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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 screen mutations and polymorphisms of AT-Ⅲ gene in the developing cerebral venous thrombosis (CVT) in women of reproductive age by denaturing high performance liquid chromatography (DHPLC). Methods The specimens were obtained from female patients with CVT in Southern Hospital during June 2006 to December 2007 and peripheral blood in 52 healthy women with strict compatibility. The whole blood DNA was extracted from all the subjects. After PCR amplification, the promoter region of antithrombin-Ⅲ (AT-Ⅲ) gene, exons 1 ~ 6 and their exons 1 to 6 were analyzed by denaturing high performance liquid chromatography (DHPLC) Gene variation in flanking sequences. Results Six cases of abnormal peak shape were found in case group by DHPLC. One case of pathogenic mutation was identified as Exon6 G13328A heterozygous mutation. One case of newly discovered synonymous mutation Exon4 + 243G> A; six SNP loci, Four of these SNP pools have reported SNP sites and two newly discovered SNP sites. The control group found an abnormal peak (triplet). Conclusions DHPLC is an automatic, rapid and high-throughput method for gene mutation and SNP screening. AT-Ⅲ gene mutation may be one of the genetic factors leading to CVT in non-pregnant women in reproductive stage. The functional SNP loci May be involved in the occurrence of VTE in this population.