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.