甲型血友病的基因诊断研究
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摘要
目的建立甲型血友病的基因诊断方法。方法对33例甲型血友病患者以一期法检测血浆凝血因子FⅧ(F8)活性。采用长距离聚合酶链式反应扩增法(LD-PCR)进行FⅧ内含子22倒位检测,并对反应体系进行优化;聚合酶链式反应(PCR)技术检测内含子1倒位,凝胶成像技术分析扩增产物。捕获测序技术检测是否存在其他突变类型。结果 33例患者的FⅧ活性检测结果均小于1%,为重型;LD-PCR技术检测出13例患者存在Int22倒位,倒位发生率为39.4%;Int1倒位患者1人,发生率3%;对13例内含子22倒位患者进行测序比对,未发现其他基因突变。结论 LD-PCR技术和多重PCR技术可以有效的检测重型甲型血友病FⅧ的基因倒位。
Objective To establish a method for direct genetic diagnosis on hemophilia A(HA).Methods The concentration of FⅧ(FⅧ:C)was detected with one stage method in 33 HA patients.Detection of factorⅧ gene inversion among 33patients with severe hemophilia A was performed by long distance-polymerase chain reaction(PCR) and 0.6%agarose gel electrophoresis.Multiple-PCR was used to detect intron 1inversions respectively,seeking other mutations with sequencing technology.Results All 33 HA who were diagnosed by FⅧ:C were severe.Intron 22 inversion was detected in 13HA patients,accounting for 39.4%(13/33).And intron 1inversion was detected in 1HA patient,for 3.0%(1/33).Other mutation was not found by sequencing technology.Conclusion LD-PCR and multiplePCR are effective method for detecting factorⅧ gene inversion.
Objective To establish a method for direct genetic diagnosis on hemophilia A(HA).Methods The concentration of FⅧ(FⅧ:C)was detected with one stage method in 33 HA patients.Detection of factorⅧ gene inversion among 33patients with severe hemophilia A was performed by long distance-polymerase chain reaction(PCR) and 0.6%agarose gel electrophoresis.Multiple-PCR was used to detect intron 1inversions respectively,seeking other mutations with sequencing technology.Results All 33 HA who were diagnosed by FⅧ:C were severe.Intron 22 inversion was detected in 13HA patients,accounting for 39.4%(13/33).And intron 1inversion was detected in 1HA patient,for 3.0%(1/33).Other mutation was not found by sequencing technology.Conclusion LD-PCR and multiplePCR are effective method for detecting factorⅧ gene inversion.
引文
[1]王学峰.血友病的基因诊断[J].内科理论与实践,2013,8(3):63-166.
[2]刁戈,马莉,林方昭,等.LD-PCR检测FⅧ基因XbaⅠ位点多态性及其在血友病A携带者诊断中的应用[J].中国输血杂志,2013,26(1):33-36.
[3]张雪芹,张梅红,张心声,等.重型血友病A患者及其携带者直接基因诊断结果分析[J].山东医药,2012,52(7):88-89.
[4]Naylor J,Brinke A,Hassock S,et al.Characteristic mRNA abnormality found in half the patients with severe haemophilia A is due to large DNA inversions[J].Hum Mol Genet,1993,2(11):1773-1778.
[5]Bagnall RD,Waseem N,Green PM,et al.Recurrent inversion breaking intron 1of the factorⅧgene is a frequent cause of severe hemopilia A[J].Blood,2002,99(1):168-174.
[6]Tizzano EF,Cornet M,Baiget M.Inversion of intron 1of the factorⅧgene for direct molecular diagnosis of hemophilia A[J].Haematologica,2003,88(1):118-120.
[7]Lakich D,Kazaziahn HH Jr,Antonarakis SE,et al.Inversions disrupting the factorⅧgene are a common cause of severe haemophilia A[J].Nat Genet,1993,5(3):236-241.
[8]郭艳丽,杨林花,王学锋,等.血友病A内含子22倒位的初步研究[J].临床血液学杂志,2003,16(1):21-22.
[9]毛江洪,汪青山,谭俊青,等.用长距离PCR技术检测江西籍重型血友病甲Ⅷ因子基因倒位基因[J].中国优生与遗传杂志,2009,17(11):10-11.
[10]薛峰,杨仁池.100例血友病A患者凝血因子Ⅷ基因突变与临床表型的研究[J].临床血液学杂志,2012,25(5):299-301.
[2]刁戈,马莉,林方昭,等.LD-PCR检测FⅧ基因XbaⅠ位点多态性及其在血友病A携带者诊断中的应用[J].中国输血杂志,2013,26(1):33-36.
[3]张雪芹,张梅红,张心声,等.重型血友病A患者及其携带者直接基因诊断结果分析[J].山东医药,2012,52(7):88-89.
[4]Naylor J,Brinke A,Hassock S,et al.Characteristic mRNA abnormality found in half the patients with severe haemophilia A is due to large DNA inversions[J].Hum Mol Genet,1993,2(11):1773-1778.
[5]Bagnall RD,Waseem N,Green PM,et al.Recurrent inversion breaking intron 1of the factorⅧgene is a frequent cause of severe hemopilia A[J].Blood,2002,99(1):168-174.
[6]Tizzano EF,Cornet M,Baiget M.Inversion of intron 1of the factorⅧgene for direct molecular diagnosis of hemophilia A[J].Haematologica,2003,88(1):118-120.
[7]Lakich D,Kazaziahn HH Jr,Antonarakis SE,et al.Inversions disrupting the factorⅧgene are a common cause of severe haemophilia A[J].Nat Genet,1993,5(3):236-241.
[8]郭艳丽,杨林花,王学锋,等.血友病A内含子22倒位的初步研究[J].临床血液学杂志,2003,16(1):21-22.
[9]毛江洪,汪青山,谭俊青,等.用长距离PCR技术检测江西籍重型血友病甲Ⅷ因子基因倒位基因[J].中国优生与遗传杂志,2009,17(11):10-11.
[10]薛峰,杨仁池.100例血友病A患者凝血因子Ⅷ基因突变与临床表型的研究[J].临床血液学杂志,2012,25(5):299-301.