摘要
目的:对FⅧ基因14号外显子poly A区缺失或插入A进行热点突变分析。方法:采用活化部分凝血活酶时间、凝血酶原时间、凝血酶时间、纤维蛋白原、凝血因子Ⅷ活性(FⅧ:C)及血管性血友病因子抗原及活性(vWF:Ag、vWF:Act)等测定进行血友病A表型诊断,用长链聚合酶链反应和双重PCR进行FⅧ基因内含子22倒位及内含子1倒位检测,采用直接核苷酸测序法检测FⅧ基因启动子区、各外显子及其侧翼序列中的突变。用AccuCopyTM多重基因拷贝数检测试剂盒对未找到突变的患者进行拷贝数检测,采用多重PCR扩增FⅧ基因内外6个短串联重复序列(FⅧUp226、FⅧUp146、FⅧInt13、FⅧInt25、FⅧDown48、DXS1073)进行遗传连锁分析。结果:在近4年检测的343个血友病A家系中,发生于FⅧ基因14号外显子poly A区的插入或缺失A突变共32例,占总血友病A家系的9.33%,占小片段插入或缺失突变的42.11%。这些家系中多数为散发(21例),无血友病家族史,其中10例先证者的FⅧ基因突变为de novo突变,且部分患者表现为轻型或中型血友病(FⅧ:C 2%～10%)。结论:FⅧ基因14号外显子A8/A9区的插入或缺失A为血友病A的突变热点,其致病机制可能与该区域的特殊结构导致的DNA聚合酶在DNA复制过程中的滑移、错配相关,而poly A区DNA复制、转录、蛋白翻译过程的二次错误又可能使患者的表型得到部分"纠正"。
Objective To analysis frameshifl mutations in poly A stretches of exon 14 in factor m （FV$） gene. Methods The APTI＇,PT,TY,Fg, Ⅷ：C, vWF：Ag, vWF：Act were tested to make phenotypic diagnosis. LD-PCR was used to detect Ⅷ gene intron 22 inversion and two sets sequence specific PCR was adopted to detect Ⅷ gene intron 1 inver- sion. The promoter and all 26 extrons of Ⅷ gene and its flank sequences were directly sequenced. The AccuCopy method was employed to investigate copy number variations （CNVs）. The multiplex PCR was performed to analysis a novel panel of the 6 short tandem repeats （ⅧUp226, FVmUp146, ⅧIntl3, FⅧInt25, ⅧDown48 and DXS1073） and amelogenin locus （the sex-typing marker） for genetic counseling. Results In all the 343 HA families, 32 were frameshifl mutations in poly A stretches of exon 14 in Ⅷ gene （9.33%）, which account for 42.11% of small deletions/insertion. In the 32 cases, 21 HA families were sporadic, and 10 unrelated hemophilia families were due to de novo mutations. Interestingly, part of the hemophilia A patients manifested as mild to moderately severe phenotype, with the clotting activities of Ⅷ （Ⅷ：C） more than 2%. Conclusions Frameshift mutations in poly A stretches at position g.96418_96425（A8） and g.95674_95683（A9） of exon 14 in Ⅷ gene are proved to be the mutation hot spots of HA. Potential mechanism of the high mutability of these regions may be due to polymerase errors or slippage during replication. At the same time, errors in DNA replication or RNA transcription/translation may result in a partial restoration of the correct reading frame, and ameliorate an expected severe phenotype.
出处
《诊断学理论与实践》
2012年第5期466-470,共5页
Journal of Diagnostics Concepts & Practice
