Bowen D J, Bowley S, John M, Collins P W
Arthur Bloom Haemophilia Center, Department of Haematology, University of Wales College of Medicine, Cardiff, S. Wales, UK.
Thromb Haemost. 1998 May;79(5):949-54.
Simultaneous genetic diagnosis of factor V (FV) Leiden (G1691A), the prothrombin variant (G20210A) and the thermolabile methylenetetrahydrofolate reductase (MTHFR) variant (C677T) has been achieved using multiplex heteroduplex analysis. All three loci are amplified in a single polymerase chain reaction (PCR) containing test DNA and three heteroduplex generators, respectively detecting the three nucleotide substitutions. After PCR, the products are analysed directly without further manipulation and the resulting heteroduplex profiles permit straightforward interpretation of the respective genotypes. The multiplex test has been used to assess the prevalence and allele frequency of each of the three nucleotide substitutions in 300 individuals (150 males and 150 females) from the local (S. Wales) population. A prevalence of 8% and an allele frequency of 0.040 +/- 0.015 (95% confidence interval) was obtained for FV Leiden; the prothrombin variant showed a prevalence of 1% and an allele frequency of 0.007 +/- 0.006 (95% confidence interval); the MTHFR mutation showed a prevalence of 60% and an allele frequency of 0.377 +/- 0.039 (95% confidence interval). This method is applicable to investigation of large cohorts of patients with arterial or venous thrombotic disease.
采用多重异源双链分析实现了对因子V（FV）Leiden（G1691A）、凝血酶原变异体（G20210A）和热不稳定亚甲基四氢叶酸还原酶（MTHFR）变异体（C677T）的同时基因诊断。在含有测试DNA和三种异源双链生成剂的单个聚合酶链反应（PCR）中对所有三个位点进行扩增，分别检测这三个核苷酸替换。PCR后，产物无需进一步处理即可直接分析，所得的异源双链图谱允许直接解读各自的基因型。该多重检测已用于评估来自当地（南威尔士）人群的300名个体（150名男性和150名女性）中这三种核苷酸替换各自的患病率和等位基因频率。FV Leiden的患病率为8%，等位基因频率为0.040±0.015（95%置信区间）；凝血酶原变异体的患病率为1%，等位基因频率为0.007±0.006（95%置信区间）；MTHFR突变的患病率为60%，等位基因频率为0.377±0.039（95%置信区间）。该方法适用于对大量动脉或静脉血栓性疾病患者队列的研究。
