直接核酸测序法进行血友病A携带者及产前诊断

Direct Sequencing in Hemophilia A Carrier Diagnosis and Prenatal Diagnosis

目的:对一血友病A(HA)家系进行携带者及产前诊断。方法:用活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、纤维蛋白原(Fg)和血浆因子Ⅷ促凝活性(FⅧ:C)测定进行表型诊断;用FⅧ内含子22倒位直接检测以及FⅧ基因内外4个位点的多态性进行遗传连锁分析;用核酸直接测序法对FⅧ各外显子及其侧翼序列进行检测。结果:表型检测证明先证者为重型HA,其妹妹FⅧ:C正常;FⅧ内含子22倒位直接检测及FⅧ基因内外4个位点的多态性遗传连锁分析均未获得有诊断意义的信息;直接核酸测序证实先证者及其妹妹FⅧ基因24号外显子第2209位氨基酸存在Arg→Gln(CGA→CAA)错义突变,酶切结果与此一致,其妹为携带者,后者胎儿羊水细胞DNA检测为正常男性。结论:FⅧ基因24号外显子错义突变Arg2209Gln(CGA→CAA)是该HA先证者发病的分子机制。此突变位点为国内首次报道,且该家系为国内首例运用直接核酸测序法进行HA携带者及产前诊断的家系。

To make the carrier detection and prenatal diagnosis in a pedigree with hemophilia A.Methods:Tests of the activated partial thromboplastin time(APTT),prothrombin time(PT),fibrinogen(Fg)and plasma FⅧactivity(FⅧ:C)were employed to make the phenotype diagnosis in a propositus and his sister.Intron22inversion in FⅧgene was examined by LD-PCR and the hereditary linkage analysis were done by analyzing the polymorphism of FⅧintragenic and extragenic loci.All the26exons and their flanks of FⅧgene were amplified by polymerase chain reaction and the products were screened by direct sequencing.Results:Phenotype diagnosis revealed that the propositus was of severe HA while his sister had normal FⅧ:C.The result of intron22inversion and the intragenic and extragenic loci hereditary linkage analysis found no valuable information for diagnosis.The missense mutation Arg2209Gln(CGA→CAA)in exon24of FⅧgene has been identified in the propositus and his sister and was confirmed by restriction enzyme analysis with TaqⅠ.The fetus of the sister was diagnosed as a normal male.Conclusion:The molecular mechanism of the hemophilia A occurred in the propositus is the missense mutation Arg2209Gln(CGA→CAA)in exon24of FⅧgene,and this mutation point was identified for the first time in China.It was also the first pedigree of hemophilia A carrier and prenatal diagnosis made by direct sequencing in China.