Rationalized DNA sequencing-based protocol for genotyping patients receiving coumarin therapy.

During the last decade genetic factors affecting coumarin therapy have been extensively investigated. The most important genes appear to be CYP2C9 and VKORC1, and different studies have shown that DNA testing can dramatically improve the safety and effectiveness of the therapy. However, the implementation of pharmacogenetic testing in everyday practice is still not a reality. Facilities and ability to get results before the start of therapy are very important. The implementation of specific methodology and equipment for particular type of diagnostics can represent a serious, even impossible, financial hurdle to overcome (especially in developing countries). For this reason, the use of every tool that contributes to rationalization of the existing methods can be a considerable asset. Therefore, we set the goal to rationalize our current DNA sequencing based protocol for analysis of the VKORC1 c.-1639G> A, CYP2C9*2 and CYP2C9*3 variant alleles, in order to obtain shorter and easier procedure. Simplification of the protocol was achieved by setting up multiplex PCR and omitting DNA extraction. This rationalization of the existing DNA sequencing based procedure allows getting results in 12 hours. The new protocol was tested on 118 samples. Obtained results have shown full accordance to those obtained with previous, non-modified protocol. Therefore, given the circumstances, we consider that protocol for pharmocogenetic testing should be made more accessible - both to doctors and patients. It is one of the prerequisites in order to make genotyping prior to the therapy common practice.

Pharmacogenetic tests could be helpful in predicting of VKA maintenance dose in elderly patients at treatment initiation.

Several studies have linked mutations in the genes encoding cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex 1 (VKORC1) to a reduced VKA dose requirement and an increased risk of bleeding. Nevertheless, implementation of genotyping as a routine practice is still controversial. Our study was conducted in order to investigate the impact of genetic factors, presence of VKORC1-c.1639A, CYP2C*2 and CYP2C*3 among outpatients referred to Anticoagulation Service due to extremely unstable anticoagulant therapy. From 2008 to 2011, 68 patients, mean age 65.9, were included in the study. They were referred from primary care physicians due to inability to sustain the therapeutic range in the period of initiation of anticoagulant therapy. Genotyping results showed that 17 (25%) of them were carriers of both CYP2C9 and VKORC1 variant alleles, 38 (55.9%) were carriers of VKORC1 c.1639AA, 6 (8.8%) were carriers of CYP2C9 variant alleles, while 7 (10.3%) of them were carriers of wild type alleles. INR control upon admission showed that 34 (50%) of them were over-anticoagulated, while 12 (17.6%) of them had subsequent bleeding complications. Among over-anticoagulated patients, 32 were carriers of mutated alleles in both CYP2C9 and VKORC1 gene or VKORC1 alone, while 2 of patients carried wild type alleles. In addition to presence of CYP2C9 or VKORC 1 alleles, older age was an important factor related to a lower dose and risk for over-anticoagulation. Genotype (CYP2C9/VKORC1) and age are the most important factors that could predispose an extreme response and subsequent bleeding complications during the initiation of VKA.