论文部分内容阅读
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 carry out hot spot mutation analysis of deletion or insertion of poly-A region of exon 14 of FⅧ gene. Methods: The activated partial thromboplastin time, prothrombin time, thrombin time, fibrinogen, factor Ⅷ activity (F Ⅷ: C) and vWF antigen and activity (vWF: Ag, vWF: Act ) For the determination of hemophilia A phenotype diagnosis, using long-chain polymerase chain reaction and double PCR FⅧ gene intron 22 inverted and intron 1 inverted detection of direct nucleotide sequencing FⅧ gene Promoter region, exons and their flanking sequences. The number of copies was detected in patients with no mutation by using AccuCopyTM multiplex gene copy number detection kit. Multiplex PCR was used to amplify the six short tandem repeat sequences (FⅧUp226, FⅧUp146, FⅧInt13, FⅧInt25, FⅧown48, DXS1073) inside and outside the FⅧ gene analysis. RESULTS: Of the 343 haemophilia A pedigrees tested in the recent 4 years, 32 cases of A mutation were found in the poly A region of exon 14 of FⅧ gene, accounting for 9.33% of the total hemophilia A pedigrees, accounting for 42.11% of small insertions or deletions. Most of these families were sporadic (21 cases), with no family history of hemophilia. FⅧ gene mutations were identified as de novo mutations in 10 probands, and some patients showed mild or moderate hemophilia (F Ⅷ: C 2% ~ 10%). CONCLUSION: Insertion or deletion of A VIII / A9 region of exon 14 of FⅧ gene is a hot spot of mutation in hemophilia A. The pathogenic mechanism may be related to the slippage of DNA polymerase during DNA replication caused by the special structure of this region , Mismatch, and poly A region DNA replication, transcription, protein translation process, the second error may make the patient’s phenotype get some “correct ”.