山西省血友病A基因诊断初步研究
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摘要
血友病A(Hemophilia A，HA)，又称先天性或遗传性因子Ⅷ缺陷症。是因凝血因子Ⅷ基因缺陷，导致因子Ⅷ分子结构异常或含量减少而产生的一种临床上较常见的遗传性出血性疾病，其遗传特点是：x染色体隐性遗传，女性为携带者，男性发病，发病率为1/5000～1/10000，且发病无种族特异性。临床表现为不同程度的皮肤、粘膜、软组织、关节、肌肉及内脏各器官的出血。出血的程度和频率与血浆因子Ⅷ活性水平高度相关。
随着分子生物学及遗传技术的深入开展，对因子Ⅷ基因结构异常的遗传机制有了较深入的认识，因子Ⅷ基因位于x染色体长臂末端，(Xq28)，长186kb，由26个外显子和25个内含子组成。因子Ⅷ基因突变具有异质性，目前已发现有500余种，主要类型有：缺失、插入、重复、点突变、倒位。探讨血友病A发病的分子机理，研究其分子生物学改变，为血友病A携带者的检出、产前诊断及遗传咨询均具有重要
山西医科大学 硕士学位论文
的临床意义。
目前国外及国内上海、北京、苏州等地己开展了血友病A的基因突
变检测及诊断，既往山西省对于血友病A仅限于粗略的统计报道和临床
对症治疗，但对于血友病A发病的分子机理未曾做过系统、深入和全面
的研究，尤其是在血友病A的基因突变的检测及基因诊断方面更是空
白。
本课题属临床分子生物学研究范畴，旨在初步研究山西省重型血友
病A内含子22倒位，并用该基因缺陷直接进行家系的携带者检测，同
时利用DNA多态性联合运用多个遗传标志进行家系遗传连锁分析，为今
后血友病A临床遗传咨询，基因诊断提供可靠依据。
研究对象选自2001年3月～2001年10月山西医科大学第二医院、
省儿童医院、省人民医院等各大医院初诊及复诊24例血友病A患者，
及部分家系的成员14人。24例患者中有明确家族史的15例，散发9
例。
运用血浆凝固法（一期法）进行凝血因子Vlll活性（Fffi汇）检测，
用EL SA方法检测VWF：Ag水平。使用QI＊GEN’E公司的试剂盒提取基
因组DNA，分别设计各种引物，进行PCR反应及产物电泳分析。
根据Fffi活性水平进行分型民m：＊1％为重型，F皿：CI％～5％为中
型，＊栅：瞩％～25％为轻型。＊m：C 25％～500／O为亚临床型肝检测的N
2
山西医科大学 硕士学位论文
例血友病A患者中有14例为重型，8例为中型，2例轻型，所有血友病
A患者 VWF：Ag均在正常范围。
应用 LD一PCR（Long Distance—一 Poly。erase Chain Reaction
LD－PCR）检测 F＼llll基因内含子 22倒位，结果 14例重型血友病 A中，7
例患者确定为内含子22倒位，占50％（7／14），与国内外有关文献报道
一致，运用该基因缺陷直接为其中两个家系的3名女性成员进行检测，
结果均为携带者。联合应用a14，＊c几i＊＊n13和h汀。吐ZSTR位点等遗
传标志对两个血友病A者及其家系成员14人进行遗传连锁分析，结果：
家系C中一名女性确定为携带者，家系D中两名女性确定为携带者，
一名为正常人。
运用长距离 DNA扩增技术（LD…PCR）可快速简便测定内含子 22
倒位这一基因缺陷，检测准确率高，为HA的基因诊断开辟了一条新途
径。中国人群中的血友病A内含子22倒位检测率为47石％，与文献报
道大致相符，本文报道的山西省重型血友病A的患者中50％为内含子22
倒位，可见该机制所致血友病在中国人群中的发病率是较高的，这样可
以通过直接检测内含子22倒位对半数的中国人重型血友病A家系进行
携带者检测与产前诊断。直接检测的最大优点在于其特异性，只要得到
阳性结果即可得出明确而肯定的诊断。被测家系只要有一名患者或携带
者存在明确倒位现象，整个家系的任一成员即可通过该检测确定其疾病
状态。散发家系基因突变的构成与有家族史者相似，对于此类家系，无
3
山西医科大学 硕士学位论文
法用多态性进行基因诊断，此时，内含子22倒位的直接检测体现出其
优越性。建议在对血友病A家系进行遗传咨询时应将内含子22倒位的
检测作为首选筛查方法。
由于Fill基因庞大，其基因突变存在显著的遗传异质性，因此如果
对每一例血友病A家系常规进行突变的直接检测，不仅费时费力，而且
实验成本高，不适合临床常规检测，临床上为血友病家系提供咨询时，
只需确定被检测者的疾病状态，而可以不明确具体突变性质，故依赖遗
传多态性连锁分析对临床血友病A家系的检测是一种较为可行的方法。
本研究联合使用RFLP、VNTR、STR遗传连锁标志，选择Fib基因内的位
点Bell（RFLP人intronl3，nitron22STR位点 DTR人以及基因外的
St14ONTR）位点，运用叱R方法对两个血友病 A家系进行家系连锁分析，
说明多个多态性位点联合使用可明显提高诊断率。
总之，内含子22倒位是山西省重型血友病A的重要遗传基因缺陷，
利用LD—一PCR技术可快速、简便、精确进行患者及携带者的检测，临
床?
Hemophilia A (HA) is caused by factor Ⅷ(FⅧ) gene defects, which is a common inherited bleeding disorder and resulted from deficiency and functional abnormality of coagulation factor Ⅷ . It is a classic example of X-linked recessive inheritance . The incidence is about one in five to ten thousand males and there is no difference in the various ethnic groups. The severity and frequency of bleeding in HA patients correlate with the activity of factor Ⅷ in plasma.
The clinical presentation of HA is hemorrhage in skin, muscle, soft tissue .joint ,and internal organs .At present , a lot of studies indicated that HA was caused by various FVDI gene defects. Factor Vffl gene located on the human X chromosome, is 186kb long and contains 26 exons and 25 introns . Gene mutation of factor Ⅷ is highly particular , the amount of mutation is more than 500 , the main type includes deletion , insertion, inversion, duplication and point mutation .To investigate the molecular changes of FⅧ gene has important clinical significance for carrier detection .prenatal diagnosis and genetic counseling. Gene diagnosis for HA has been performed at abroad and Peking,
Shanghai , Suzhou in our country .But the research only limited to simple case reports and clinical treatment in Shanxi . No paper has been published in gene diagnosis. Our aim is to provide a reliable detection methods in lab for HA genetic counseling and gene diagnosis in the future. Severe HA patients were analyzed intron 22 inversion ,directly detected carriers in the two pedigrees using long distance - polymerase chain reaction (LD-PCR) ;and the pedigree linkage analysis were performed by use polymorphic genetic markers.
24 HA patients and 14 related pedigree members were selected from the Second Hospital of Shanxi Medical University, Children's Hospital of Shanxi Province and People's Hospital of Shanxi Province . Among them , 15 cases were positive in family history , 9 sporadic.
The concentration of factor VII were detected with one stage method , the antigen of von-Willebrand factor with enzyme linked immunosorbent assay ( ELISA )method , DNA extraction was performed by QIAamp DNA mini kit purchased from QIAGEN company. After PCR, the production was performed by electrophoresis analysis.
The diagnosis standard :The concentration of factor Ⅷ below 1% was severe; 1% ~ 5% moderate;5% - 25% mild; 25% ~ 50% sub clinical .
Among 24 patients, 14 cases were severe, 8 moderate and 2
mild .The level of vWF :Ag were all normal.
Intron 22 inversion was detected by application of LD-PCR, 7 patients were positive in 14 severe cases, the positive rate was 50%, which is similar to relevant reports .Three female members in the two pedigrees were defined as carrier by direct detection intron 22 inversion. Two HA pedigrees were analyzed by utilization of entragenic and intragenic markers including Stl4 VNTR locus, BclI RFLP locus and two STR loci in intron 13 and 22. All results showed that one female member was a carrier in pedigree C, and another two females defined carriers in pedigree D.
LD-PCR is a new method for HA gene diagnosis especially to analyze intron 22 inversion ,this method was rapidly, simply and accurately. The detection rate of inversion was 47.6% in Chinese population, same as other reports. Through this study , 50% severe HA patients in our province were caused by intron 22 inversion, carrier detection and prenatal diagnosis can be made by detection inversion in half of HA pedigree . The greatest advantage of direct detection was its speciality. Any member can be diagnosed if one patient or carrier has been detected with intron 22 inversion in this pedigree.
The constitution of gene mutation in sporadic HA pedigree was same as that in positive family history. The direct detection of inversion has more priority in sporadic pedigree when polymorphism can't be used .
Thus , the search for inversion of factor Ⅷ should be considered as the first DNA diagnostic option in genetic counseling of HA.
The gene mutation of factor Ⅷ has ma
随着分子生物学及遗传技术的深入开展，对因子Ⅷ基因结构异常的遗传机制有了较深入的认识，因子Ⅷ基因位于x染色体长臂末端，(Xq28)，长186kb，由26个外显子和25个内含子组成。因子Ⅷ基因突变具有异质性，目前已发现有500余种，主要类型有：缺失、插入、重复、点突变、倒位。探讨血友病A发病的分子机理，研究其分子生物学改变，为血友病A携带者的检出、产前诊断及遗传咨询均具有重要
山西医科大学 硕士学位论文
的临床意义。
目前国外及国内上海、北京、苏州等地己开展了血友病A的基因突
变检测及诊断，既往山西省对于血友病A仅限于粗略的统计报道和临床
对症治疗，但对于血友病A发病的分子机理未曾做过系统、深入和全面
的研究，尤其是在血友病A的基因突变的检测及基因诊断方面更是空
白。
本课题属临床分子生物学研究范畴，旨在初步研究山西省重型血友
病A内含子22倒位，并用该基因缺陷直接进行家系的携带者检测，同
时利用DNA多态性联合运用多个遗传标志进行家系遗传连锁分析，为今
后血友病A临床遗传咨询，基因诊断提供可靠依据。
研究对象选自2001年3月～2001年10月山西医科大学第二医院、
省儿童医院、省人民医院等各大医院初诊及复诊24例血友病A患者，
及部分家系的成员14人。24例患者中有明确家族史的15例，散发9
例。
运用血浆凝固法（一期法）进行凝血因子Vlll活性（Fffi汇）检测，
用EL SA方法检测VWF：Ag水平。使用QI＊GEN’E公司的试剂盒提取基
因组DNA，分别设计各种引物，进行PCR反应及产物电泳分析。
根据Fffi活性水平进行分型民m：＊1％为重型，F皿：CI％～5％为中
型，＊栅：瞩％～25％为轻型。＊m：C 25％～500／O为亚临床型肝检测的N
2
山西医科大学 硕士学位论文
例血友病A患者中有14例为重型，8例为中型，2例轻型，所有血友病
A患者 VWF：Ag均在正常范围。
应用 LD一PCR（Long Distance—一 Poly。erase Chain Reaction
LD－PCR）检测 F＼llll基因内含子 22倒位，结果 14例重型血友病 A中，7
例患者确定为内含子22倒位，占50％（7／14），与国内外有关文献报道
一致，运用该基因缺陷直接为其中两个家系的3名女性成员进行检测，
结果均为携带者。联合应用a14，＊c几i＊＊n13和h汀。吐ZSTR位点等遗
传标志对两个血友病A者及其家系成员14人进行遗传连锁分析，结果：
家系C中一名女性确定为携带者，家系D中两名女性确定为携带者，
一名为正常人。
运用长距离 DNA扩增技术（LD…PCR）可快速简便测定内含子 22
倒位这一基因缺陷，检测准确率高，为HA的基因诊断开辟了一条新途
径。中国人群中的血友病A内含子22倒位检测率为47石％，与文献报
道大致相符，本文报道的山西省重型血友病A的患者中50％为内含子22
倒位，可见该机制所致血友病在中国人群中的发病率是较高的，这样可
以通过直接检测内含子22倒位对半数的中国人重型血友病A家系进行
携带者检测与产前诊断。直接检测的最大优点在于其特异性，只要得到
阳性结果即可得出明确而肯定的诊断。被测家系只要有一名患者或携带
者存在明确倒位现象，整个家系的任一成员即可通过该检测确定其疾病
状态。散发家系基因突变的构成与有家族史者相似，对于此类家系，无
3
山西医科大学 硕士学位论文
法用多态性进行基因诊断，此时，内含子22倒位的直接检测体现出其
优越性。建议在对血友病A家系进行遗传咨询时应将内含子22倒位的
检测作为首选筛查方法。
由于Fill基因庞大，其基因突变存在显著的遗传异质性，因此如果
对每一例血友病A家系常规进行突变的直接检测，不仅费时费力，而且
实验成本高，不适合临床常规检测，临床上为血友病家系提供咨询时，
只需确定被检测者的疾病状态，而可以不明确具体突变性质，故依赖遗
传多态性连锁分析对临床血友病A家系的检测是一种较为可行的方法。
本研究联合使用RFLP、VNTR、STR遗传连锁标志，选择Fib基因内的位
点Bell（RFLP人intronl3，nitron22STR位点 DTR人以及基因外的
St14ONTR）位点，运用叱R方法对两个血友病 A家系进行家系连锁分析，
说明多个多态性位点联合使用可明显提高诊断率。
总之，内含子22倒位是山西省重型血友病A的重要遗传基因缺陷，
利用LD—一PCR技术可快速、简便、精确进行患者及携带者的检测，临
床?
Hemophilia A (HA) is caused by factor Ⅷ(FⅧ) gene defects, which is a common inherited bleeding disorder and resulted from deficiency and functional abnormality of coagulation factor Ⅷ . It is a classic example of X-linked recessive inheritance . The incidence is about one in five to ten thousand males and there is no difference in the various ethnic groups. The severity and frequency of bleeding in HA patients correlate with the activity of factor Ⅷ in plasma.
The clinical presentation of HA is hemorrhage in skin, muscle, soft tissue .joint ,and internal organs .At present , a lot of studies indicated that HA was caused by various FVDI gene defects. Factor Vffl gene located on the human X chromosome, is 186kb long and contains 26 exons and 25 introns . Gene mutation of factor Ⅷ is highly particular , the amount of mutation is more than 500 , the main type includes deletion , insertion, inversion, duplication and point mutation .To investigate the molecular changes of FⅧ gene has important clinical significance for carrier detection .prenatal diagnosis and genetic counseling. Gene diagnosis for HA has been performed at abroad and Peking,
Shanghai , Suzhou in our country .But the research only limited to simple case reports and clinical treatment in Shanxi . No paper has been published in gene diagnosis. Our aim is to provide a reliable detection methods in lab for HA genetic counseling and gene diagnosis in the future. Severe HA patients were analyzed intron 22 inversion ,directly detected carriers in the two pedigrees using long distance - polymerase chain reaction (LD-PCR) ;and the pedigree linkage analysis were performed by use polymorphic genetic markers.
24 HA patients and 14 related pedigree members were selected from the Second Hospital of Shanxi Medical University, Children's Hospital of Shanxi Province and People's Hospital of Shanxi Province . Among them , 15 cases were positive in family history , 9 sporadic.
The concentration of factor VII were detected with one stage method , the antigen of von-Willebrand factor with enzyme linked immunosorbent assay ( ELISA )method , DNA extraction was performed by QIAamp DNA mini kit purchased from QIAGEN company. After PCR, the production was performed by electrophoresis analysis.
The diagnosis standard :The concentration of factor Ⅷ below 1% was severe; 1% ~ 5% moderate;5% - 25% mild; 25% ~ 50% sub clinical .
Among 24 patients, 14 cases were severe, 8 moderate and 2
mild .The level of vWF :Ag were all normal.
Intron 22 inversion was detected by application of LD-PCR, 7 patients were positive in 14 severe cases, the positive rate was 50%, which is similar to relevant reports .Three female members in the two pedigrees were defined as carrier by direct detection intron 22 inversion. Two HA pedigrees were analyzed by utilization of entragenic and intragenic markers including Stl4 VNTR locus, BclI RFLP locus and two STR loci in intron 13 and 22. All results showed that one female member was a carrier in pedigree C, and another two females defined carriers in pedigree D.
LD-PCR is a new method for HA gene diagnosis especially to analyze intron 22 inversion ,this method was rapidly, simply and accurately. The detection rate of inversion was 47.6% in Chinese population, same as other reports. Through this study , 50% severe HA patients in our province were caused by intron 22 inversion, carrier detection and prenatal diagnosis can be made by detection inversion in half of HA pedigree . The greatest advantage of direct detection was its speciality. Any member can be diagnosed if one patient or carrier has been detected with intron 22 inversion in this pedigree.
The constitution of gene mutation in sporadic HA pedigree was same as that in positive family history. The direct detection of inversion has more priority in sporadic pedigree when polymorphism can't be used .
Thus , the search for inversion of factor Ⅷ should be considered as the first DNA diagnostic option in genetic counseling of HA.
The gene mutation of factor Ⅷ has ma
引文
1. Geoffrey KC, Edward GDT, AdamI W, et al. The factor Ⅷ structure and mutation resource site: HAMSTeRS Version 4.Nucleic Acids Research. 1998; 26(1): 216～219
2. Lakich D, Kazazian HH, Antonarakis SE et al. inversions disrupting the factor Ⅷgene as a common cause of severe haemophilia A. Nature Genetic. 1993; 5: 236～241.
3. Naylor JA, Brinke A, Hassock S, et al. Characteristic mRNA abnormality found in half the patients with severe Hemophilia A is due to large DNA inversion. Hum Mol Genet. 1993; 2: 1773.
4. Liu JZ, Liu Q, Liang Y, et al. PCR assay for the inversion causing severe hemophilia A and its application. Chinese Medical Journal. 1999, 112(5): 419～423.
5. Liu. Q, Nozari G, Sommer SS, et al. Single-Tube Polymerase Chain Reaction for Rapid Diagosis of the Inversion Hotspot of Mutation in Hemophilia A. Blood. 1998; 92(4): 1458～1459.
6．王学锋，刘元昉，李志广等．血友病A携带者检测与产前诊断。中华血液学杂志，2001．22(3)：117～120．
7. Windsor S, Taylor SAM, Lillicrap D, et al. Direct detection of a common inversion mutation in the genetic diagnosis of severe Hemophilia A. Blood. 1994, 84(7): 2202～2205.
8. Jenkins PV, Collins P.W, Golden E, et. al. Analysis of intron22 inversion of the factor Ⅷgene in severe hemophilia A: Implication for Genetic Counseling. Blood. 1994;84(7):2197～2201.
9. Peake I. Molecular Genetics and Counselling in Haemophilia. Thrombosis
and Haemostasis. 1995,74(1): 40～44.
10. Antonarakis SE, Rossiter JP, Young M, et al. Factor Ⅷ gene inversion in severe Hemophilia A: Results of an international Consortoum study. Blood. 1995; 86(6): 2206～2212.
11. Peake I. Molecular Genetics and Counselling in Haemophilia. Thrombosis and Haemostasis. 1995,74(1): 40～44.
12. Richards B, Heilig R, Oberle I, et al. Rapid PCR analysis of the St14(DXS52)VNTR Nucleic Acids Research. 1991,19(8):206.
13. Lalloz MRA, McVey JH, Pattinson JK, et al. Haemophilia A diagnosis by analysis of a hypervariable dinucleotide repeat within the factor Ⅷ gene. Lancet. 1991, 338: 207～211.
14. Winder S, Taylor SAM, Liuierap D, et al. Mutiplex analysis of two intragenic microsatellite repeat polymorphisms in the genetic diagnosis of haemophilia A. Br J. Haemotol. 1994, 86: 810～815.
15．陈维之，周道斌，武永吉。DXS52 VNTR位点多态性频率分布的研究。中华血液学杂志，1998，19(10)：209～210．
16．王晓冬，储小红，阮长耿。国人St14(Dxs52)VNTR及其对血友病甲基因诊断价值的探讨。苏州医学院学报，1996，16(1)：38～40。
17．金春莲，林长坤，宋军等。东北地区正常人群DXS52 VNTR位点在甲型血友病基因诊断中的应用。中华遗传学杂志，1998，15：31～33。
18．卞美璐，吴冠芸，马林生等。甲型血友病产前诊断应用DNA聚合酶链反应(PCR)进行家系RFLP连锁分析。中国医学科学院学报，1993，15：102。
19. Lalloz, M R.A, Schwaab R, McVey JH, et al. Haemophila A diagnosis by
simultaneous analysis of two Variable dinucleotide tandem repeats whithin the factor Ⅷ gene. Br J Haematol. 1994, 86: 804～809,
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