German Red Cross Blood Service Baden-Württemberg/Hesse and Institute for Transfusion Medicine and Immunohematology, Goethe University, Frankfurt, Germany.
Mol Diagn Ther. 2011 Feb 1;15(1):13-9. doi: 10.1007/BF03257189.
Variant alleles of vitamin K epoxide reductase complex subunit 1 gene (VKORC1), the target molecule of vitamin K antagonists, and of cytochrome P450 (CYP) 2C9, an enzyme involved in coumarin metabolism, affect the anticoagulant response of coumarins, which have a narrow therapeutic window. Genotyping for these variants allows for prediction of therapeutic drug doses. The discussion of the clinical role of genotype-guided coumarin dosing is ongoing. For pharmacogenetic information to be useful, results must be available quickly.
Here we report on the establishment of an allele-specific amplification (ASA)-PCR assay for the three most relevant polymorphisms for coumarin pharmacogenetics. The assay was validated against sequencing data on 100 random samples from Caucasian blood donors, incorporating all genotypes. Divergent results were confirmed by repeating the analysis with both methods. One hundred percent congruence with DNA sequencing was determined as the 'pass' criterion for the assay.
The ASA-PCR assay reproducibly identified the three informative single nucleotide polymorphisms. Discrepancies between ASA-PCR and sequencing were clarified by retrospective analysis as being due to erroneous analysis or documentation. In summary, the congruence of sequencing and duplex ASA-PCR was 100%.
ASA-PCR is significantly faster and less expensive than sequencing. We expect that pharmacogenetics-based dosing decisions may reduce the frequency of over- and undertreatment with vitamin K antagonists, especially during drug initiation, and thus improve patient safety.
维生素 K 环氧化物还原酶复合物亚基 1 基因（VKORC1）的变异等位基因是维生素 K 拮抗剂的作用靶点，细胞色素 P450（CYP）2C9 是香豆素代谢中涉及的一种酶，这两种物质均影响香豆素的抗凝反应，而香豆素的治疗窗较窄。对这些变异进行基因分型有助于预测治疗药物剂量。目前正在讨论基因型指导的香豆素给药的临床作用。为了使遗传药理学信息有用，必须快速获得结果。
我们在此报告了一种用于香豆素药物遗传学的三个最相关多态性的等位基因特异性扩增（ASA）-PCR 检测方法的建立。该检测方法在 100 名随机来自白种人献血者的样本的测序数据中进行了验证，其中包含所有基因型。通过两种方法重复分析以确认不一致的结果。将与 DNA 测序 100%一致确定为该检测的“通过”标准。
ASA-PCR 检测方法可重复性地鉴定出三个有意义的单核苷酸多态性。通过回顾性分析，ASA-PCR 和测序之间的差异被澄清为分析或记录错误。总的来说，测序和双管 ASA-PCR 的一致性为 100%。
ASA-PCR 比测序更快且更经济。我们预计，基于遗传药理学的给药决策可能会减少维生素 K 拮抗剂过度和治疗不足的频率，尤其是在药物起始阶段，从而提高患者安全性。
