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.