甲型血友病家系的直接和间接基因诊断

The direct and indirect gene diagnosis for hemophilia A families

目的该研究针对温州地区甲型血友病(HA)家系,首先进行直接基因诊断,然后根据可变数目串联重复序列(VNTR)多态性、短串联重复序列(STR)多态性和限制性片段长度多态性(RFLP)进行间接基因诊断,为基于HA症状前、携带者基因诊断结果的遗传咨询和生育指导提供依据。方法针对HA先证者及其有关家系成员,首先采用倒位位移PCR(IS-PCR)进行凝血因子Ⅷ(FⅧ)inv22、del22、dup22、inv1的检测。然后进行单体型基因连锁分析。采用PCR检测FⅧ基因外的DXS52(St14)位点的VNTR多态性,检测FⅧ基因外的DXS15(CA)n、DXS9901(GT)n、DXS1073(GT)n位点和内含子1(GT)n、13(CA)n、22(GT)n(AG)n、24(GT)n位点的STR多态性并经毛细管电泳确证。另外采用PCR产物限制酶切检测FⅧ基因的内含子18、19、22位点的RFLP多态性。结果以2个HA家系为例报道研究结果。家系三的先证者为远端Inv22患者,先证者母亲为远端inv22携带者,先证者父亲为正常者。先证者、先证者母亲、先证者父亲均未发生del22或dup22,并且均未发生inv1。家系五先证者外婆肯定不是携带者,先证者的X染色体来自外公,但已知外公不是患者,那么按照最大风险估计,母亲的那条来自外公的X染色体在外公生殖细胞中FⅧ基因发生了突变,因此母亲是携带者。结论该研究的HA家系的直接和间接基因诊断,特别是对症状前男孩的诊断、对未曾有患病后代的女性携带者的检出,具有非常重要的实际意义,可以为遗传咨询和生育指导提供可靠依据。

Objective：First to develop direct gene diagnosis,then to develop indirect gene diagnosis based on variable number tandem repeat（VNTR）polymorphism,short tandem repeat（STR）polymorphism and restriction fragment length polymorphism（RFLP）for hemophilia A（HA）pedigrees in Wenzhou,further to provide evidences for genetic counseling and family planning guidance according to the gene prognosis results of males and female carriers of HA. Methods：For the HA probands and related family members,first,inverse shifting-PCR（IS-PCR）was used to detect inv22,del22,dup22 and inv1 of clotting factor Ⅷ（FⅧ）gene. Then,haplotype gene linkage analysis was processed. PCR was used to analyze the VNTR polymorphism in extragenic site DXS52（St14）of FⅧ gene,to analyze the STR polymorphism in extragenic sites DXS15（CA）n,DXS9901（GT）n,DXS1073（GT）n and in intragenic sites intron 1（GT）n,13（CA）n,22（GT）n（AG）n,24（GT）n of FⅧgene,and the STR polymorphism was identified by capillary electrophoresis. Also PCR/RFLP was used to analyze the RFLP in intragenic sites intron 18,19 and 22 of FⅧ gene. Results：The results of two HA pedigrees were taken as examples. In pedigree 3,the proband is inv22 distal pattern hemophiliac,his mother is inv22 distal pattern carrier and his father is non-inv22 individual. The proband,his mother and father are all non-del22,non-dup22 and non-inv1 individuals. In pedigree 5,the grandmother of proband is not carrier,the X chromosome of proband was interited from his grandfather. Since the grandfather is not HA patient,supposing the most serious situation,it was deduced that a mutation of FⅧ gene had happened to the X chromosome of the mother which interited from the grandfather when it was in germ cell of grandfather,thus the mother of proband is carrier. Conclusion：The direct and indirect gene diagnosis of HA pedigrees of this study,especially the presymptomatic gene diagnosis of males,the gene diagnosis of female carriers without HA offspring,would have their very important clinical implications,could provide reliable evidences for genetic counseling and family planning guidance.