应用变性高效液相色谱筛查颅内静脉系统血栓形成AT-Ⅲ基因突变及多态性  

Denaturing high-performance liquid chromatography for screening antithrombin Ⅲ gene mutation and polymorphisms in patients with cerebral venous thrombosis

作　　者：汪丽萍[1] 裘毓雯[1] 尹爱兰[1] 马云燕[1] 刘柯玲[1] 熊丽[1] 余艳红[1] 钟梅[1] 王辰[2] 

机构地区：[1]南方医科大学南方医院妇产科,广东广州510515 [2]首都医科大学附属北京朝阳医院呼吸内科,北京100020

出　　处：《南方医科大学学报》2009年第10期1982-1986,共5页Journal of Southern Medical University

基　　金："十五"国家科技攻关项目(2001BA703B15);广东省科技计划基金资助项目(2007B030502015)

摘　　要：目的应用变性高效液相色谱(DHPLC)方法筛查生育期女性颅内静脉系统血栓形成(CVT)AT-Ⅲ基因的突变及多态性。方法标本来自于2006年6月～2007年12月南方医院生育期女性CVT患者,及52例严格配伍的健康女性外周静脉血。提取所有受试者全血DNA,PCR扩增后应用变性高效液相色谱(DHPLC)技术分析抗凝血酶-Ⅲ(AT-Ⅲ)基因的启动子区,第1～6外显子及其侧翼序列的基因变异情况。结果DHPLC技术分析发现病例组出现6种异常峰形,经测序证实1例致病突变为Exon6 G13328A的杂合突变,1例新发现的同义突变Exon4+243G>A;SNP位点6个,其中4个SNP库已报道的SNP位点,2个新发现的SNP位点。对照组发现一种异常峰型(三峰)。结论DHPLC是一种自动、快速、高通量的基因突变及SNP位点的筛查方法,AT-Ⅲ基因突变可能是导致生育期非妊娠女性CVT的遗传因素之一,功能性SNP位点也可能参与了该人群VTE的发生。Objective To identify antithrombin HI （AT- Ⅲ） gene mutation and polymorphisms in pregnant women and parturients with cerebral venous thrombosis （CVT） using denaturing high-performance liquid chromatography （DHPLC）. Methods The genomic DNA was extracted fi＇om the blood samples of 50 pregnant women and parturients with CVT and 52 matched healthy women for molecular analysis using a PCR/DHPLC assay followed by DNA sequence analysis. Ten primer pairs were designed for amplifying the AT- Ⅲ promoter region and exons 1-6 including the exon/intron boundaries. A rapid screening assay based on DHPLC was established to screen the mutation and polymorphisms of AT- Ⅲ gene. Results Six abnormal peaks were detected in 40 of the patients by DHPLC. Direct DNA sequencing was performed on representative samples detected by DHPLC profiling. One pathogenic heterozygous G13328A missense mutation in exon 6, and a novel silent mutation in exon 4＋243 G〉A were identified. Six single nucleotide polymorphism （SNP） sites were found, including 4 previously reported ones in the SNP library and two were novel SNP sites. An abnormal peak was detected in the control group by DHPLC. Conclusion DHPLC allows automated and rapid high-throughput detection of AT- Ⅲ gene mutation and polymorphisms in the clinical setting and prenatal diagnosis. Our findings suggested that AT- Ⅲ gene mutation, as well as its polymorphisms, contributes to the occurrence of CVT in pregnant women and parturients.

关 键 词：变性高效液相色谱 静脉血栓栓塞 单核苷酸多态性 基因突变 AT-Ⅲ基因 

分 类 号：R743.3[医药卫生—神经病学与精神病学]