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Biohellenika

The oral anticoagulant treatment (with Warfarin, Acenocoumarol, Ρhenprocoumon Fluindione), is responsible for the 10% of all side effects connected with drugs and lead to emergency hospitalisation.

The causes of these hospitalizations are attributed to minor or major hemorrhagic manifestations (about 1% of the cases appear with major complications), and at the same time as failures in the right adjustment of the oral anticoagulation treatment, that often comes up with recurrent thrombotic events.
Bodin et al, Blood 2005

For this failure various acquired or hereditary factors have been implicated

We have problems with adjusting the treatment due to the fact that in some patients an augmented resistance was observed. This resistance is connected with the mutation of the vitamin K epoxidase (VKORC1),

In the same time some patients were observed with augmented sensitivity for which a number of polymorphisms were implicated.

Liver dysfunction Metabolic syndromes

Vitamin K epoxide reductase complex subunit 1 (VKORC1)

Backround :

The oral treatment with antivitamin K is being applied in clinical practice almost exclusively for the prevention and treatment of thromboembolic disease. This treatment often comes with haemorrhagic or thrombotic complications that are connected with the right adjustment of the dosage and the follow up of the treatment due to number of factors that are involved such as age, body weight, nutrition habits and genetic factors.

The detection of this mutation between greek patients receiving oral anticoagulant treatment.

We studied 145 subjects, 105 were the control group with out receiving any anticoagulant and (Χ=35±15 years old) 40 patients in oral anticoagulant treatment with antivitamin K. (Χ=42 ± 17 years old)

• DNA extraction from whole blood with the Qiagen kit (Blood mini kit) • Real-Time PCR was applied with SYBR Green in order to detect possible mutation in the gene of VKORC1 between the patients • Also in order to confirm some clinical data (increased sensitivity in small doses of Sintrom) We used the strip assay of ViennaLab that detects the polymorphism VKORC1 1639 G>A

For the RT-PCR we used: 1. a set of primers for the amplification of the wild type of the gene (PCR Primer Set for Human
VKORC1, SuperArray)

2. a mixture of reagents for the PCR that included the pigment SYBR Green
(SYBR Green real-time PCR master mix, SuperArray)

For the RT-PCR we used: 1. a set of primers for the amplification of the wild type of the gene (PCR Primer Set for Human
VKORC1, SuperArray)

2. a mixture of reagents for the PCR that included the pigment SYBR Green
(SYBR Green real-time PCR master mix, SuperArray)

Melting curve analysis
The melting curve analysis is done after the PCR and enables the splitting ability Of the fluorescence fraction that comes from the original product , from The fraction that comes from the primer’s dimmers.

Our results are demonstrated in figures 1 and 2. We note that it was not possible to detect some kind of mutation in the VKORC1 gene between our subjects.This fact is explained from the rarity of appearance of the specific gene mutation and mainly those that are connected with the increased resistance to the oral anticoagulant treatment (1-3% of the patients).

Fig 1. melting curve of the subjects with out receiving anticoagulant. Fig 2. melting curve of the subjects under oral anticoagulant treatment with antivitaminn–Κ. The common peak confirms the presence of the wild type of VKORC1 gene in all the subjects.

From the appliance of RT PCR we did not have important findings. We had though an important clinical finding : a patient with a very low dose(1 mg/daily) had a very high INR (5- 5.5). In order to have a guide for the rest of our research we applied the specialized for the polymorphism -1639 G>A strip assay of ViennaLab.

Melting curve analysis of the specific patient. For the first time we found a low ratio of fluorescence (as shown in the figure) in comparison with a normal subject. So now we how to detect the differences when searching for the mutations.

Indeed the specific patient is a homozygote for this polymorphism. We repeated the method 3 times and our findings are shown in the figure. We have shown that this polymorphism is related with (the homozygote form) with a very high INR in a low dose of Warfarin.

The research for this mutation and the polymorphism of VKIRC1 can provide us with answers in for the problems with come across in the therapeutic follow up of patients under oral anticoagulant treatment.