Evaluation of the role of three thrombophilic mutations (FV Leiden, Prothrombin G20210A and MTHFR C677T) in recurrent fetal losses.
Foka Z*,
Lambropoulos A, Saravellos C, Agorastos , Zournatzi V, Bontis J,
Kotsis A, Makris PE.
Thrombosis and
Haemostasis Unit AHEPA University Hospital,
Molecular Biology
Department and A Gynaecologic Clinic,
Aristotelian
University of Thessaloniki, Greece.
Background:
There is strong evidence that thrombophilia is related with poor
pregnancy outcome. A number of reports is suggesting that
anti-phospholipid syndrome (APS), AT, PC, PS deficiency and aPC
resistance with or without the FV Leiden mutation is related with
recurrent fetal losses, due to poor feto-maternal circulation.
Since all the cases of inherited thrombophilic disorders may lead
to predisposition to frequent fetal losses, we speculated that
the prothrombin G 20210A and the MTHFR C677T polymorphism could
be responsible for a number of them. To our knowledge, there has
been no evidence yet that the prothrombin G20210A polymorphism or
MTHFR C677T is related with recurrent miscarriages.
Aim:
The aim of this study was to confirm the relationship of
recurrent miscarriages with the factor V Leiden mutation and to
investigate if there is any relationship with the prothrombin
G20210A and the MTHFR C677T polymorphism.
Methods: Case-
Control study: We studied 111 subjects, 63 consecutive patients
(cases) aged 33.4±4.12 years old who were referred for recurrent
miscarriages (2-6). The patients had been tested for
auto-antibodies, hormonal status, coagulation disorders other
than factor V Leiden and Prothrombin G20210A polymorphism,
karyotype and anatomical anomalies at least 4-6 month after
delivery or miscarriage. We also studied 48 age-matched women of
the same ethnic group who had two or more consecutive
uncomplicated pregnancies (controls).
Results:
Our results are shown in the following table.
|
Patients
(n=63) |
Controls
(n=48) |
Odds
Ratio |
|
|
|
no (%) |
|
|
|
|
Factor
V Leiden mutation, +/- |
10
(15.8) |
2
(4.0) |
3.95 |
|
|
Prothrombin
G20210A, +/- |
6
(9.5) |
1
(2.0) |
4.75 |
|
|
MTHFR
C677T, +/+ |
4
(6.8) |
7
(14.5) |
0.46 |
|
|
* +/- denotes heterozygous state, +/+ homozygous state. There were not combined mutations among the patients.
Conclusion:
Factor V Leiden and prothrombin G20210A polymorphism, but
not MTHFR C677T is an additional risk factor for recurrent
miscarriages.
Co-existence of haemorragic hereditary
defect and thrombolic mutations in Greek haemophiliacs.
Foka Z, Lampropoulos A, Gerotziafas GT,
Pithara E, Kotsis A, Makris PE.
Haemostasis and Thrombosis Unit, AHEPA
University Hospital
Molecular Biology Department of AUT.
Background: The clinical
phenotype of haemophilia syndromes is quite variable even in
patients sharing the same level of deficient factor or the same
molecular abnormality. Modulation of clinical expression and
disease severity by different interacting factors has been
recently proposed. Moreover, it is well known that the
haemophiliacs do not bleed all the time, but only after trauma,
surgical operation and tooth extraction. may be due to a
regulatory mechanism.
Aim: The aim of the study was
to evaluate the prevalence of factor V Leiden and the prothrombin
G20210A polymorphism in Greek haemophiliacs and to compare it
with the prevalence in thrombophilic unrelated subjects and in
normal controls.
Materials: 78 unrelated
haemophiliacs A or B, aged 20-45 years old. We also studied 100
age-matched thrombophilic patients with one or more episode of
thrombosis and 100 healthy men of the same ethnic group.
Methods: We performed a hot
start PCR following by digestion with the restriction enzymes
Mnl1 for the factor V Leiden and Hind III for the Prothrombin
G20210A polymorphism.
Results: Our results are shown
in the following table:
|
Haemophiliacs (n=63) |
Thrombophiliacs
(n=48) |
Controls (n= ) |
|
|
no (%) |
|||
Factor
V Leiden mutation, +/- |
|
|
|
|
Prothrombin
G20210A, +/- |
|
|
|
|
Partial
deficiency of factor XII as a cause for thrombophilic tendency.
PE. Makris, Z.
Foka, E Pithara.
Thrombosis &
Haemostasis Unit, A’ Pathologic Propaedeutic Clinic,
Aristotelian University of Thessaloniki, Greece.
In spite of the
progress in the domain of thrombosis, there is a number of
episodes which remains unexplainable. I an attempt to examine as
many as possible of thrombosis factors, we determined the levels
of factor XII in a group of thrombophilic patients. We studied 78
patients, 37 males and 41 females, agec 34±7 and 31±5 years
correspondingly. In these patients we examined the levels of
factor XII, the factor V Leiden mutation, the prothrombin G20210A
mutation, and the new indice HAT, which checks the in vitro
resistance of the complex heparin- Antithrombin. We found that 6
patients or 7.6% had reduced levels of FXII. 22 patients or 28.1%
were heterozygous of the factor V Leiden mutation, while 1 of
them was double heterozygote for the Prothrombin G20210A mutation
and another one had abnormal the HAT test.. 2 patients or 2.5%
had the G20210A mutation, and 3 patients or 3,3% had abnormal the
HAT ratio. Conclusion: A substantial number of patients may
present thrombophilic tendency as a result of deficiency of
factor XII.
THE HEPARIN-ANTITHROMBIN (HAT) COAGULATION TEST IN CLINICAL PRACTICE
Makris PE, Foka Z.
E Pithara
A’ Med. Prop.
Clinic, Haemostasis & Thrombosis Unit University of
Thessaloniki Greece.
The role of
AT pathway is especially important in the regulation of
heamostatic process.To our knowledge, it has not been applied yet
a screening test for the efficiency of the complex AT/Heparin.
For this reason we have undertaken the present study. Materials:
We studied 459 individuals divided in three groups.a) 136
apparently healthy persons (controls) free of medication (80
males aged 36.9±11.95 years old and 56 females aged 37.01±
11.87 years old). b) 273 patients (127 males aged 44.5± 14.06
years old and 146 females aged 44.52 ± 13.59 years old) with
unexplained thrombophilic tendency who had received or not
treatment with antivitamines K. All of these patients (pts) had
been previously checked for all the known indices of
thrombophilia (as Protein C, S, AT, Hc-II, A2-Apl,
PAI,). At the time of the study they were examined for APCR and
for factor V Leiden. In 144 patients we also examined the levels
of factor VIII:c. c) 50 subjects (44 males 55±6 years old and 6
females 59±9years old) who were under heparin treatment in case
of angioplasty or cardiac catheterization. Methods: For
all the coagulation tests venipuncture was performed according to
the rules. Principle of the method: Measurement of the APTT in
the presence and absence of AT/Heparin and determination of a new
ratio. Description of the new assay: Two mixtures were made: One
(M1) with equal parts of buffer Owren- Koller and Cephaline-
Kaolin reagent, and another one (M2) with equal parts of
AT/Heparin and Cephalin-Kaolin reagent. Addition of 0,1 ml PPP to
0,1 ml M1 or M2. Incubation for 3 min at 37°C and measurement of
the clotting times soon after the addition of CaCl2
(0,025M). Two clotting times are obtained: one in the absence and
one in the presence of AT/Heparin. The final concentration of AT
(human Aantithrombin concentrate produced by Biotransfusion) in
the mixture was 0,5 iu/ml and that of heparin was 1 iu/ml (Leo).
These concentrations were chosen among a series of dilutions from
0,1 to 2,0 iu/ml because they gave the most reliable results and
offered the best discrimination between the two clotting times.
The maternal mixture was made by the addition of 1000 iu Heparin
to 500 iu AT and kept frozen in aliquots. Reference values for
the new test were determined by testing the 136 healthy controls.
Reproducibility was checked by testing the same sample 20 times.
After the above proceedings we applied the new test to our
thrombophilic pts. All the clotting times were performed on the
BCT-coagulation instrument of Behring co. Results: a) Reproducibily:
The variation of coefficient for 20 determinations done on the
same sample were CV: 1,9% and after 20 days 3,5%. We also checked
the reproducibility over time in 60 out of 136 subjects (samples
taken after 2 or 3 months after the initial venipuncture) and it
was 2.1%. b) Our findings concerning the 136 healthy persons: The
mean value of the HAT ratio in the control subjects was 2.26+
0.38. Values less than two standard deviations below the mean (ie
<1.50, CL 95%) were arbitrarily considered abnormal. Five out
of the 136 control persons had a ratio less than 1.50, or 3.6%. 2
out of these five persons have abnormal aPCr- test and factor V
Leiden. c) Our findings concerning the 273 thrombophiliacs: 26
individuals had abnormal HAT ratio or 9.52%. 11 out of these 26
patients had also abnormal aPCr- test and 2 of them had factor V
Leiden mutation. In the 18 out of 26 we found the same
abnormality in the members of their families. We also examined
the influence of the elevated levels of factor VIII:c in the HAT
ratio: r= 0.161, p<0.1. This finding suggests that there is
not any influence of the elevated factor VIII:c in the HAT ratio.
d) 5 subjects out of 50 under heparin treatment had abnormal HAT
ratio before the administration of heparin; all of them corrected
their ratio after the administration of heparin. There was also
an increase in the already normal HAT ratio to the rest of them.
e) Finally, we examined if the addition of normal plasma,
purified factor IX, Xa, VIIa or VIII had the ability to correct
the abnormal ratio. All the pts corrected the HAT ratio by the
addition of normal plasma. More analytically, 7 pts corrected
with purified factor IX, 2 with FVIII and 1 with FXa. In
conclusion, this new assay might reveal new causes of
thrombophilic tendency related with the AT system of inhibition.
The HAT ratio is not influenced by the elevated levels of factor
VIII:c, but is influenced by the presence of heparin. The
correction of the ratio after the addition of purified factors
may lead to the search for new mutations at certain positions in
the genes of these factors.
Τέσσερις εργασίες που παρουσιάστηκαν
στο International Meeting of Europe and African division of
International Society of Haematology, Durban 19/09/1999.