F8基因大片段缺失所导致的一例重型血友病A的分子发病机制研究  

Studies on the molecular mechanisms of large deletion in the F8 gene in a severe haemophilia A patient

作　　者：游国岭[1] 陆晔玲[2] 戴菁[2] 王学锋[2] 丁秋兰[2] 许冠群[2] 张利伟[2] 王鸿利[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院上海血液学研究所医学基因组学国家重点实验室,200025 [2]上海交通大学医学院附属瑞金医院检验科

出　　处：《中华检验医学杂志》2013年第6期534-537,共4页Chinese Journal of Laboratory Medicine

基　　金：上海市公共卫生重点学科建设计划资助项目（12GWZX0202）

摘　　要：目的对F8基因大片段缺失所导致的重型血友病A患者进行基因断裂点分析，探讨其分子发病机制。方法筛查患者凝血功能相关指标，明确血友病A表型诊断。采用长距离PCR及序列特异PCR进行内含子22和内含子1倒位的检测，并对F8基因的所有外显子及其侧翼序列直接测序。基因步移的方法确定F8基因断裂点。断裂点特异双重PCR对携带者进行诊断。多重荧光PCR法检测6个微卫星DNA位点（F8Up226、F8Up146、F8Int13、F8Int25、F8Down48、DXS1073）多态性对家系进行遗传连锁分析验证。结果先证者FⅧ活性〈l％，诊断为重型血友病A。患者F8内含子22及内含子1倒位均为阴性。F8基因4～7号外显子多次PCR扩增后电泳未见相应条带，其余外显子测序未发现突变。基因步移PCR扩增产物经测序发现F8基因共缺失27685个碱基，并在断裂点融合处存在2个微小同源碱基，且插入7个碱基（CTCTrCC）。断裂点特异性双重PCR结果显示胎儿及该患者的女儿均为缺失携带者，与基因连锁分析结果相吻合。结论F8基因4—7号外显子缺失导致患者发生重型血友病A，微小同源介导的末端连接等机制可能介导了缺失的发生。断裂点特异双重PCR为携带者的诊断提供了一个可靠的方法。Objective To disclose the molecular mechanisms of large deletion in the F8 gene in a sever haemophilia A patient and to screen the potential carriers. Methods Coagulation assay was performed to establish the phenotype. LD-PCR and sequence-specific PCR were adopted for detecting intron 22 and intron 1 inversion of F8 gene. All the exons of F8 gene and its flanking sequences were amplified and sequenced directly. Deletion breakpoints were identified by primer walking strategy. Breakpoint-specific assays were performed for the identification of carriers. Multiplex iluoreseent PCR was used to detect the polymorphism of six STR locus （F8Up226, F8Up146, F8Intl3, F8Int25, F8Down48 and DXS1073） for gene linkage analysis. Results The proband had a significantly reduced activity of factorⅧ（ 〈 1% ）. Inversion of intron 22 and intron 1 showed negative results in the proband. Deletion of exons 4 -7 in the F8 gene in the proband was suspected by the repeated failure to amplify the corresponding exons by PCR. Sequencing the products of prime walking showed that the patient had a deletion of 27 685 bp comprising exons 4 -7, a 2-bp microhomology （AT） was observed at the breakpoint junction. The results of breakpoint-specific PCR showed that the fetal and the proband＇s daughter were deletion carriers, consistent with the results of the genetic linkage analysis. Conclusions The deletion of exons 4-7 in the F8 gene results in a sever haemophilia A. Microhomology-mediated end-joining mediates the formation of detected deletion. The breakpoint-specific PCR provids a reliable diagnostic tool for identification of carrier status.

关 键 词：血友病A 因子Ⅷ 突变 外显子 多重聚合酶链反应 

分 类 号：R735.34[医药卫生—肿瘤]