G20210A prothrombin gene mutation identified in patients with venous leg ulcers

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1 J.Cell.Mol.Med. Vol 5, No 4, 2001 pp G20210A prothrombin gene mutation identified in patients with venous leg ulcers Gh. Jebeleanu, Lucia Procopciuc * Department of Medical Biochemistry, University of Medicine and Pharmacy "Iuliu Hatieganu" Cluj Napoca, Romania Received: September 6, 2001; Accepted: December 4, 2001 Abstract The G20210A mutation variant of prothrombin gene is the second most frequent mutation identified in patients with deep venous thrombosis, after factor V Leiden. The risk for developing deep venous thrombosis is high in patients identified as heterozygous for G20210A mutation. In order to identify this polymorphism in the gene coding prothrombin, the 345bp fragment in the 3'- untranslated region of the prothrombin gene was amplified using amplification by polymerase chain reaction and enzymatic digestion by HindIII (restriction endonuclease enzyme). The products of amplification and enzymatic's digestion were analized using agarose gel electrophoresis. We investigated 20 patients with venous leg ulcers and we found 2 heterozygous (10%) for G20210A mutation. None of the patients in the control group had G20210A mutation. Our study confirms the presence of G20210A mutation in the Romanian population. Our study also shows the link between venous leg ulcers and this polymorphism in the prothrombin gene. Keywords: coagulation mutation analysis prothrombin gene venous leg ulcers venous thrombosis Introduction Several deficiencies are associated with an increased risk for thrombophilic complicationsdeficiencies of protein C, protein S, antithrombin III. The prevalence of these deficiencies in patients with venous thromboembolism is only 5 to 10% [1,2,3]. Resistance to activated protein C (rapc) is *Correspondence to: Dr. Lucia PROCOPCIUC, Department of Medical Biochemistry, University of Medicine and Pharmacy "Iuliu Hatieganu" Cluj Napoca, 6 Pasteur St., 3400 Cluj Napoca, Romania. Tel. 064/ Lucia.p@clujnapoca.ro a major risk factor in thrombosis, accounting for 21 to 60% of patients with thromboembolic disorders [4]. In 95% of cases, resistance to activated protein C is associated with factor V Leiden mutation [5,6,7, 8,9]. Recently a new genetic polymorphism located in the 3 - untranslated region of the prothrombin gene has been described [10,11,12]. Prothrombin is the precursor of the thrombin, a serine protease, a key enzyme in the process of haemostasis and thrombosis with procoagulant, anticoagulant and antifibrinolytic effects.

2 The polymorphism in the prothrombin gene has been associated with increased prothrombin levels, resulting in an increased risk for venous thrombosis [11]. This polymorphism is an autosomal dominant inherited defect and involves a single base pair substitution, a guanine to adenine substitution at position in the 3 - untranslated region of the prothrombin gene (G20210A) [12]. This abnormality is the second most frequent factor found in patients with thromboembolism (with prevalence varying between 5% and 19%), after factor V Leiden mutation. It is slightly prevalent (1% - 2%) in the general population. The prevalence of G20210A mutation is 1% to 5% in Caucasian populations (Rosendaal 1998) [11] but this mutation is very rare in individuals from Asia or Africa, as is factor V Leiden mutation [12,13]. The prevalence of heterozygotes is 2.3% in healthy controls, 6.2% in venous thrombosis patients and 18% in selected thrombophilia patients with a positive family history [11,14]. On the other hand de Moerloose shows that no statistically significant differences were observed between patients and controls regarding prothrombin gene G20210A mutation [15]. The aim of our study was to analyze the presence of this mutation in the Romanian population, to try establish a link between G20210A mutation and patients with venous leg ulcers, compared with a control group. Another objective was to confirm if the pathogenesis of venous leg ulcers involved genetic and acquired risk factors. Materials and method Patients Consecutive patients younger than 70 years old who had venous leg ulcers, were selected from the Department of Dermatology between We studied 20 patients (10 men and 10 women) who had venous leg ulcers and recurrent leg ulcerations. We also studied 10 blood donors, with no personal or familial history of thrombosis and/ or venous leg ulcers. All the subjects were informed about the study and gave their written consent. Method In order to identify a G20210A polymorphism in the prothrombin gene we utilised whole genomic DNA isolated from blood leucoytes using the Lahiri method [16]. Genotyping analysis using the polymerase chain reaction (PCR) technique was performed using a Eppendorf thermocycler. In order to amplify the 345bp fragment in the prothrombin gene we used the primers described by Poort [10]. The forward primer was 5 - TCTAGAAACAGTTGCCTGGC- 3. The reverse primer has the sequence 5 - ATAGCACTGGGAGCATTGAA-3. This primer creates a new restriction enzyme cleavage site introduced in the amplified fragment. The primers were purchased from Pharmacia. The amplification was performed in a final volume of 100µl and included the following constituents: 500ng of genomic DNA, 2 µl of 10mM solution of each of the deoxynucleoside triphosphates (datp, dgtp, dctp, dttp), 1µl of 10µM of each primer, 8 µl of 20mM magnesium chloride and 2 units of Taq polymerase (Promega). The DNA was denatured at 95ºC for 10 minutes, followed by 35 cycles of denaturation at 94ºC for 30 seconds, annealing of primers at 59ºC for 30 seconds, extension of primers by Taq DNA polymerase at 72ºC for 1 minute and 30 seconds and a final extension of primers at 72ºC for 10 minutes. The amplified 345bp product was enzyme digested with HindIII for mutations detection. The enzymatic digestion was performed in a final volume of 10µl, using the following components: 6µl of polymerase chain reaction product, 100µg bovine serum albumin and 2 U HindIII /3µl of polymerase chain reaction product. The reaction was incubated at 37ºC for 3 hours. The digestion products were then analysed by agarose gel electrophoresis stained with ethidium bromide, at 105V for one hours. The normal allele with G at position gives an undigested fragment of 345bp, while the mutant allele with A at position gives 2 fragments of 322 and 23bp [10] (Fig. 1). A normal subject has a single band for a 345bp fragment, a homozygous patient has 2 bands for 322 and 23bp fragments, while a heterozygous patient has 3 bands for 345, 322 and 23bp fragments. In agarose gel electrophoresis it is possible to see the 345 and 322bp fragments. Results In order to identify the G20210A mutation we studied 20 patients with venous leg ulcers using the polymerase chain reaction and enzymatic digestion with HindIII. Table 1 summarises the results of this study. This mutation in the prothrombine gene was detected in 2 of 20 patients with venous leg ulcers. 398

3 J.Cell.Mol.Med. Vol 5, No 4, 2001 Table 1 Inherited and acquired risk factors in patients with venous leg ulcers. These patients were heterozygous for G20210A mutation. We identified no patients homozygous for G20210A mutation. The frequency of heterozygous was 10% in this group. No individual of the control group was found to be heterozygous or homozygous for G20210A mutation. This is a preliminary study and more cases are required in order to corroborate these observations. Discussion It is known that venous thrombosis is one of most common diseases, after myocardial infarction and stroke, in general population identified in 1:1000 of patients [17]. It is also known that venous leg ulcers affects approximatively 1% of the population and 40% to 80% are caused by postthrombotic disease [3,18]. 345 bp 322 bp Fig. 1 HindIII restriction enzyme digestion of the amplified 345 bp fragment of the prothrombin gene. Lane 1 - DNA molecular marker. Lane 2- patient normal for G20210A mutation: fragment of 345bp. Lane 3, 4- patients heterozygotes for G20210A mutation: fragments of 345 and 322bp. Lane 5- amplified 345bp fragment of the prothrombin gene. 399

4 Table 2 G20210A mutation in patients with venous leg ulcers and controls. In a recent study we established the relationship between the factor V Leiden mutation and the predisposition for deep venous thrombosis and venous leg ulcers. Thirty percent of patients with deep venous thrombosis (DVT) and 45% of patients with venous leg ulcers were positive for the Leiden mutation [19]. The second inherited defect associated with venous thrombosis is mutation in the prothrombin gene [10, 11, 12]. Heterozygous carriers are known to have a risk of DVT that is two times higher than in the general population and six times higher when patients carry both the Leiden mutation and prothrombin gene mutation [12, 13]. But it is still not clear if there is an association between venous leg ulcers and prothrombin gene mutation. In our study we identified 2 patients (10%) heterozygous for prothrombin gene mutation. Subject 1 presented at the age of 61 with multiple venous leg ulcerations on the right leg. She had had 4 major surgeries and after those she developed DVT. Later she developed venous leg ulcers. She had a family history of thrombosis: both her brother and father had DVT and venous leg ulcers. She took no contraceptives orally and had no alcohol consumption, but she was obese. She was identified heterozygous for G20210A mutation. Anticoagulant (dicumarinic) treatment was given, resulting in good evolution of ulcerations. Subject 2 was a man - 42 years old. He was referred to the Department of Dermatology for evaluation for reccurent venous leg ulcers. He had developed post traumatic venous leg ulcers 10 years ago. He had a family history of thrombosis; his mother and father had venous leg ulcers and his brother had resistance to activated protein C (rapc). Another brother was identified with thrombophlebitis. The index patient was a smoker; he had smoked cigarettes for 15 years daily. In a recent study he was identified heterozygous for factor V Leiden mutation [19]. In our study this patient was also identified heterozygous for G20210A mutation. Both these patients heterozygous for G20210A presented multiple ulcerations, resistance to treatment and had a recurrence of venous leg ulcers. Pathogenesis of venous leg ulcers also involves acquired risk factors such as: major surgery, pregnancy, immobilisation and use of oral contraceptives. Of the 20 patients clinically identified with venous leg ulcers, 6 patients (30%) developed the first event of DVT after surgery, 3 patients (15%) associated the first thrombotic event with pregnancy, 4 patients (20%) developed the first event of DVT after immobilisation because of trauma, while 7 patients (35%) had venous leg ulcers without acquired risk factors (Table 2). Epidemiology studies show that obesity is a risk factor for thrombosis. In our study 3 patients were found to be obese. Of the 20 patients investigated we identified 10 patients (50%) with a family history of thrombosis. The chronic consumption of alcohol has a significant role in pathogenesis of venous leg ulcers by causing a deficit syndrome. We identified 4 patients with a history of alcohol consumption. None of these patients with venous leg ulcers used oral contraceptives. 400

5 J.Cell.Mol.Med. Vol 5, No 4, 2001 In conclusion, our study shows that prothrombin gene mutation exists in Romanian patients with venous leg ulcers. In the pathogenesis of venous leg ulcers there were identified acquired risk factors such as: surgery, immobilisation, pregnancy, obesity, smoking. Both patients identified with G20210A mutation had an acquired risk factor associated with the first thrombotic event. Confirmation of the prothrombin gene polymorphism is necessary for patients that present these risk factors or when this polymorphism is associated with other inherited risk factors such as factor V Leiden mutation. We also recommend that patients with personal and familial history of DVT and/or venous leg ulcers, healthy patients before exposure to acquired risk factors must be tested for these abnormalities. References 1. Olds R.J., Fitches A.C., Geary C.P., The multigenic basis for venous thrombosis, BJ Haematol., 109: , Dahlback B., Inherited thrombophilia: resistance to activated protein C as a pathogenic factor of venous thromboembolism, Blood, 85,: , Simioni P., Prandoni P., Lensing W.A., The risk of recurrent venous thromboembolism in patients with an Arg 506 Gln mutation in the gene for factor V (factor V Leiden), N Engl J Med., 336: , Dahlback B., Resistance to activated protein C, the Arg506 to Gln mutation in the factor V gene and venous thrombosis. Functional tests and DNAbased assays, pros and cons, Thromb Haemost, 74: , Zoller B., Svensson P.J., He X., Identification of the same factor V gene mutation in 47 out of 50 thrombosis- prone families with inherited resistance to activated protein C, J Clin Invest, 94: , Bertina R.M., Koeleman B.P., Koster T., Mutation in blood coagulation factor V associated with resistance to activated protein C, Nature, 369:64-67, Greengard J.S., Sun X., Xu X., Activated protein C resistance caused by Arg506Gln mutation in factor Va, Lancet, 343: , Voorberg J., Roelse J., Koopman R., Association of idiopathic venous thromboembolism with single point mutation at Arg 506 of factor V, Lancet, 343: , Lensen R., Rosendaal F., Vandenbroucke J., Bertina R., Factor V Leiden: the venous thrombotic risk in thrombophilic families, B J Haematol, 110: , Poort S.R., Rosendaal F.R., Reitsma P.H., Bertina R.M., A common genetic variation in the 3 - untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis, Blood, 88: , Rosendaal F.R, Doggen C.J., Geographic distribution of the G to A prothrombin variant, Thromb Haemost, 79(4): 706-8, Rosendaal F.R., Siscovick D.S., Schwartz S.M., Raghunathan T.E., Vos H.L., A common prothrombin variant (20210G to A) increases the risk of myocardial infarction in young women, Blood, 90 (5): , Martinelli I., Bucciarelli M., Margaglione M., De Stefano V., Castaman G., Mannucci P.M., The risk of venous thromboembolism in family members with mutations in the genes of factor V or prothrombin or both, BJ Haematol, 111: , Castoldi E., Simioni P., Kalafatis M., Lunghi B., Tormene D., Girelli D., Combinations of 4 mutations (FV R506Q, FV H1299R, FV Y1702C, PT20210G/A) affecting the prothrombinase complex in a thrombophilic family, Blood, 96 (4): , de Moerloose P., Reber G., Perrier A., Perneger T., Bounameaux H., Prevalence of factor V Leiden and prothrombin G20210A mutations in unselected patients with venous thromboembolism, BJ Haematol., 110: , Lahiri D.K., Nurnberger J.I. Jr, A rapid nonenzymatic method for the preparation of HMW DNA from blood for RFLP studies, Nucleic Acids Res., 19: 5444, Ridker M., Miletich J.P., Hennekens C.H., Buring J.E., Ethnic distribution of factor V Leiden in 4047 men and women, implications for venous thromboembolism screening, JAMA, 277: , Peus D., Heit J.A., Pittelkow M.R., Activated protein C resistance caused by factor V gene mutation: common coagulation defect in chronic venous leg ulcers, J Am Acad Dermatol, 36: , Procopciuc L.M., Drugan C., Has C., Jebeleanu Gh., Detection of factor V Leiden mutation using polymerase chain reaction, J Med Biochem, 3: ,