A genotyping method for VKORC1 1173C>T by Pyrosequencing^® technology

Abstract

Vitamin K epoxide reductase complex subunit 1 (VKORC1) is the site of inhibition by warfarin and other anti‐vitamin K drugs during oral anticoagulant therapy. The SNP rs9934438 in intron 1 of VKORC1 (c.173+1000C>T or 1173C>T) discriminating the VKORC1*2 haplotype is associated with low warfarin dose requirement and unstable prothrombin time – international normalized ratio. To genotype this SNP, we have developed a rapid method using Pyrosequencing^® technology. The proposed method takes a post‐PCR sample preparation of less than 1 h and a DNA sequencing time of less than 15 min to genotype 96 samples. The current method was compared with a dHPLC method that we reported previously. Genotype frequencies at VKORC1 1173C>T for our Swedish population were 38 % wild‐type, 40 % heterozygote and 22 % homozygote. The frequency of the T‐allele was 0.42, which exactly matches the frequency previously reported for Germans. The current method can be used to determine whether patients initiating warfarin therapy are carriers of SNP 1173 C>T that is strongly associated with low warfarin dose requirement.

• Anticoagulation
• pharmacogenetics
• polymorphism
• vitamin K epoxide reductase
• warfarin

Acknowledgements

We thank Sören and Inger Hanssen, Christer Kihlström, Leif Engquist, Torbjörn Wallén and their staff and patients in the anticoagulation clinics at the hospitals in Eksjö, Linköping, Motala, Värnamo and Västervik. We are also grateful to Kerstin Gustafsson for technical assistance. The study was supported by the county council of Östergötland and the Medical Research Council of Southeast Sweden (FORSS).