Warfarin Pharmacogenetics – Real-Time PCR Genotyping

Pharmacogenetics is the science that studies the effect of genetic diversity on patients’ response to drugs. Other factors, such as the environment, preexisting pathogenic conditions and combination treatment with other medications can also influence to some extent the heterogeneity response to drugs. A portion of 50% of patients either do not respond at all to a pharmaceutical treatment or experience unwanted side effects. As a result, their treatment is delayed until an alternative treatment with desirable clinical outcomes can be found.

Millions of people, daily take warfarin, an anticoagulant drug used for the treatment and prevention of serious thrombolytic conditions. Although this drug is considered to be very effective, it is difficult to administer the appropriate dose because of the narrow therapeutic window of the drug, meaning that there are limited margins between optimal (effective) and toxic doses. According to the FDA (U.S. Food and Drug Administration), hemorrhage, a side effect induced by chronic treatment of heart patients using warfarin, is the leading cause of death in developed countries. The detection of genetic polymorphisms (DNA sequence segment that responses to different variants) that exist in at least one of the two genes involved in the warfarin processing mechanism can assist the doctor in offering personalized treatment by accurately determining the dose and the duration of the treatment for each organism to excrete the drug.

Warfarin acts as a Vitamin K antagonist by inhibiting the synthesis of several blood clotting proteins. Research has shown the association of polymorphisms in specific genes such as CYP2C9, VKORC1 and CYP4F2 between patients’ response and the requirement of different dose of warfarin. CYP2C9 gene encodes for the enzyme involved in warfarin metabolism whilst single nucleotide polymorphisms (SNPs; sequences differing in a single nucleotide) have been identified that reduce the enzymatic action and increase the risk of hemorrhage. VKORC1 gene encodes for the enzyme target of warfarin, which is responsible for the conversion of vitamin K epoxide to vitamin K, while ten single nucleotide polymorphisms in this gene are associated with warfarin resistance.

At the molecular department of A. EVANGELOU LABORATORIES, a simple blood sample is received, and it is tested using a pioneering pharmacogenetic diagnostic test, Warfarin Pharmacogenetics REAL-TIME PCR genotyping, which detects the genetic polymorphisms in the genes involved in individual response to warfarin therapy. The results of the test are used by the doctor to provide a safe and effective dose of the drug, reducing dangerous side effects while saving money in the health system, by reducing hospital admissions with such incidents.

This method is based on the quantification of fluorescence signal in each amplification cycle. The method utilises the Taqmanprobe, which when hybridised to the target sequence, it is hydrolysed by the Taq-polymerase, enabling the fluorescent detector and quencher to dislocate and separate, emitting a fluorescent signal directly proportional to the target sequence, namely CYP2C9, CYP4F2 and VKORC1. The HEX fluorophore detects any alleles with polymorphisms for each of the genes while the FAM fluorophore detects the other gene without any polymorphisms. Cy5 fluorescent label it is used to detect the internal control. The Internal Process Control (IPC), is detected in the same reaction though the Cy5 fluorophore and permits the quantification of DNA isolated from the biological sample during the process of DNA extraction, therefore dismissing false negative results due to insufficient amount of genetic material or improper sample transport. The advantage of this type of PCR is that by using three different fluorescent dyes in a single reaction, it simultaneously detects two alleles of the genes, but also quantifies the genetic material of the biological specimen. The results are analysed by the qualified and experienced staff, increasing the reliability of the results given to the candidates under examination.

This pharmacogenetic test, allows the doctor to provide a more effective treatment to each patient individually, bringing us a step closer to personalised medicine.