摘要
目的 建立单管多重聚合酶链反应(PCR)方法检测我国缺失型与非缺失型α-地中海贫血(α-地贫)基因型。方法 应用两套单管多重PCR方法，分别对146例Hb H病样本和74例绒毛膜或羊水胎儿DNA样本进行α-地贫基因型分析，并应用经典α-地贫基因分析方法和DNA测序做对照。结果 单管多重PCR方法一次即可成功地检测出东南亚缺失型(--~(SEA))α-地贫1，右侧缺失型(-α~(3.7))和左侧缺失型(-α~(4.2))α-地贫2，及Hb Constant
Spring(Hb CS)和Hb Quong Sze(Hb QS)基因突变。结果与经典(α-地贫基因分析方法及DNA测序完全相符。146例Hb H病样本中，检出非缺失型Hb H病83例(--~(SEA)／α~(CS)α81例，--~(SEA)／α~(QS)α1例，--~(SEA)／α~Tα(未明突变)1例)，占56.9％；缺失型Hb H病63例(--~(SEA)／-α~(3.7)41例，--~(SEA)／-α~(4.2)22例)，占43.2％。74例绒毛膜或羊水胎儿DNA样本中，检出α珠蛋白基因正常(αα／αα)40例，东南亚缺失型α-地贫1杂合子(--~(SEA)／αα)25例，Hb Bart’s胎儿水肿综合征(--~(SEA)／--~(SEA))4例，缺失型Hb H病4例(--~(SEA)／-α~(4.2)3例，--~(SEA)／-α~(3.7)1例)，Hb CS杂合子(αα／α~(CS)α)1例。结论 单管多重PCR方法能快速检测α-地贫基因型，尤其是Hb H病的双重α-地贫基因型，操作简便，结果准确，可应用于临床上α-地贫的基因型检测，尤其适用于大规模人群的α-地贫基因型筛查。
Objective To develop techniques based on single-tube multiplex polymerase chain reaction (PCR) for diagnosis of common a- thalassemia (a-thal) genotypes in china. Methods 146 Hb H disease samples and 74 fatel DNA from chorionic villi samples (CVS) or amniocyte samples were detected by the two sets of single-tube multiplex polymerase chain reaction (PCR) and classic PCR methods which had been commonly used respectively. Results The single-tube PCR can detect SEA α-thal-1, -α3.7 α-thal-2, -α4.2 a-thal-2, Hb Constant Spring (Hb CS) and Hb Quong Sze (Hb QS) genotypes, and the results were the same as those detected by classic PCR methods. Among 146 Hb H disease patients, 83 cases were riondeletional Hb H disease , accounting for 56.9% (81 cases of -SEA/αcsα, 1 case of --SEA/αQSα, 1 case of -SEA/αTα unknown mutation); 63 cases were deletional Hb H disease, accounting for 43.2% ( 41 cases of -SEA/-α 3.7, 22 cases of -SEA/-α4.2) Among 74 fatel DNA samples, 40 cases were normal (αα/αα), while a-thal-1 het
erozygotes 25, Hb Bart's hydrops fetalis syndrome 4, deletional Hb H disease 4(3 for -SEA/-α4.2, 1 for -SEA /-α3.7) and Hb CS heterozygotes 1. Conclusion The single-tube multiplex PCR for detecting a-thal genotypes especially Hb H disease was accurate and sensitive. It was simple and easy to be practice for screening a-thal genotypes.
引文
1. Higgs DR, Vickers MA, Wilkie AOM, et al. A review of the molecular genetics of the human alpha -globin gene cluster. Blood, 1989, 73(5): 1081-1104.
2．全国血红蛋白病研究协作组．20省、市自治区60万人血红蛋白病调查．中华医学杂志．1983，63(6)：382－385．
3．杜传书．地中海贫血研究的现状与未来．中华医学遗传学杂志．1996，13(5)：257-258．
4．吴冠芸，王申五主编．基因诊断．人民卫生出版社．1988，140-142．
5. Amold SC, C. Tan, Samuel S. thong, et al. A rapid and reliable 7-deletion Multiplex polymerase chain reaction assay for α-Thalassemia. Blood, 2001,98(1):250-251.
6. EBaysal, T.H.J. Huisman. Detection of Deletional α-thalassemia-2 Deteminants by PCR.Am.J. Hematology, 1994, 46(:3):208-213.
7．林伟雄，龙桂芳，李卫，等．广西地区α-地中海贫血高危胎儿产前诊断．广西科学，1998，5(2)：81-82．
8．段山，李洪义，陈争，等．中国南方α地中海贫血基因突变型研究．中国实验血液学杂志，2003，11(1)：54-60．
9. Winichagoon Pranee, Fucharoen Suthat. Kanokpongsakdi S, et al. Detection of α-thalassemia-1(southeast Asian type) and its application for prenatal diagnosis. Clin Genet, 1995, 47(6): 318-320.
10．文晓军，梁徐，金琪，等．应用PCR技术进行非缺失型Hb H病的基因筛查．中华医学遗传学杂志，1992，9(5)：271-273．
11．肖维威，徐湘民，徐铃．中国人缺失型α-地中海贫血的分子基础和产前诊断 第一军医大学学报，1998，18(1)：68-72．
12．周玉球，李莉艳，肖鸽飞，等．珠海市户籍人群中α-地中海贫血的分子流行病学调查．中华医学遗传学杂志，2002，19(4)：358-360．
13．蔡禾念，李莉艳，梁日斤，等．柳州市城镇人群α和β地中海贫血的发生率调查和基因型鉴定．中华流行病学杂志，2002，23(4)：281-285．
14．周玉球，肖鸽飞，李莉艳，等．Gap-PCR作为临床—线α-地中海贫血携带者筛查技术的应用评价。第一军医大学学报，2002，22(5)：434-436
15. Chang JG, Lee LS, Lin CP, et al. Rapiddiagnosis of α-thalassemia-1 of Southeast Asia type andhydeops fetalis by polymerase chain reaction. Blood, 1991, 78(3):853-854.
16. Bowden OK, Vickers MA, Higgs DR. A PCR-based strategy to detect the common severe determinants of alpha thalassaemia. Br J Haematol 1992 May;81(1):104-108.
17．徐湘民，马维芳，李上奎，等．用PCR直接检测缺失型α中海贫血携带者及产前诊断一例报告．第一军医大学学报，1992，12(3)：183－187．
18. Ko TM, Tseng LH, Hsieh Fi, Hsu PM, et al. Detection and prenatal diagnosis of alpha-thalassemia of Southeast Asian deletion by polymerase chain reaction.Hum Genet, 1992 Jan; 88(3): 245-248.
19．文晓军，梁徐，林伟雄，等．多聚酶链反应在α地中海贫血基因分析及产前诊断中的应用．中华血液学杂志，1992，13(2)：629-631．
20．阎志杰，吴冠芸，王荣新，等．非缺失型Hb H病基因突变类型的研究．中华血液学杂志，1994，15(8)：393－395．
21. Liang S, X J. Wen, WK Lin. Detection of Hb Quong Sze mutation by selective amplification of α2-globin germ and restriction analysis with Mspl in a Chinese family. Hemoglobin, 1991, 15(6): 535-540.
22. hebo RV,Saiki RK,Swanson K, et al. Diagnosis of alpha-thalassemia by polymerase chain reaction and dual restriction enzyme analysis. Hum Genet,1990 Aug; 85 (3): 293-299.
23. Chen TP, Lin SF, Chang JG, et al. Molecular characterization of Hb H disease by polymerase chain reaction. Acta Haematol,1993, 90(4):177-181.
24. Chang JG, Liu TC, Perng LI, et al. Rapid molecular characterization of Hb H disease in Chinese by polymerase chain reaction. Ann Hematol, 1994 Jan; 68(1):33-37.
25．徐湘民，张基增，李上奎．应用限制性内切酶分析PCR产物快速诊断Hb CS基因突变．中华血液学杂志，1994，15(8)：40－403．
26．李巍，蔡志明，林坚，等．ARMS法快速鉴定中国人非缺失型α地中海贫血基因型．中华血液学杂志，1999，20(4)：219～221．
27. gng B, Patterson M, Walker L, et al. A single-tube multiplex ARMS assay. Detection of severe nondeletional alpha-thalassemia mutations using a single-tube multiplex ARMS assay. Genet Test, 2001 Winter;5(4):327-329.
28. Chan V, Yam I, Chen FE, et al.A reverse dot-blot method for rapid detection of non-deletion (alpha) thalassaemia. Br J Haematol, 1999, 104(3): 513-515.
29. Foglietta E, Bianco Ⅰ, Maggio A. et al. Rapid detection of six common Mediterranean and three non-Mediterranean alpha-thalassemia point mutations by reverse dot blot analysis. Am J Hematol. 2003 Nov; 74(3): 191-195.
30．李莉艳，莫秋华，徐湘民．反向点杂交快速诊断非缺失型α－地中海贫血．中华医学遗传学杂志，2003，20(4)，345－347．
31．赵永忠，徐湘民，徐铃．PCR－LIS－SSCP快速分析非缺失型α地中海贫血点突变．中华医学遗传学杂志，1999，16(2)：113－115．
32. Harteveld KL, Heister AJ, Oiordano PC, et al. Rapid detection of point mutations and polymorphisms of the alpha-globin genes by DGGE and SSCA. Hum utat, 1996, 7(2): 114-22.
33．刘敬忠，周桔，王力荣．缺失型α地中海贫血的基因诊断新技术研究．中华血液学杂志，2001，22(4)：193－196．
34．肖维威，徐湘民，刘忠英．东南亚缺失型α地中海贫血的聚合酶链式反应快速诊断技术及产前诊断．中华血液学杂志，2000，21(4)：192-194．
35. Chan V, Yip B, Lam YH, Tse HY, et al. Quantitative polymerase chain reaction for the rapid prenatal diagnosis of homozygous alpha-thalassaemia (Hb Barts hydrops fetalis). Br J Haematol, 2001 Nov; 115(2): 341-346.
36. Lemuel J Bowie, P. L Reddy, M Nagabhushan, et al. Detection of α－Thalassemia by multiplex polymerase chain reaction. Clin. Chem, 1994, 40(12): 2260-2266
37．区采莹，蒙晶，郑诗华．多重PCR筛查高发群体缺失型α地中海贫血基因的研究．中国热带医学，2002，2(2)：129－131．
38. Y.T. Liu, J.M. Old, K. Milex, et. al. Rapid detection of α－thalassemiadeletions and α－globin gene thiplication by multiplex polymerase chain reactions. Br J Haematol, 2000, 108(2): 295-299
39. Samuel S. Chong, Corinne D. Boehm, Garry R. Cutting, et al. Simplified Multiplex-PCR Diagnosis of Common Southeast Asian Deletional Determinants of α－Thalassemia.Blood, 2000,95(1): 360-362.
40. Samuel S. Chong, Corinne D. Boehm, Garry R. Cutting. Simplified Multiplex-PCR Diagnosis of Common Southeast Asian Deletional Determinants of α－Thatassemia,Clinical Chemistry. 2000 Oct; 46(10): 1692-1695.
41．[美]K．B穆里斯，F．费里，R．吉布斯等著．陈受宜等译．PCR聚合酶链式反应：科学出版社，1997，37－44．
42. Woltgang aenke, Kerstin Herdel,Klaus Jung, et al. Betaine improves the PCR amplification of OC-rich DNA sequences Nucleic Acids Research, 1997 Oct; 35(19): 3957-3958.
43．伍欣星，聂广，胡继鹰．医学分子生物学原理与方法．科学出版社2000，155-159．
44. Liang R., Liang S., N H Jiang, et al. α and β thalassemia among Chinese children in Guangxi Province, P.R. China: molecular and haematological
characterization. Br J Haematol, 1994, 86(1): 351-354.
45. Liu TC, Chiou SS, Lin SF, Chen TP, et al. Molecular basis and hematological characterization of Hb H disease in southeast Asia. Am J Hematol, 1994 Apr:45(4): 293-297.
46．陈坚，孙琼，陈美玉，等．124例Hb H患者α珠蛋白基因的分析．上海医学，1999，22(2)：83-86．
47．曾瑞萍，胡彬，金龙金．广东地区血红蛋白H病基因型分析及高危胎儿基因诊断．中华医学遗传学杂志，1996，13(5)：266－268．
48. Chen FE, Ooi C, Ha SY, Cheung BM, et al. Genetic and clinical features of hemoglobin H disease in Chinese patients. N Engl J Med 2000 Aug 24: 343(8):544-550.
49．潘红飞，李强，赵国章．桂西地区Hb H病的基因型特征和产前诊断．中国优生与遗传杂志，1999，7(4)：21，19