血友病A／B的携带者和产前诊断研究

目的：建立一套简易、快速且准确率高的血友病携带者和产前诊断体系。方法：对血友病A（HA）家系采用长距离聚合酶链反应（LD-PCR）及序列特异PCR进行F8基因内含子22及内含子1倒位检测。对有家族史的HA家系可联合F8基因内外的8个多态性位点进行遗传连锁分析．产前诊断加测性别位点（Amelo）。而对散发家系，则通过直接核苷酸测序查找先证者的基因突变，继而对家系女性成员进行携带者与产前诊断。对血友病B（HB）家系，采用直接核苷酸测序法确定其基因突变．结果：100个HA家系中。22例先证者的F8基因内含子22倒位检测结果为阳性；2例患者母亲为F8基因内含子1倒位的携带者：对有家族史的家系，综合应用倒位检测和遗传连锁分析，携带者与产前诊断率均为100％。34个HA散发家系除2个家系外均可找到致病突变：发现可能携带者105例，产前诊断65例，总诊断率为95．9％：23个HB家系中19个家系通过直接测序可找到突变．而联合F9基因外6个STR位点对HB家系进行遗传连锁分析，发现31例可能携带者及需产前诊断者16例，总诊断率为95．7％。结论：F8基因内含子22及1倒位筛检联合F8基因内、外多个位点的遗传连锁分析．可作为HA的携带者检测及产前诊断的方法；直接核苷酸测序可确定F9基因突变．而联合基因外多个多态性位点检测并进行遗传连锁分析．是HB携带者检测及产前诊断的简便、快速方法。

Carrier and prenatal diagnosis of haemophilia

Objective To establish a simple, rapid carrier detection and prenatal diagnosis system on hemophilia. Methods Haemophilia A （HA） cases were tested for screening the F8 gene intron 22 and 1 inversions by LD-PCR and PCR. The remaining inversions negative families with family history, were screened using linkage analysis with eight combined polymorphie markers, add the amelo site for prenatal diagnosis. For sporadic families,the whole gene sequencing was applied to detect the mutation directly. For haemophilia B （HB） families, linkage analysis with six STRs was applied to get a quick diagnostic information. The whole gene sequencing was used to get the final diagnosis. The rapid fluorescent PCR method with combined polymorphism markers were applied for linkage analysis in HA and HB families respectively. Results In all investigated 100 HA families, intron 22 inversion was found in 22 probands and two probands was positive in intron 1 inversion detection. Combined inversions detection with linkage analysis,the carrier and prenatal diagnosis of families with HA family history were all successfully diagnosed. Gene alter were been detected in the 34 sporadic families, except 2 families. The total diagnostic rate of 170 carriers and prenatal was 95.9%. Nineteen mutations were detected in 19 families of 23 HB families. Combined with the linkage analysis, the total diagnostic rate was 95.7%. Conclusions Intron 22 and 1 inversions screening combined with the linkage analysis using these highly informative polymorphic markers are available for earrier detection and prenatal diagnosis in Chinese HA families,while the direct sequencing of F IX with the linkage analysis can be successfully applied for carrier and prenatal diagnosis of HB families.