Rh 血型是臨床輸血醫學上相當重要且具有多型性的血型系統。在西方國家中，Rh陰性約佔15%，但在亞洲地區Rh陰性卻只有0.1~0.5%，而在台灣的Rh陰性族群中約有30%為RhDel，這是一種須以傳統的吸附沖出方法才可測得出其極弱D抗原的血型。過去一些研究報告指出，多重分子機轉可能是造成RhDel血型的原因，因此本研究的主要目的，希望能對造成中國人RhDel血型的分子調控機轉進行探討。本論文共分為三個部份，首先我們收集294個Rh陰性的血液檢體，以PCR-SSP的方法進行RHD基因exon 4、5、7、9、10的偵測，證明了94個以吸附沖出法鑑定為RhDel的檢體，均存在有完整的RHD基因、且同時具有RHD 1227A的多型性，因此我們認為RHD 1227A 的變異可能是台灣RhDel血型一個很重要的基因標記。為了建立實驗室RhDel血型快速測定流程，我們以395個Rh陰性捐血者的血液檢體進行分析，首先篩檢出171個RhC(+)檢體，再比較這些檢體，同時以傳統血清學吸附沖出法與PCR-SSP方法分析RHD 1227A，分析比較兩種方法發現，以RHD 1227A測定RhDel之敏感度和特異性分別為96.9%和97.5%，其中有126個檢體在兩種測試方法均呈現陽性反應，結果並顯示RHD 1227A與Rh phenotype Ce具有高度關聯性(95.2%)，因此利用血清學測定RhＣ phenotype加上RHD 1227A基因型的分析，將可運用在臨床實驗室以便於快速偵測RhDel血型。最後則進行RHDel基因表現的分子特性研究，當利用 nested RT-PCR 方法分析RhDel 紅血球基因表現的情形，發現RhDel因RHD gene 1227的位置發生了G>A的變異，可能會使轉錄時發生了Exon 9 skipping，若根據DNA序列推論，這種變異的cDNA序列會因而發生了移碼 (frame shift)現象，形成了不同的RHDel基因的序列。所以，我們利用分生技術分別構築了RHD 及RHDel 之 cDNA 表現在含有GFP的載體，將其轉染到HEL 細胞，並以流式細胞儀 比較RhD and RhDel 蛋白質的差異，結果分析值顯示在相同的轉染效率下，RhDel蛋白質表現確實少於RhD蛋白質表現量，但與單株抗體（LOR-15C9）的反應結果，證明RhDel仍具有與RhD相類似的抗原性。總而言之，由此研究我們證實RHDel 之RHD 1227A的變異可以導致 mRNA 產生變異，而此種變異所產生之RhDel蛋白質表現量會減少，但仍具有相類似的抗原性。

　The Rh blood type is the most polymorphic human blood group system, and is clinically significant in transfusion medicine. About 15% of Caucasoid people are RhD-negative, whereas in the Asian population the RhD-negative blood type only occurs in 0.1 to 0.5%. In addition, approximately 30% of apparently RhD-negative Taiwanese people actually were RhDel which has a weak D antigen on RBC membrane only detected by serological adsorption-elution test. Several studies have indicated that the RhDel traits might be generated by multiple molecular mechanisms. The aim of this study is to investigate the genetic status of RHD gene and molecular characteristics among RhDel in Taiwan population. In the beginning, 294 RhD-seronegative blood samples were collected to evaluate the status of five RHD-specific exons (4, 5, 7, 9 and 10) by polymerase chain reaction with sequence-specific primers (PCR-SSP). All 94 RhDel samples (32.0%) were found carrying intact RHD gene and having RHD 1227A polymorphism. We proposed that RHD 1227A might be an important genetic marker for RhDel genotypes in Taiwan. For setting up the rapid protocol to detect RhDel in clinical laboratory, RhC (+) phenotypes were selected by serological tests among 395 RhD-negative subjects. One hundred and twenty six of all 171 RhC (+) samples were positive for both adsorption/elution for RhD detection and PCR-SSP assay for RHD 1227A. The sensitivity and specificity were 96.9% and 97.5%, respectively for RHD 1227A detection as compared to the traditional adsorption/elution test. The results indicated that RHD 1227A was highly linked to Ce haplotypes (95.2%). We concluded that combined RhC phenotyping and RHD 1227A analysis can be used as a simple and accurate laboratory screening method for RhDel detection in RhD-negative population. In the third part of this study, molecular characterization of RHDel gene expression was investigated by nested reverse transcriptase-polymerase chain reaction. The transcript demonstrated a deletion of exon 9 in RHDel gene, that is probably caused by the G>A transition at RHD 1227. The RHD and RHDel cDNAs were thus constructed and transfected into HEL cells to clarify the effects of alterative splicing mutation on RhD protein expression and antigenicity. Our results showed less RhDel protein expression than RhD protein by flow cytometry, but RhDel antigenicity still could be detected by LOR-15C9 monoclonal antibody like RhD protein. In conclusion, the silent mutation in RHD 1227 is associated with altered splicing and exon skipping, and the putative RhDel is decreased in protein expression but still have the antigenicity.

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