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系統識別號 U0026-0812200912095435
論文名稱(中文) RHD基因的裁剪變異與DEL血型的相關性
論文名稱(英文) Splicing mutation of RHD gene associated with DEL phenotype
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 94
學期 2
出版年 95
研究生(中文) 楊毓棻
研究生(英文) Yu-Fen Yang
學號 t3693404
學位類別 碩士
語文別 中文
論文頁數 71頁
口試委員 口試委員-陳彩雲
指導教授-林尊湄
口試委員-傅子芳
口試委員-余榮熾
中文關鍵字 剪接變異  血型 
英文關鍵字 splicing  RHD  DEL 
學科別分類
中文摘要 Rh血型是一個重要且複雜的血型系統,因種族不同而有差異。亞洲地區的Rh陰性族群中,有30 %實際上是帶有完整RHD基因的DEL血型,其D抗原表現微弱無法以傳統血清學方法測出,唯有吸附沖出試驗才能鑑定。在過去的研究中我們發現位在RHD基因exon 9的RHD 1227A的變異是中國人DEL血型中一個重要的基因標記。在本篇第一部份研究中發現DEL個體有三種不同於Rh陽性的剪接變異,分別為RHD基因的 (1) exon 9 跳躍;(2) exon 8及exon 9跳躍;(3) exon 7和exon 8之間插入一段170 bp的cryptic exon,且exon 9跳躍。除了確認RHD 1227A存在於DEL個體中,我們又發現了位在170 bp的cryptic exon中的另一個變異點RHD IVS7+923C>T。第二部份的實驗,針對台灣地區294名Rh陰性個體與399名一般人口,分析RHD 1227A、hybrid Rhesus box、RHD IVS7+923T基因型,結果顯示同時具有RHD 1227A與RHD IVS7+923T allele的個體在DEL族群與一般人口中的分佈有顯著的差異 (P<0.0001),並且RHD 1227G>A與RHD IVS7+923C>T以連鎖不平衡 (linkage disequilibrium)的狀態存在於DEL個體。DEL的剪接變異造成移碼 (frameshift)作用,影響了RhD蛋白的C端序列。由於之前的報導已知Rh抗原在紅血球膜上的表現與Rh complex組成有關,且Rh和RhAG的C端藉由和ankyrin-R的D2 domain的作用,在紅血球細胞膜和細胞骨架之間扮演連結的角色,以利於Rh蛋白在細胞膜上的表現。因此,在第三部分的研究中,我們以GST-pull down的方法證明DEL產生序列差異的C端蛋白失去了與ankyrin-R D2 domain的連結。由本篇的數據及實驗結果我們推測DEL個體因為RHD 1227A與RHD IVS7+923T基因型導致剪接變異,而這些DEL蛋白因為無法形成穩定的complex以至於無法表現於紅血球細胞膜上或是使表現量下降。


英文摘要 The Rh blood group is the most polymorphic and clinically important blood group system in transfusion medicine. About 30 % of apparently Rh-negative subjects in Chinese are actually DEL, which has a weak D antigen demonstrated only by absorption and elution test. In our previous report, genomic analysis found an intact RHD gene and RHD 1227G>A polymorphism in DEL individuals. In this study, we further analyze and confirm the molecular basis of the DEL phenotype in Chinese. There are three RHD transcript variants detected and sequence analysis revealed that the two shorter cDNA fragments were products of exon 9 and exons 8-9 skipping, whereas the longer fragment was the product of exon 9 skipping as well as insertion of a 170-bp cryptic exon between exon 7 and exon 8. Further study of the 170-bp cryptic exon as assayed by polymerase chain reaction and direct sequence revealed one polymorphism IVS7+923C>T in all DEL individuals. The prevalence of RHD 1227A, hybrid Rhesus box, and RHD IVS7+923T alleles were investigated in 294 Rh-negative individuals and 399 unrelated healthy blood donors. The frequency of RHD 1227A combined with RHD IVS7+923T allele in subjects of DEL is significantly higher than those of normal population (p<0.0001). These results indicate that linkage disequilibrium of RHD 1227G>A and RHD IVS7+923C>T causes splicing mutation accompanied by frame-shifts in the C-terminal domain of D protein to be associated with DEL phenotype. Rh membrane complex that includes the non-glycosylated Rh proteins and Rh-associated glycoprotein (RhAG) is strictly required for cell surface expression of Rh. Several studies suggested that the C-terminal cytoplasmic domains of Rh and RhAG are sufficient to mediate interaction with the erythroid adaptor protein, ankyrin-R. Using GST-pull-down assays, we demonstrate that the interaction between the C-terminal domain of putative DEL polypeptides and ankyrin-R D2 domain is impaired. We propose that the disruption of the interaction of Rh/RhAG-ankyrin-R complex resulted in alternation of the routing and/or stability of Rh membrane complex may be the main cause of RhD deficiency in Chinese DEL phenotype.


論文目次 中文摘要 I
英文摘要 II
致謝 IV
目錄 V
表目錄 VII
圖目錄 VIII
縮寫檢索表 IX
儀器及藥品 X
第一章 緒論
第一節 Rh血型系統 1
第二節 RH基因的構造與變異 1
第三節 Rh抗原的表現 2
第四節 DEL血型與其分子特性 3
第五節 Rh complex與細胞骨架的連結 5
第二章 研究動機與實驗設計 7
第三章 材料與方法
第一節 RHD與DEL mRNA分析 9
第二節 RHD 1227、hybrid Rhesus box、RHD IVS7+923基因型分析 16
第三節 質體重組工程 (Recombinant plasmid construction) 19
第四節 蛋白質表現 23
第五節 in vitro translation反應 28
第六節 GST-pull down反應 32
第四章 結果
第一節 RHD與DEL mRNA分析 34
第二節 RHD exon 7、8、9、10及cryptic exon前後序列分析 34
第三節 台灣Rh陰性族群與一般人口的RHD 1227基因型與
RHD IVS7+923基因型分析 35

第四節 DEL的C端變異蛋白與ankyrin-R D2 domain之間的作用 36
第五章 討論 38
第六章 參考文獻 44
表 52
圖 59
附錄 67
個人資料 70







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