臺灣博碩士論文加值系統

中文摘要: RhD抗原是血庫作業中的一個重要血型系統之一，臨床上有可能造成溶血性輸血反應或新生兒溶血性疾病。在台灣，有99.65%的人為RhD(+)，而在其餘的0.35%中有三分之一為所謂Del的弱陽性。據文獻報告，Del仍會誘發Anti-D而造成溶血性輸血反應。但是，在現階段捐血中心在做RhD血型判定時，並無法分辨Del和trueD(-)。由於臨床上要判定Del唯有利用吸附-洗脫測試來檢測，這是一種既費材又費時的方法。發展一種簡便的方法以判讀真正RhD(-)的血液是相當有需要的。由於白種人的RhD(-)都是RHD整段缺失，是在Rh boxes發生不等交換所致；本篇即想利用Hybrid Rh box相關技術以快速分辨出Del和真正RhD陰性的案例。我們針對60個真正RhD(-)台灣人測其CE表現型，並以PCR分析其RHD的10個exons，然後再以PCR-RFLP (並佐以定序觀察) hybrid Rh box的方法分析其是否具有hybrid Rh box之存在。我們發現這60個真正RhD(-)的台灣人全都是RHD全基因缺失，有52位的CE表現型為C-c+E-e+(86.6%)。有59位適用PCR-RFLP的方式偵測到Hybrid Rh box存在(98.4%)，只有1位(1.6%)在Hybrid Rh box上的PstI site發生突變而無法適用此法，而此人的CE表現型為C+c-E-e+，並非一般的C-c+E-e+。這顯示RHD與RHCE間之聯結效果。此結果也證實了RhD(-)台灣人中，是因Rh boxes產生不等交換而產生hybrid Rh box進而導致RHD的完全缺失。

Rh D antigen is an important antigen system in blood transfusion services. It could cause hemolytic transfusion reaction or hemolytic disease of newborns. In Taiwanese, 99.65% is D(+).One third of the remainder 0.35% belongs to a weak RhD category, so-call Del, which has shown to induce anti-D in D(-) individuals. However, Del blood units are not distinguished from the D(-) units in current practice of the blood centers. In addition, adsorption and elution test is both expansive and time-consuming. An easy alternative for detection of a true D(-) blood is warranted. Since genomic D(-) structure for Caucasian D(-) individuals are total deletion of RHD with a hybrid Rh box as a hallmark from unequal cross-over, the purpose of this study is to evaluate the hybrid Rh box as an hallmark of RHD total deletion for detection of a true D(-) blood. In 60 serologically true-D(-) individuals, we phenotyped the CcEe antigens and tested the presence of all RHD exons by polymerase chain reaction (PCR) and analyzed the Rh boxes by PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing. The C-c+E-e+ phenotypes were found in 52 (86.6%)of 60 D(-) Taiwanese. The RHD total deletion types were observed in 60 cases(100%).The results of PCR-RFLP demonstrated the presence of a hybrid Rh box in 59 (98.4%)of 60 D(-) Taiwanese. There is only 1(1/60,1.6%) D(-) individual with a total deletion of RHD to have a negative PCR-RFLP for 1Rh box. Sequencing result indicated a mutated hybrid Rh box at the 3rd PstI site. This individual has a C+c-E-e+ phenotype, in stead of the common C-c+E-e+ phenotypes for D(-) in Taiwan. The result suggested the linkage effect between RHCE and RHD . Our data showed that the presence of Rh box may be a useful marker for true D(-) and suggested the unequal cross-over and total deletion of RHD as a mechanism for Taiwanese D(-) individuals.

目 錄
指導教授推薦書------------------------------------------------------ i
口試委員會審定書---------------------------------------------------- ii
授權書------------------------------------------------------------ iii
誌謝-------------------------------------------------------------- iv
中文摘要----------------------------------------------------------- v
英文摘要----------------------------------------------------------- vi

I. Introduction--------------------------------------------------- 1
1.1 Rh blood group system--------------------------------------- 1
1.1.1 Rh 血型抗原------------------------------------------------ 1
1.1.2 RH gene locus------------------------------------------ 4
1.2 RhD antigen會導致許多臨床方面的問題 -------------------- 5
1.2.1 RhD antigen造成臨床問題的原因 ------------------------- 5
1.2.2 RhD(-)血型及其基因之結構----------------------------------- 7
1.3 RhD血型的診斷------------------------------------------- 8
1.3.1 尋找新RhD分子診斷的動機 ------------------------------- 8
1.3.2 發現新RhD分子診斷的緣由 ------------------------------- 8
1.4 研究目標 ------------------------------------------------- 10
II.Materials and methods -------------------------------------- 12
2.1 Serological method ----------------------------------- 12
2.2 SSP-PCR for RHD deletion ------------------------------- 12
2.3 RHD zygosity determination by PCR-RFLP------------------ 13
2.4 Sequencing analyses of the Rhesus boxes ------------- 15
III.Results --------------------------------------------------- 17
3.1 60位RhD(-)台灣人的CE phenotype分析------------------ 17
3.2 60個trueD(-)samples皆屬RHD gene total deletion. ------ 17
3.3 證實PCR-RFLP是否適用於RHD total deletion的60個
true D(-) samples ---------------------------------------------- 18
3.4 Sequencing證實hybrid Rhesus box存在及發現mutated
hybrid Rhesus box.------------------------------------------------ 19
IV.Discussion ------------------------------------------------- 21
V. References ------------------------------------------------ 28
圖表 --------------------------------------------------------- 32

圖表目錄
Figure 1. RhAG,RhCE和RhD的蛋白質結構及所在的
chromosme位置 ----------------------------------------------- 32
Figure 2. RH gene locus的結構 ------------------------------- 33
Figure 3. 此圖為transfusion 2005;45;345-352這篇中實驗的
組織架構及流程 ----------------------------------------------- 34
Figure 4. 三種Rhesus boxes的結構------------------------------ 35
Figure 5. Rhesus box造成RHD gene不見的遺傳機制--------------- 36
Figure 6. Serological methods的流程圖------------------------- 37
Figure 7. PCR-RFLP如何RHD zygosity的機制---------------------- 38
Figure 8. 用在PCR-RFLP的primers在chromosome上的位置--- 39
Figure 9. True D(-)台灣人的分子診斷技術之分枝圖 --------------- 40
Figure 10. PCR針對RHD gene上的exon 1所做出的結果圖----- 41
Figure 11. PCR針對RHD gene上的exon 2所做出的結果圖----- 42
Figure 12. PCR針對RHD gene上的exon 3所做出的結果圖----- 43
Figure 13. PCR針對RHD gene上的exon 4所做出的結果圖----- 45
Figure 14. PCR針對RHD gene上的exon 5所做出的結果圖----- 46
Figure 15. PCR針對RHD gene上的exon 6所做出的結果圖----- 48
Figure 16. PCR針對RHD gene上的exon 7a所做出的結果圖 -- 49
Figure 17. PCR針對RHD gene上的exon 7b所做出的結果圖---- 50
Figure 18. PCR針對RHD gene上的exon 8所做出的結果圖 ---- 52
Figure 19. PCR針對RHD gene上的exon 9所做出的結果圖 ---- 53
Figure 20. PCR針對RHD gene上的exon 10所做出的結果圖---- 54
Figure 21. PCR-RFLP分析60個true D(-)samples之結果圖 ---- 55
Figure 22. Standard hybrid Rhesus box及mutated hybrid
Rhesus box sequencing的作用流程圖----------------------- 59
Figure 23. 證明hybrid Rhesus box的第三個PstI切點存在 --------- 60
Figure 24. 第47號true D(-)sample的hybrid Rhesus box上3rd
PstI site變異處-------------------------------------------------- 61
Figure 25. Downstream Rhesus box的第5161 nt之變異圖---------- 62
Figure 26. 造成mutated hybrid Rhesus box產生之機制圖----------- 63

Table 1. SSP-PCR condition for 10 exons of RHD gene ----------- 64
Table 2. 用在PCR-RFLP和sequencing的primers的序列及
專一性----------------------------------------------------------- 66
Table 3. 來自林口長庚醫院的32位true D(-)台灣人之
CE phenotype ---------------------------------------------------- 67
Table 4. 來自新竹捐血中心的28位true D(-)台灣人
CE phenotype----------------------------------------------------- 68

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