臺灣博碩士論文加值系統

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。

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.

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

1.Levine P and Stetson RE, Landmark article July 8, 1939. An unusual case of intra-group agglutination. By Philip Levine and Rufus E Stetson, Vol. 251, 1984.
2.Landsteiner K and Wiener A, An agglutinable factor in human blood recognized by immune sera for rhesus blood. Proc Soc Exp Biol Med 43: 223-224, 1940.
3.Cherif-Zahar B, Mattei MG, Le Van Kim C, Bailly P, Cartron JP and Colin Y, Localization of the human Rh blood group gene structure to chromosome region 1p34.3-1p36.1 by in situ hybridization. Hum Genet 86(4): 398-400, 1991.
4.MacGeoch C, Mitchell CJ, Carritt B, Avent ND, Ridgwell K, Tanner MJ and Spurr NK, Assignment of the chromosomal locus of the human 30-kDal Rh (rhesus) blood group-antigen-related protein (Rh30A) to chromosome region 1p36.13----p34. Cytogenet Cell Genet 59(4): 261-3, 1992.
5.Colin Y, Cherif-Zahar B, Le Van Kim C, Raynal V, Van Huffel V and Cartron JP, Genetic basis of the RhD-positive and RhD-negative blood group polymorphism as determined by Southern analysis. Blood 78(10): 2747-52, 1991.
6.Mouro I, Colin Y, Cherif-Zahar B, Cartron JP and Le Van Kim C, Molecular genetic basis of the human Rhesus blood group system. Nat Genet 5(1): 62-5, 1993.
7.Simsek S, de Jong CA, Cuijpers HT, Bleeker PM, Westers TM, Overbeeke MA, Goldschmeding R, van der Schoot CE and von dem Borne AE, Sequence analysis of cDNA derived from reticulocyte mRNAs coding for Rh polypeptides and demonstration of E/e and C/c polymorphisms. Vox Sang 67(2): 203-9, 1994.
8.Okuda H, Suganuma H, Kamesaki T, Kumada M, Tsudo N, Omi T, Iwamoto S and Kajii E, The analysis of nucleotide substitutions, gaps, and recombination events between RHD and RHCE genes through complete sequencing. Biochem Biophys Res Commun 274(3): 670-83, 2000.
9.Wagner FF and Flegel WA, RHD gene deletion occurred in the Rhesus box. Blood 95(12): 3662-8, 2000.
10.Wagner FF, Frohmajer A and Flegel WA, RHD positive haplotypes in D negative Europeans. BMC Genet 2: 10, 2001.
11.Wagner FF and Flegel WA, Review: the molecular basis of the Rh blood group phenotypes. Immunohematol 20(1): 23-36, 2004.
12.Westhoff CM, The Rh blood group system in review: a new face for the next decade. Transfusion 44(11): 1663-73, 2004.
13.Singleton BK, Green CA, Avent ND, Martin PG, Smart E, Daka A, Narter-Olaga EG, Hawthorne LM and Daniels G, The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in africans with the Rh D-negative blood group phenotype. Blood 95(1): 12-8, 2000.
14.Sun CF, Chou CS, Lai NC and Wang WT, RHD gene polymorphisms among RhD-negative Chinese in Taiwan. Vox Sang 75(1): 52-7, 1998.
15.Lan JC, Chen Q, Wu DL, Ding H, Pong DB and Zhao T, Genetic polymorphism of RhD-negative associated haplotypes in the Chinese. J Hum Genet 45(4): 224-7, 2000.
16.Avent ND and Reid ME, The Rh blood group system: a review. Blood 95(2): 375-87, 2000.
17.Okuda H and Kajii E, The evolution and formation of RH genes. Leg Med (Tokyo) 4(3): 139-55, 2002.
18.Ansart-Pirenne H, Asso-Bonnet M, Le Pennec PY, Roussel M, Patereau C and Noizat-Pirenne F, RhD variants in Caucasians: consequences for checking clinically relevant alleles. Transfusion 44(9): 1282-6, 2004.
19.Mayne KM, Allen DL and Bowell PJ, 'Partial D' women with anti-D alloimmunization in pregnancy. Clin Lab Haematol 13(3): 239-44, 1991.
20.Kormoczi GF, Legler TJ, Daniels GL, Green CA, Struckmann R, Jungbauer C, Moser S, Flexer M, Schonitzer D, Panzer S and Gassner C, Molecular and serologic characterization of DWI, a novel "high-grade" partial D. Transfusion 44(4): 575-80, 2004.
21.Wagner FF, Gassner C, Muller TH, Schonitzer D, Schunter F and Flegel WA, Molecular basis of weak D phenotypes. Blood 93(1): 385-93, 1999.
22.Mak KH, Yan KF, Cheng SS and Yuen MY, Rh phenotypes of Chinese blood donors in Hong Kong, with special reference to weak D antigens. Transfusion 33(4): 348-51, 1993.
23.Denomme GA, Wagner FF, Fernandes BJ, Li W and Flegel WA, Partial D, weak D types, and novel RHD alleles among 33,864 multiethnic patients: implications for anti-D alloimmunization and prevention. Transfusion 45(10): 1554-60, 2005.
24.Fukumori Y, Hori Y, Ohnoki S, Nagao N, Shibata H, Okubo Y and Yamaguchi H, Further analysis of Del (D-elute) using polymerase chain reaction (PCR) with RHD gene-specific primers. Transfus Med 7(3): 227-31, 1997.
25.Chang JG, Wang JC, Yang TY, Tsan KW, Shih MC, Peng CT and Tsai CH, Human RhDel is caused by a deletion of 1,013 bp between introns 8 and 9 including exon 9 of RHD gene. Blood 92(7): 2602-4, 1998.
26.Singleton B, Green C, Kimura K, Minami A, Okubo Y and Daniels G, Two new RHD mutations associated with the Del phenotype. Transfus Clin Biol 8(suppl 1): 9s, 2001.
27.Shao CP, Maas JH, Su YQ, Kohler M and Legler TJ, Molecular background of Rh D-positive, D-negative, D(el) and weak D phenotypes in Chinese. Vox Sang 83(2): 156-61, 2002.
28.Chen JC, Lin TM, Chen YL, Wang YH, Jin YT and Yue CT, RHD 1227A is an important genetic marker for RhD(el) individuals. Am J Clin Pathol 122(2): 193-8, 2004.
29.Gassner C, Doescher A, Drnovsek TD, Rozman P, Eicher NI, Legler TJ, Lukin S, Garritsen H, Kleinrath T, Egger B, Ehling R, Kormoczi GF, Kilga-Nogler S, Schoenitzer D and Petershofen EK, Presence of RHD in serologically D-, C/E+ individuals: a European multicenter study. Transfusion 45(4): 527-38, 2005.
30.Wagner T, Kormoczi GF, Buchta C, Vadon M, Lanzer G, Mayr WR and Legler TJ, Anti-D immunization by DEL red blood cells. Transfusion 45(4): 520-6, 2005.
31.Huang CH, Molecular insights into the Rh protein family and associated antigens. Curr Opin Hematol 4(2): 94-103, 1997.
32.Eyers SA, Ridgwell K, Mawby WJ and Tanner MJ, Topology and organization of human Rh (rhesus) blood group-related polypeptides. J Biol Chem 269(9): 6417-23, 1994.
33.Cartron JP, Defining the Rh blood group antigens. Biochemistry and molecular genetics. Blood Rev 8(4): 199-212, 1994.
34.Huang CH, The human Rh50 glycoprotein gene. Structural organization and associated splicing defect resulting in Rh(null) disease. J Biol Chem 273(4): 2207-13, 1998.
35.Bhattacharyya R, Das AK, Moitra PK, Pal B, Mandal I and Basu J, Mapping of a palmitoylatable band 3-binding domain of human erythrocyte membrane protein 4.2. Biochem J 340 ( Pt 2): 505-12, 1999.
36.Nunomura W, Takakuwa Y, Parra M, Conboy J and Mohandas N, Regulation of protein 4.1R, p55, and glycophorin C ternary complex in human erythrocyte membrane. J Biol Chem 275(32): 24540-6, 2000.
37.Huang CH, Chen Y, Reid ME and Seidl C, Rhnull disease: the amorph type results from a novel double mutation in RhCe gene on D-negative background. Blood 92(2): 664-71, 1998.
38.Cherif-Zahar B, Matassi G, Raynal V, Gane P, Mempel W, Perez C and Cartron JP, Molecular defects of the RHCE gene in Rh-deficient individuals of the amorph type. Blood 92(2): 639-46, 1998.
39.Huang CH, Liu PZ and Cheng JG, Molecular biology and genetics of the Rh blood group system. Semin Hematol 37(2): 150-65, 2000.
40.Kato-Yamazaki M, Okuda H, Kawano M, Omi T, Iwamoto T, Ishimori T, Hasekura H and Kajii E, Molecular genetic analysis of the Japanese amorph rh(null) phenotype. Transfusion 40(5): 617-8, 2000.
41.Nicolas V, Le Van Kim C, Gane P, Birkenmeier C, Cartron JP, Colin Y and Mouro-Chanteloup I, Rh-RhAG/ankyrin-R, a new interaction site between the membrane bilayer and the red cell skeleton, is impaired by Rh(null)-associated mutation. J Biol Chem 278(28): 25526-33, 2003.
42.Bennett V and Baines AJ, Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues. Physiol Rev 81(3): 1353-92, 2001.
43.Lux SE, John KM and Bennett V, Analysis of cDNA for human erythrocyte ankyrin indicates a repeated structure with homology to tissue-differentiation and cell-cycle control proteins. Nature 344(6261): 36-42, 1990.
44.Lambert S, Yu H, Prchal JT, Lawler J, Ruff P, Speicher D, Cheung MC, Kan YW and Palek J, cDNA sequence for human erythrocyte ankyrin. Proc Natl Acad Sci U S A 87(5): 1730-4, 1990.
45.Otto E, Kunimoto M, McLaughlin T and Bennett V, Isolation and characterization of cDNAs encoding human brain ankyrins reveal a family of alternatively spliced genes. J Cell Biol 114(2): 241-53, 1991.
46.Michaely P and Bennett V, The membrane-binding domain of ankyrin contains four independently folded subdomains, each comprised of six ankyrin repeats. J Biol Chem 268(30): 22703-9, 1993.
47.Goguel V, Liao XL, Rymond BC and Rosbash M, U1 snRNP can influence 3'-splice site selection as well as 5'-splice site selection. Genes Dev 5(8): 1430-8, 1991.
48.Asselta R, Montefusco MC, Duga S, Malcovati M, Peyvandi F, Mannucci PM and Tenchini ML, Severe factor V deficiency: exon skipping in the factor V gene causing a partial deletion of the C1 domain. J Thromb Haemost 1(6): 1237-44, 2003.
49.James PD, O'Brien LA, Hegadorn CA, Notley CR, Sinclair GD, Hough C, Poon MC and Lillicrap D, A novel type 2A von Willebrand factor mutation located at the last nucleotide of exon 26 (3538G>A) causes skipping of 2 nonadjacent exons. Blood 104(9): 2739-45, 2004.
50.Reese MG, Eeckman FH, Kulp D and Haussler D, Improved splice site detection in Genie. J Comput Biol 4(3): 311-23, 1997.
51.Yeo G and Burge CB, Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol 11(2-3): 377-94, 2004.
52.Rogozin IB and Milanesi L, Analysis of donor splice sites in different eukaryotic organisms. J Mol Evol 45(1): 50-9, 1997.
53.Kormoczi GF, Gassner C, Shao CP, Uchikawa M and Legler TJ, A comprehensive analysis of DEL types: partial DEL individuals are prone to anti-D alloimmunization. Transfusion 45(10): 1561-7, 2005.
54.Wang YH, Chen JC, Lin KT, Lee YJ, Yang YF and Lin TM, Detection of RhD(el) in RhD-negative persons in clinical laboratory. J Lab Clin Med 146(6): 321-5, 2005.
55.Burge CB, Tuschi T and Sharp PA, The RNA World II. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press., 1999.
56.Will CL and Luhrmann R, Spliceosomal UsnRNP biogenesis, structure and function. Curr Opin Cell Biol 13(3): 290-301, 2001.
57.Krawczak M, Reiss J and Cooper DN, The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet 90(1-2): 41-54, 1992.
58.Moore MJ, Query CC and Sharp PA, The RNA World. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press., 1993.
59.Robberson BL, Cote GJ and Berget SM, Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol Cell Biol 10(1): 84-94, 1990.
60.Philips AV and Cooper TA, RNA processing and human disease. Cell Mol Life Sci 57(2): 235-49, 2000.
61.Stoilov P, Meshorer E, Gencheva M, Glick D, Soreq H and Stamm S, Defects in pre-mRNA processing as causes of and predisposition to diseases. DNA Cell Biol 21(11): 803-18, 2002.
62.Nakai K and Sakamoto H, Construction of a novel database containing aberrant splicing mutations of mammalian genes. Gene 141(2): 171-7, 1994.
63.Stenson PD, Ball EV, Mort M, Phillips AD, Shiel JA, Thomas NS, Abeysinghe S, Krawczak M and Cooper DN, Human Gene Mutation Database (HGMD): 2003 update. Hum Mutat 21(6): 577-81, 2003.
64.Chen W, Kubota S and Seyama Y, Alternative pre-mRNA splicing of the sterol 27-hydroxylase gene (CYP 27) caused by a G to A mutation at the last nucleotide of exon 6 in a patient with cerebrotendinous xanthomatosis (CTX). J Lipid Res 39(3): 509-17, 1998.
65.Ozkara HA and Sandhoff K, A new point mutation (G412 to A) at the last nucleotide of exon 3 of hexosaminidase alpha-subunit gene affects splicing. Brain Dev 25(3): 203-6, 2003.
66.Claes K, Poppe B, Machackova E, Coene I, Foretova L, De Paepe A and Messiaen L, Differentiating pathogenic mutations from polymorphic alterations in the splice sites of BRCA1 and BRCA2. Genes Chromosomes Cancer 37(3): 314-20, 2003.