Association of the Prothrombin G20210A Mutation With Factor V ...

Coagulation and Transfusion Medicine / PROTHROMBIN G20210A MUTATION AND FACTOR V LEIDEN

Association of the Prothrombin G20210A Mutation With Factor V Leiden in a Midwestern American Population Neil R. Bavikatty, MD,1 Anthony A. Killeen, MD, PhD,1 Nahida Akel, MT, MBA,1 Daniel Normolle, PhD,3 and Alvin H. Schmaier, MD1,2 Key Words: Thrombosis; Thrombophilia; Factor V Leiden; Prothrombin 20210

Abstract The prothrombin G20210A mutation has been identified as a risk factor for thrombosis. We studied the relationship between prothrombin G20210A and factor V Leiden mutations in patients with thrombophilia. The first 264 patients for whom these molecular diagnostic studies were requested at our institution were included in the study. For 116 of the 264 patients, additional coagulation test results were available in the laboratory database. The prothrombin G20210A mutation was found in 16 (6.1%) of the patients and the factor V Leiden mutation in 44 (16.7%). Of the 16 patients with the prothrombin G20210A mutation, 8 also carried factor V Leiden; this association was significant. In contrast, only 2 patients of the 116 with additional coagulation testing harbored more than 1 prothrombotic risk factor. These data support the hypothesis that thrombophilia is a multigenic disorder. Among unselected samples from a Midwestern population evaluated for thrombotic risk factors, the prevalence of factor V Leiden and prothrombin G20210A mutations are similar to those found in other populations in the Western world.

A G to A transition at position 20210 within the 3′untranslated region of the prothrombin gene has been identified as a risk factor for thrombosis.1-5 Poort et al1 found the mutant prothrombin G20210A allele in 18% of patients with familial thrombophilia, 6% of patients with a first deep venous thrombosis, and 1% to 2% of healthy control subjects. Geographic differences in the frequency of carriership of the mutation exist, with the highest frequencies seen in southern Europe and the Middle East.6 The mechanism by which the mutation causes a predisposition to thrombosis is not fully understood but may be related to increased levels of plasma prothrombin.1 Patients with multiple genetic defects in the hemostaticthrombotic system may have an increased risk of thrombosis compared with patients with single defects.7 While several studies have demonstrated a frequent association of the prothrombin G20210A mutation and the factor V Leiden mutation in patients with thrombophilia,1,8-10 another study did not find this association.11 We sought to study the frequency of coexistence of prothrombin G20210 A and factor V Leiden mutations in patients with thrombosis and their family members in our patient population in the midwestern United States. We also performed a retrospective analysis of our coagulation laboratory database to determine the frequency of a patient having 2 risk factors for thrombosis other than the prothrombin G20210A and factor V Leiden mutations.

Materials and Methods The study population consisted of the first 264 patients referred for molecular diagnostic studies for thrombophilia. They were examined in 2 groups. The first group consisted of 272

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135 consecutive patients for whom blood samples were sent to our laboratory for prothrombin G20210A testing between September 1997 and May 1998. For most of these patients, previous or concurrent results for factor V Leiden were available; when unavailable, their frozen DNA samples were examined for factor V Leiden. The second group consisted of an additional 129 consecutive patients identified between April 1996 and August 1997 for whom molecular testing for the factor V Leiden mutation had been ordered before the availability of testing for the prothrombin G20210A mutation in our institution. These patients’ frozen DNA samples also were tested for the prothrombin G20210A mutation. Demographic and clinical data were collected by review of medical records. DNA for factor V Leiden and prothrombin G20210A testing was extracted from whole blood by standard techniques.12 Examination for prothrombin G20210A was performed by polymerase chain reaction using a mutagenic primer as previously described by Poort et al.1 Testing for factor V Leiden was performed by polymerase chain reaction according to the method of Bertina et al.13 Clottable fibrinogen (reference range, 0.15-0.45 g/dL [4.4-13.2 µmol/L]) was measured by thrombin clotting time determination using diluted plasma according to the method of Clauss.14 Fibrinogen antigen (reference range, 0.15-0.45 g/dL [4.4-13.2 µmol/L]) and antithrombin III antigen (reference range, 24-36 mg/dL [240-360 mg/L]) were determined by radial immunodiffusion using commercially available reagents obtained from The Binding Site Limited, Birmingham, England. Plasminogen antigen levels (reference range, 7.513.9 mg/dL [75-139 mg/L]) also were determined by radial immunodiffusion (Helena Laboratories, Beaumont, TX). Antithrombin III activity (reference range, 80%-120% [0.81.2]) and protein C activity (reference range, 62%-126% [0.62-1.26]) were determined by chromogenic assay using reagents from Baxter Diagnostics, Deerfield, IL. Plasminogen activity (reference range, 88%-136% [0.88-1.36] for females, 80%-124% [0.80-1.24] for males) was measured by chromogenic assay using reagents from Chromogenix (Diapharm, Franklin, OH). Free protein S antigen (reference range, 43%132%) [0.43-1.32] was assayed by Laurell rocket electrophoresis after removal of bound protein S by precipitation with 2.5% polyethylene glycol followed by centrifugation. The dilute Russell viper venom time was performed by using reagents from American Diagnostica, Greenwich, CT, according to the manufacturer’s instructions. Resistance to activated protein C was determined using reagents from Chromogenix according to the manufacturer’s instructions. The plasma homocysteine level (reference range, 4-15 µmol/L) was measured by high-performance liquid chromatography with fluorescent detection following reduction and fluorescent labeling. A diagnosis of a protein deficiency was made on any value, after excluding the influence of anticoagulants, © American Society of Clinical Pathologists

demonstrated twice to be outside the assay’s 95% confidence interval. In the present study, values described as possible protein defects are values that were demonstrated to be outside the 95% confidence interval only once. Statistical analysis was performed using SAS software (SAS Institute, Cary, NC). The independence of pairs of categorical variables was tested using the Fisher exact test. The value of patient sex and age as a predictor of the presence or absence of prothrombin G20210A and factor V Leiden mutations was evaluated by logistic regression.

Results The mean age of the entire patient population included in this analysis was 40.4 years (range, 7 days-81 years). There were 152 females and 112 males (F/M ratio, 1.4). Information about race was available for 224 of 264 patients: 203 (76.9%) were white; 19 (7.2%) were African Americans; 2 (0.8%) were Asian, and none were Hispanic. Retrospective chart review provided a specific medical diagnosis for 214 patients ❚Table 1❚. Of these, 110 patients, the largest single group, had a diagnosis of deep venous thrombosis and/or pulmonary embolism. The results of factor V Leiden and prothrombin G20210A studies in this population are summarized in ❚Table 2❚. The factor V Leiden mutation was found in 44 (16.7%) of the 264 patients. Among the 44 patients with the factor V Leiden mutation, 8 (18% of the patients with the factor V Leiden mutation, 3.0% of the total group) also had the prothrombin ❚Table 1❚ Clinical Diagnoses Diagnosis

No. of Patients

Deep venous thrombosis and/or pulmonary embolism Cerebrovascular accident Family history of thrombosis Noncerebral arterial thrombosis Thrombosis of mesenteric or portal vasculature Transient ischemic attack Dural sinus thrombosis Retinal thrombosis Superficial venous thrombosis or thrombophlebitis Budd-Chiari syndrome Migraine Angina pectoris Early myocardial infarction Family history of early myocardial infarction Recurrent miscarriage Right atrial thrombus Clots in extracorporeal membrane oxygenation circuit Failed renal transplant Idiopathic pulmonary hypertension Thrombotic renal disease Superior vena cava syndrome Thrombosis of vascular graft Not available Total

110 20 20 10 9 8 6 6 4 3 3 2 2 2 2 1 1 1 1 1 1 1 50 264

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❚Table 2❚ Results for Factor V Leiden and Prothrombin G20210A* Prothrombin G20210A Mutation

Factor V Leiden mutation Positive Negative Total

Positive

Negative

8 (3.0) 8 (3.0) 16 (6.1)

36 (13.6) 212 (80.3) 248 (93.9)

Total

44 (16.7) 220 (83.3) 264 (100.0)

* Data are given as number (percentage).

G20210A mutation and 36 (82% of the patients with the factor V Leiden mutation, 13.6% of the total group) did not. Of the 36 patients with only the factor V Leiden mutation, 16 were female and 20 male (F/M ratio, 0.8). The mean age of this patient subset was 39.2 years (range, 3-75 years). Twenty-five patients were white and 2 African American; race was not available for 9 patients. Fourteen patients had a diagnosis of deep venous thrombosis and/or pulmonary embolism, 6 a family history of thrombosis, 2 cerebrovascular accidents, 2 arterial thrombosis, and 1 Budd-Chiari syndrome. No diagnosis was available for the remaining 11 patients. Two patients with the factor V Leiden mutation also had additional defects that could contribute to thrombosis: 1 had antiphospholipid antibody syndrome and another had a dysfibrinogenemia. The prothrombin G20210A mutation was found in 16 patients (6.1%). Among the 16 patients with the prothrombin G20210A mutation, 8 (50% of prothrombin G20210A group, 3.0% of the entire population) had the factor V Leiden mutation. Of the 8 patients having only the prothrombin G20210A mutation, 5 were female and 3 male (F/M ratio, 1.67). The mean age of the prothrombin G20210A group alone was 47.1 years (range, 10-77 years). Six were white, and none were African American or Asian; race was unavailable for 2. Five of these patients had a deep venous thrombosis or pulmonary embolism, and 1 had a mesenteric artery thrombosis. No diagnosis was available for the remaining 2 patients. No patient had another reason for thrombosis. Eight patients had both factor V Leiden and prothrombin G20210A mutations. There was a significant interaction between factor V Leiden and prothrombin G20210A mutations; subjects with 1 mutation were more likely to have the other (P < .01). Of these 8 patients, 7 were female and 1 male (F/M ratio, 7). A disproportionate number of subjects with both factor V Leiden and prothrombin G20210A mutations were female. Of 112 males, the expected number of men with both mutations, given the marginal distributions of each, was 0.75; 1 male had both mutations. The expected number of females expressing both mutations, from a total of 152, was 1.8; 7 females had both mutations, significantly more than expected (P < .01). The mean age of these patients was 28.5 274

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years (range, 19-52 years). Although the subjects with both mutations averaged 10 years younger than subjects with 0 or 1 mutation, age at diagnosis was not a significant predictor of patients with both mutations. The lack of statistical significance may have been a function of the relatively small number of patients (8) with both mutations. Five of the patients with 2 mutations were white, and none were African American or Asian; race was unavailable for the other 3. Three patients had a history of deep venous thrombosis and/or pulmonary embolism, 1 a history of migraine, and 1 a family history of thrombosis. Historic data were unavailable for the other 3 patients. A retrospective search of the University of Michigan coagulation laboratory database revealed additional coagulation test results for 116 patients (75 females and 41 males; F/M ratio, 1.83). The mean age of these patients was 37.3 years (range, 7 days-78 years). Of the 116 patients with additional laboratory results, 15 had abnormalities that were well documented, and 6 had abnormalities without repeated studies to confirm the findings. Of the 15 patients (5.7% of the total patient population) with well-documented abnormalities, 7 had protein S deficiency, 5 had lupus-like anticoagulants, 2 had elevated serum homocysteine levels, and 1 had a dysfibrinogenemia. The 6 unconfirmed diagnoses (2.3% of the total population) included 4 protein S deficiencies and 2 dysfibrinogenemias. No patient in this entire group had more than 1 abnormality other than the 2 aforementioned patients who also had the factor V Leiden mutation.

Discussion Of the 264 patients tested, 16 (6.1%) had the prothrombin G20210A mutation, a prevalence figure similar to that found by Poort et al1 in consecutive unselected patients with a first episode of deep venous thrombosis (6.3%). Of the 264 patients, 44 (16.7%) were heterozygous for factor V Leiden; this prevalence was similar to that reported by Bertina,15 who found factor V Leiden in 19% of consecutive patients with deep venous thrombosis.15 Thus, in our midwestern population, the prevalence of prothrombin G20210A and factor V Leiden mutations was similar to that found in other populations. Half (8/16) of the patients with the prothrombin G20210A mutation also harbored the factor V Leiden mutation. This association was statistically significant (P < .01). Interestingly, a significantly larger number of females (7) expressed both mutations than the number expected (P < .01). This finding was not seen in the study by De Stefano.17 Whether this observation reflected referral patterns by patients and physicians is not known. Although patients with combined factor V Leiden and prothrombin G20210A mutations had a mean age of onset (28.5 years) that was lower than © American Society of Clinical Pathologists

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the entire group (40.4 years) or any of the other subgroups with thrombophilia (37.3-47.1 years), this difference did not achieve statistical significance. Also, there were no statistically significant differences in age or sex in the patients with single mutations for factor V Leiden or prothrombin G20210A. The association of factor V Leiden and prothrombin G20210A mutations in this population is unlike that seen with any other coagulation protein defect. In the subset of patients with test results for additional causes of thrombosis, only 2 (1.7%) of 116 had more than 1 abnormality compared with the patients with prothrombin G20210A mutation, 50% of whom had defects associated with factor V Leiden. However in both cases, the second abnormality associated with the 116 patients was factor V Leiden. The most common documented or suspected diagnosis for thrombophilia in the 116 patients was protein S deficiency in 11 patients (9.5%). These data suggest that finding more than 1 biochemical or molecular risk factor for thrombosis in general is not a frequent occurrence. The clinical significance of having multiple coagulation defects is not completely established. In a study by Lindmarker et al,16 the risk for recurrent venous thrombosis in heterozygous carriers for factor V Leiden or prothrombin G20210A was not greater than for noncarriers who have had venous thrombosis. However, 2 studies indicated that the risk for spontaneous and recurrent venous thromboembolism in persons carrying both factor V Leiden and prothrombin G20210A mutations is significantly higher (34.6%) compared with persons without any gene or protein defect (14.5%), factor V Leiden alone (16.2%), or prothrombin G20210A alone (20%).17,18 These data indicate that the combined mutation defect may be more serious and require a different approach to the duration of anticoagulation therapy. Furthermore, these data suggest that the combined defect may be as serious a thrombosis problem as deficiencies of certain natural anticoagulation proteins such as antithrombin or protein C or antiphospholipid antibodies. 18 Whether patients with combined mutations need to receive anticoagulation longer than other groups is a subject for future study. From the Departments of 1Pathology and 2Internal Medicine and the 3Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor. Address reprint requests to Dr Schmaier: University of Michigan, 5301 MSRB III, 1150 W Medical Center Dr, Ann Arbor, MI 48109-0640.

References 1. Poort SR, Rosendaal FR, Reitsma PH, et al. 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. 1996;88:3698-3703. © American Society of Clinical Pathologists

2. Margaglione M, Brancaccio V, Giuliani N, et al. Increased risk for venous thrombosis in carriers of the prothrombin G → A sup 20210 gene variant. Ann Intern Med. 1998; 129:89-93. 3. Martinelli I, Sacchi E, Landi G, et al. High risk of cerebral vein thrombosis in carriers of a prothrombin gene mutation and in users of oral contraceptives. N Engl J Med. 1998;338:1793-1797. 4. Brown K, Luddington R, Williamson D, et al. Risk of venous thromboembolism associated with a G to A transition at position 20210 in the 3′-untranslated region of the prothrombin gene. Br J Haematol. 1997;98:907-909. 5. Ferraresi P, Marchetti G, Legnani C, et al. The heterozygous 20210 G/A prothrombin genotype is associated with early venous thrombosis in inherited thrombophilias and is not increased in frequency in artery disease. Arterioscler Thromb Vasc Biol. 1997;17:2418-2422. 6. Rosendaal FR, Doggen CJM, Zivelin A, et al. Geographic distribution of the 20210 G to A prothrombin variant. Thromb Haemost. 1998;79:706-708. 7. Seligsohn U, Zivelin A. Thrombophilia as a multigenic disorder. Thromb Haemost. 1997;78:297-301. 8. Howard TE, Marusa M, Boisza J, et al. The prothrombin gene 3′-untranslated region mutation is frequently associated with factor V Leiden in thrombophilic patients and shows ethnic-specific variation in allele frequency [letter]. Blood. 1998;91:1092. 9. Ehrenforth S, Ludwig G, Klinke S, et al. The prothrombin 20210 A allele is frequently coinherited in young carriers of the factor V arg 506 to gln mutation with venous thrombophilia [letter]. Blood. 1998;91:2209-2210. 10. Zöller B, Svensson PJ, Dahlbäck B, et al. The A20210 allele of the prothrombin gene is frequently associated with the factor V arg 506 to gln mutation but not with protein S deficiency in thrombophilic families [letter]. Blood. 1998;91:2210-2211. 11. Alhene-Gelas M, Le Cam-Duchez V, Emmerich J, et al. The A20210 allele of the prothrombin gene is not frequently associated with the factor V arg 506 to gln mutation in thrombophilic families [letter]. Blood. 1997;90:1711. 12. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989. 13. Bertina R, Koeleman B, Koster T, et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature. 1994;369:64-67. 14. Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol. 1957;17: 237-246. 15. Bertina R. Factor V Leiden and other coagulation factor mutations affecting thrombotic risk. Clin Chem. 1997;43:1678-1683. 16. Lindmarker P, Schulman S, Sten-Linder M, et al. The risk of recurrent venous thromboembolism in carriers and noncarriers of the G1691A allele in the coagulation factor V gene and the G20210A allele in the prothrombin gene. Thromb Haemost. 1999;81:684-693. 17. De Stefano V, Martinelli I, Mannucci PM, et al. The risk of recurrent deep venous thrombosis among heterozygous carriers of both factor V Leiden and the G20210A prothrombin mutation. N Engl J Med. 1999;341:801-806. 18. Margaglione M, D’Andrea G, Colaizzo D, et al. Coexistence of factor V Leiden and factor II A20210 mutations and recurrent venous thromboembolism. Thromb Haemost. 1999;82:1583-1587. Am J Clin Pathol 2000;114:272-275

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