G 突變、A79V 突變之 RFLP 分析…......9 七、 包含突變的表現子片段的選殖與製備……………………....10 (一) 瓊脂膠體純化 DNA 片段……………………………….......10 (二) 接合反應…………………………………………………......11 (三) 大腸桿菌轉形勝任細胞 (competent cell) 的製備………..11 (四) 大腸桿菌的轉形作用 (transformation)……………...…..11 (五) 重組質體的篩選與製備……………………………...….....12 八、 IDUA cDNA 重組質體的構築與製備……………………......14 九、 重組質體的轉移……………………………………………....14 十、 細胞液 (cell lysate) 的製備與 IDUA 活性的測定……...15 (一) 細胞的收集及細胞液的製備………………………….….....15 (二) 總蛋白質的定量…………………………………………......15 (三) IDUA 酵素活性的測定……………………………….…......16 十一、 北方轉漬法分析 (Northern blotting)…………………..17 (一) 探針的製備…………………………………………….….....17 (二) RNA 的分離與轉漬…………………………………...….....17 (三) 雜合反應…………………………………………………......19 十二、 西方轉漬法分析 (Western blotting)…………………...20 (一) 電泳及轉漬………………………………………………......20 (二) 免疫染色…………………………………………………......20 參、結果……………………………………………………………….21 一、 患者 1413、174 的 IDUA 基因突變分析…………………...21 (一) 患者 1413……………………………………………...…....21 (二) 患者 174…………………………………………………......21 二、 突變對 IDUA 酵素活性的影響……………………………....22 (一) pcDNA3-IDUA/134del12、pcDNA3-IDUA/Q584X、 pcDNA3-IDUA/A79V 重組質體的構築與確認.……..........22 (二) 重組質體轉移效率的測定……………………………….....24 (三) 突變對 IDUA mRNA 表現量的影響…..………………….....24 (四) 突變對 IDUA 蛋白質表現量的影響…………………........24 (五) 突變對 IDUA 酵素活性的影響………………………........25 肆、討論…………………………………………………………….…26 伍、參考文獻………………………………………………………….31 圖一 ~ 十七………………………………………………………….35 表一 ~ 五…………………………………………………………...54

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