Combined Factor V Leiden and Prothrombin Genotyping in Patients Presenting With Thromboembolic Episodes

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1 Combined Factor V Leiden and Prothrombin Genotyping in Patients Presenting With Thromboembolic Episodes John A. Friedline, MD; Ejaz Ahmad, MD; Diana Garcia, MD; Deborah Blue, MD; Noel Ceniza, MD; Joan C. Mattson, MD; Domnita Crisan, MD Background. Several genetic defects are associated with increased risk of venous thrombosis. The factor V Leiden (FVL) and prothrombin GA mutations are the most frequent causes of inherited thrombophilia. Objectives. To evaluate combined genotyping for these mutations in patients presenting with thromboembolic episodes and to correlate genotypic findings with clinical characteristics. Results. Blood specimens were collected from 4 patients presenting with thromboembolic disease between January 998 and September 998, and genotyping for both FVL and prothrombin mutations was performed. Thirty-two patients (8%) were heterozygous for FVL, 4 (%) were homozygous for FVL, and (5%) were heterozygous for the prothrombin mutation. Two cases (.5%) were identified with combined FVL and prothrombin mutations. The most common clinical presentation was lower-extremity deep vein thrombosis with or without pulmonary embolism. Arterial events were rare. The thromboembolic episodes were often precipitated by additional risk factors. Recurrent disease was found in 73.9% of FVL carriers and 5.9% of prothrombin mutation carriers; 5% of the patients with FVL and 5% of prothrombin mutation carriers had a first thrombotic episode before age 45 years. The cases with combined genetic defects demonstrate amplified thrombotic risk. In the first case this was effected in thrombosis at a young age, and recurrence of thrombotic events even in the absence of precipitating factors. A complex interplay between genetic and additional risk factors was seen in the second case. Conclusions. Identification of both FVL and prothrombin mutations is important in the overall assessment and management of patients with thrombophilia. Detection of these mutations can identify patients at high risk and help evaluate the interaction of genetic and acquired risk factors. (Arch Pathol Lab Med. ;5:5 ) Venous thromboembolism (VTE) is a leading cause of morbidity and mortality, ranking third among the most common cardiovascular diseases in the United States. Pulmonary embolism (PE) affects an estimated 3 or per Americans annually. Of those patients diagnosed, % will die on the first day, and % will have recurrence; 45% of recurrent cases will end in death.,3 In recent years, a growing number of studies have supported the contention that venous thromboembolism is a multifactorial disease, the result of an interplay of both genetic and acquired risk factors. Pregnancy, use of oral contraceptives, antiphospholipid antibody syndrome, lupus anticoagulants, postoperative states, myeloproliferative diseases, previous thrombosis, and prolonged immobilization are some of the conditions that predispose to VTE. Accepted for publication August 4,. From Kennewick, Wash (Dr Friedline); the Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Mich (Drs Ahmad, Blue, Mattson, and Crisan); the Associated Pathologist Laboratories, Las Vegas, Nev (Dr Garcia); and the Northern Michigan Hospital, Petoskey, Mich (Dr Ceniza). Presented at the Ninth Annual William Beaumont Hospital D Technology Symposium, D Technology in the Clinical Laboratory, Royal Oak, Mich, April 3 5,. Reprints: Domnita Crisan, MD, PhD, Department of Clinical Pathology, William Beaumont Hospital, 36 West 3 Mile Rd, Royal Oak, MI ( dcrisan@beaumont.edu). Several genetic defects have been associated with an increased risk of venous thrombosis. These mutations involve genes that control anticoagulant production (ie, antithrombin, protein C, and protein S) and genes responsible for the production of fibrinogen and certain procoagulant factors. All of these defects result in increased thrombin generation. 4 The most common genetic risk factor identified is a point mutation in the factor V gene; this mutation is characterized by a substitution of adenine for guanine at nucleotide 69, which results in substitution of arginine (R) with glutamine (Q) at amino acid 56 (factor V Leiden, FV R 56 Q, FV:Q 56 ). 5 The factor V Leiden (FVL) mutation is most prevalent in Caucasians, particularly in Europeans and in Americans of European descent, occurring in 5% to % of individuals in the general population, in approximately 5% to % of unselected patients with thrombophilia, and in up to 5% of patients with a family history of inherited thrombophilia. It is extremely rare in Asians and Africans. 4,6 The mutation results in the production of factor V protein that is resistant to the action of activated protein C (APC), causing over 9% of APC resistance. Heterozygotes have a fivefold to -fold increased risk of thrombosis, and homozygotes have a 5-fold to -fold increased risk of thrombosis. 7,8 In 996, investigators from Leiden reported that a mutation in the 3 untranslated region of the prothrombin gene, a guanine to adenine substitution at nucleotide, is associated with increased levels of prothrombin Arch Pathol Lab Med Vol 5, January Factor V Leiden and Prothrombin Mutations Friedline et al 5

2 and an increased risk of venous thrombosis. 9 In the Leiden Thrombophilia Study (LETS), the prevalence of this mutation was.3% in healthy controls and 6.% in patients; in selected patients with familial thrombosis, the prevalence is 8%. 9, Thus, it is the second most common genetic defect predisposing to venous thrombosis, and it increases the risk of thrombosis by a factor of.7 to In the past, familial or inherited thrombophilia was thought to be a disease caused by a single genetic defect with a simple Mendelian inheritance. With advances in molecular diagnostic techniques and results of more recent studies, this view has changed. It is now believed that familial thrombophilia is a complex disorder often caused by coseggregation of or more gene defects in a family. 4 Although double homozygosity is rare, 5 FVL mutation has been reported to occur with defects in the antithrombin, protein C, or protein S genes, as well as in the prothrombin gene. Poort et al 9 reported co-inheritance of both FVL and prothrombin gene mutation in 4% of their cases; Ehrenforth et al 6 found co-inheritance of the mutations in.4% of their cases with history of juvenile VTE. Patients who are heterozygous for both FVL and prothrombin gene mutations probably have a greater than 4-fold increased risk of thrombosis. 5 Some studies suggest that carriers of genetic defects often develop thrombosis at an early age, at unusual sites, and often spontaneously in the absence of any acquired or environmental precipitating risk factor.,6 We present our experience on the incidence of the FVL mutation and its association with the prothrombin variant allele in symptomatic patients at William Beaumont Hospital, a large 96-bed tertiary care hospital. MATERIALS AND METHODS Case Identification A total of 4 patients who had either active thrombosis or recent thrombotic episodes were tested for FVL and prothrombin GA mutations between January 998 and September 998. Peripheral blood samples anticoagulated with EDTA were collected and used for genotyping. Patient charts and laboratory records were reviewed. Genotyping Methods Genomic D was extracted from peripheral blood leukocytes by a non-organic method, using the Puregene D isolation system (Gentra Systems, Inc, Research Triangle Park, NC) according to the manufacturer s instructions. Factor V genotyping was performed by polymerase chain reaction (PCR) amplification using the primers originally reported by Bertina et al 5 and reaction conditions modified by Voelkerding et al. 7,8 Subsequent restriction fragment length polymorphism (RFLP) analysis was performed by amplicon digestion with MnlI (New England Biolabs, Beverly, Mass) at 37 C for hours. Restriction fragments were separated by agarose gel electrophoresis in.5% NuSieve 3: agarose (FMC BioProducts, Rockland, Me). The band pattern seen on ethidium bromide stained agarose gels is allele-specific: a 63 base pair (bp) band corresponds to the wild-type allele and a -bp band corresponds to the mutant (FVL) allele, since the mutation destroys an MnlI restriction site. The 67-bp amplicon contains a second MnlI restriction site, generating a 67-bp band in all D samples, which serves as an internal control for D digestion. Other controls were included in every experimental run: uncut D (internal control for PCR amplification), negative (no-d) control, a known-positive heterozygous D control, and a known-negative wild-type D control. Prothrombin genotyping was performed by PCR amplification using the primers and reaction conditions originally reported by Poortetal. 9 A 345-bp amplicon was generated. Subsequent restriction analysis was performed by amplicon digestion with HindIII (New England Biolabs, Beverly, Mass) at 37 C for.5 hours. Restriction fragments were separated by agarose gel electrophoresis as described previously. The band pattern seen on ethidium bromide stained agarose gels is allele-specific. The wild-type allele lacks a HindIII restriction site and therefore generates only a 345-bp band; a new HindIII site is introduced by the GA mutation, generating 3-bp and 3-bp bands. Controls were included in every experimental run: uncut D, negative (no-d) control, a known-positive heterozygous D control, and a known-negative wild-type D control. Guidelines for avoiding PCR contamination were strictly followed, 9 with specimen processing, D extraction, quantitation, and PCR reaction setup performed in a laboratory that was physically separate from the laboratory used for PCR amplification and amplified D manipulation. RESULTS Prevalence of Factor V Leiden and Prothrombin Mutation In the study population of 4 patients genotyped for both factor V mutation and prothrombin mutation between January 998 and September 998, 3 patients (8.%) were heterozygous for the FVL mutation, and 4 patients (.%) were homozygous, representing an overall prevalence rate of 9.%. For the prothrombin mutation, patients (5.%) were heterozygotes and no patients were homozygotes. Two cases (.5%) with combined FVL mutation and prothrombin mutation were identified. Age, Sex, and Race Distribution The overall male-female ratio for individuals with the FVL mutation was :. (heterozygotes : and homozygotes :3). The overall male-female ratio for patients with the prothrombin mutation was :.6. All patients with the FVL mutation and/or prothrombin mutation were Caucasians. The patient demographics for the FVL and prothrombin mutations were analyzed after excluding the patients with combined mutations (Table ). The age at the time of analysis for individuals having only the FVL mutation ranged from 5 to 89 years (mean, 5.; median, 47.5) for heterozygous women and 3 to 8 years (mean, 54.8; median, 57.) for heterozygous men. For patients who were homozygous for the FVL mutation, the ages at the time of analysis were 3 and 67 years (mean, 49.) for the female patients. The single homozygous man was 58 years old at the time of analysis. For patients with only the prothrombin mutation, the age range was 3 to 83 years (mean, 59.8; median, 57.) for heterozygous women and 4 to 7 years (mean, 54.4; median, 5.) for heterozygous men. When the age at the time of the initial thrombotic episode was analyzed, it ranged from 3 to 73 years (mean, 4.; median, 33.) for FVL heterozygous women and 3 to 77 years (mean, 48.3; median, 43.) for heterozygous men. Historical information was only available for of the homozygous women. The age at first thrombotic episode was 6 years for this patient. The single homozygous man developed his first thrombotic event at the age of 36 years. In cases with only prothrombin mutation, the age at first thrombotic episode ranged from to 8 years (mean, 46.6, median, 4.) for heterozygous women and 38 to 7 years (mean, 5.8; median, 49.) for heterozygous men. Two patients had both FVL and prothrombin mutations and they represent 5.9% of all FVL mutations and.% of all prothrombin mutations. The overall prevalence of 6 Arch Pathol Lab Med Vol 5, January Factor V Leiden and Prothrombin Mutations Friedline et al

3 Table. Homozygous for FVL only Women (n ) Men (n ) Heterozygous for FVL only Women (n 6) Men (n 5) Heterozygous for PT only Women (n ) Men (n 7) Age Distribution for Patients With Factor V Leiden and Prothrombin Mutations* Age at Time of Analysis, y Mean Range * Patients with combined mutations are excluded. FVL indicates factor V Leiden; PT, prothrombin. Single patient with history available. Age at First Thrombotic Event, y Mean Range Table. Site of Thrombosis DVT, No. (%) DVT PE, No. (%) PE, No. (%) Other sites TIA CVA, No. (%) Superior mesenteric, No. (%) Arterial, upper extremity, No. (%) MI, No. (%) Unknown, No. (%) Sites of Thrombosis in Patients With Factor V Leiden and Prothrombin Mutations* FVL Homozygotes (n 3) (66.7) (33.3) FVL Heterozygotes (n 3) 3 (4.9) 3 (9.7) (6.4) (3.) (3.) (6.4) 9 (9.) PT Heterozygotes (n 8) 8 (44.4) (.) (5.6) 3 (6.7) (5.6) (5.6) (.) * Patients with combined mutations are excluded. FVL indicates factor V Leiden; PT, prothrombin; CVA, cerebrovascular accident; DVT, deep venous thrombosis; MI, myocardial infarction; PE, pulmonary embolism; and TIA, transient ischemic attack. these combined mutations in our study was.5%. The details of these cases are reported below. Characteristics of First Episode The characteristics of the first thrombotic episode in the 34 subjects with the FVL mutation alone and 8 subjects with the prothrombin mutation alone are summarized in Table. In patients with available clinical histories, the most common clinical presentation was lower-extremity deep vein thrombosis (DVT) with or without pulmonary embolism; this event occurred in /34 cases (58.8%) with the FVL mutation and in /8 cases (6.%) with the prothrombin mutation. Pulmonary embolism without documented DVT was found in patients who were heterozygous for the FVL mutation (6.4%) and in patient who was heterozygous for the prothrombin mutation (5.6%). Arterial events were rare. A single case of documented peripheral arterial thrombosis was found in a 44-year-old woman who was heterozygous for the FVL mutation. The woman developed an upper-arm arterial thrombosis and presented with her first thrombotic episode while taking oral contraceptives. No peripheral arterial event was seen in cases with the prothrombin mutation. In addition, patient who was heterozygous for the FVL mutation and 3 patients who were heterozygous for the prothrombin mutation presented with transient ischemic attacks. Also, patients who were heterozygous for the FVL mutation and patient who was heterozygous for the prothrombin mutation presented with myocardial infarction. Incidence and Characteristics of Recurrent Thrombotic Episodes Both patients with combined FVL and prothrombin mutations had recurrent thrombotic disease. Out of the 3 patients who were homozygous for the FVL mutation but had no prothrombin mutation, history was available on. At the time of analysis, both of these patients had recurrent thrombotic disease. Patients who were heterozygous for either the FVL mutation alone or the prothrombin mutation alone showed a lower incidence of recurrent events. History was available for patients who had the FVL mutation without the prothrombin mutation; 6 (8.6%) of these patients had a single thrombotic episode, and 5 (7.4%) had recurrent thrombosis. The overall frequency of recurrent thrombosis ( or more episodes) for patients (heterozygous and homozygous patients combined) with the FVL mutation alone was 7 out of 3 (73.9%) in cases for which adequate medical records were available for review. It is interesting to note that among these 3 patients, (5%) had their first thrombotic episode before age 45 years (age range, 3 43 years), with a male-female ratio of :. Out of these patients with early initial thrombosis, 8 (66.7%) patients had recurrent disease, and 6 (5%) had additional risk factors. For the 8 patients who were heterozygous for the prothrombin mutation but had no FVL mutation, history was available for 7. At the time of analysis, 7 (4.%) of the 7 heterozygous patients had a single thrombotic episode, 9 (5.9%) had recurrent thrombotic disease, and had no documented thrombosis. Among the 6 patients with a history of documented thrombosis, 8 (5%) had their first thrombotic event before age 45 years (range 43 years). Out of these 8 patients with early initial thrombosis, 7 (87.5%) had recurrent disease, compared with only patients of the 8 (5%) with initial thrombosis after age 45 years. Additional risk factors were present in 6 of 8 patients in both age groups. Arch Pathol Lab Med Vol 5, January Factor V Leiden and Prothrombin Mutations Friedline et al 7

4 Table 3. Predisposing Risk Factors Pregnancy/postpartum, No. OC, No. Surgery, No. Immobilization, No. Malignancy, No. Trauma, No. IV line, No. No risk factor, No. Incomplete history, No. Additional Predisposing Risk Factors in Patients With Factor V Leiden and Prothrombin Mutations* FVL Homozygotes Men (n ) Women (n ) FVL Heterozygotes Men (n 5) Women (n 6) 7 4 PT Heterozygotes Men (n 7) 5 Women (n ) * Patients with combined mutations are excluded. FVL indicates factor V Leiden; PT, prothrombin; OC, oral contraceptives; IV, intravenous; and, not applicable. Some patients had multiple risk factors. 3 3 Incidence of Additional Predisposing and Precipitating Factors Additional precipitating factors at the time of thrombosis were present in of the patients who were homozygous for the FVL mutation and had an available history. For patients who were heterozygous for the FVL mutation, 6 had verifiable information concerning the presence of predisposing or precipitating factors, and 9 (35%) were positive. These factors are tabulated in Table 3 and included oral contraceptive use, pregnancy or the immediate postpartum period, surgery, trauma, malignancy, prolonged immobilization, and an indwelling venous line. In patients with the FVL mutation, the additional risk factors also included protein S deficiency ( cases) and antiphospholipid syndrome ( case). Sixteen of 8 patients who were heterozygous for the prothrombin mutation had adequate information available on the presence of predisposing or precipitating events. Nine of these 6 patients (56%) were positive. The patients with the prothrombin mutation had the additional inherited risk factors of protein S deficiency ( cases) and protein C deficiency ( case). For both patients heterozygous for the FVL mutation and patients heterozygous for the prothrombin mutation, the age at first event was earlier in the patients with additional risk factors. For the patients who were heterozygous for the FVL mutation, the mean age for the first thrombotic event in patients with additional risk factors (n 8) was 43.3 years, compared with 54.5 years for patients with no additional risk factors (n 3). The results were similar for the prothrombin mutation: patients who were heterozygous and had additional risk factors (n ) were generally younger at the time of the first thrombotic event (mean age, 48.8 years) when compared with patients who were heterozygous and had no additional risk factors (n 6) (mean age, 54.8 years). When female patients with early initial thrombotic events (before age 45) were analyzed for additional risk factors, the most common were oral contraceptive use and pregnancy-related events (pregnancy, postpartum state, and cesarean section). Five women were heterozygous for the FVL mutation alone and had a first thrombotic event before age 45 years; of these women were taking oral contraceptives, woman had protein C deficiency, and women had no additional risk factors. Similarly, among women who were heterozygous for the prothrombin mutation alone, 5 had thrombotic events associated with oral Figure. Case. Agarose gel electrophoresis for detection of the factor V Leiden mutation by polymerase chain reaction and MnlI restriction fragment length polymorphism. Lanes: Uncut indicates D amplicon (67 base pair [bp] band); M, molecular size marker; F3, heterozygous control (-bp and 63-bp bands); Cut, digested wild-type control (63-bp band); F948 F96, patients Ds; and dh O, negative (no-d) control. The patient s D in lane F954 shows heterozygosity (-bp and 63-bp bands). 8 Arch Pathol Lab Med Vol 5, January Factor V Leiden and Prothrombin Mutations Friedline et al

5 Figure. Case. Agarose gel electrophoresis for detection of the prothrombin mutation by polymerase chain reaction and HindIII restriction fragment length polymorphism. Lanes: HET/U indicates uncut D amplicon (345 base pair [bp] band); M, molecular size marker; HET/C, heterozygous control cut (345-bp and 3-bp bands); WT, wild-type control (345-bp band); H O, negative (no-d) control; and F947, F95, F957, F958, F959, and F96, patients Ds. The patient s D in lane F947 shows heterozygosity (345-bp and 3-bp bands). contraceptive use or pregnancy related events. These 5 patients had multiple thrombotic events with the following associations: oral contraceptive use (3), uncomplicated pregnancies (), cesarean sections (), and postpartum state (). Cases with Combined FVL and Prothrombin Mutations Case. A 36-year-old Caucasian man presented to the emergency room with right lower-extremity swelling; Doppler confirmed right occlusive popliteal vein DVT and also showed left non-occlusive superficial femoral vein thrombosis with collateral circulation. The patient was admitted and anticoagulant therapy was started. His past medical history was positive for a previous DVT episode at age 3 years, involving his left lower extremity. No additional risk factors for thrombosis were found prior to the thrombotic events. His family history was unknown. A hypercoagulable state workup included genotyping for the FVL and prothrombin mutations; the patient was found to be heterozygous for both mutations (Figures and ). The other tests for hypercoagulability were noncontributory due to active thrombosis and anticoagulation at the time of testing (protein C activity was decreased at 53%, protein S activity was normal at %, and antithrombin III was normal at 87%). This case demonstrates the increased and compounded risk of thrombosis in patients carrying both FVL and prothrombin mutations, with a first thrombotic event at a young age and recurrent thrombosis in the absence of any precipitating events. Case. A 77-year-old Caucasian woman presented with lower-extremity thrombosis, confirmed by venous duplex scan, with bilateral involvement of the common femoral, superficial femoral, and popliteal veins. The patient s past medical history was significant for hypertension, atrial fibrillation, and breast carcinoma. The carcinoma had been treated with modified radical mastectomy and the patient had been on tamoxifen for years. Her history was also positive for recurrent thrombosis, with 7 previous episodes of DVT of the lower extremities, episode of left upper-extremity thrombosis, and episodes of superficial venous thrombosis of the left lower extremity. Therapy with heparin and coumadin was started, and her condition stabilized. Prothrombin genotyping was performed and showed heterozygosity of the GA mutation, and factor V genotyping demonstrated homozygosity for the FVL mutation (Figures 3 and 4). An anticardiolipin antibody test was negative. This case exemplifies the amplified risk of thrombosis in patients having both mutations and the interaction with additional risk factors. COMMENT The frequency of the FVL mutation in our study (9%) is lower than previously published frequencies in unselected patient populations (4 to 9%), and significantly lower than reported values (up to 5%) from studies performed on patients with inherited thrombophilia. 4,6 It is also lower than the frequency we obtained from a previous, larger patient group study (3.4%). This may reflect the smaller size of the current study population. The overall male-female ratio (:.) is in keeping with anticipated results for genetic defects that have an autosomal dominant transmission. The data presented here further lend credence to the growing belief that thrombosis in patients with the FVL mutation, as with other causes of inherited thrombophilia, is often precipitated by additional risk factors, both genetic and acquired, and that under these circumstances, thrombosis may occur at an earlier age. 7,, 3 Recurrent thrombosis in individuals diagnosed with the FVL mutation is an issue that has brought disagreement between investigators. Some studies report relative risks of recurrence ranging from.4 to 4. in patients who have the mutation compared with patients who do not have the mutation,,4,5 whereas others have failed to find any association. 6 8 In our study, patients heterozygous for the FVL mutation alone and the prothrombin mutation alone often had histories of recurrent thrombotic events (73.9% and 5.9%, respectively). We also found a slight increase in recurrent disease associated with the presence of additional risk factors in patients with the prothrombin mutation, but not in patients with the FVL mutation. In a retrospective study, De Stefano et al did not find a sig- Arch Pathol Lab Med Vol 5, January Factor V Leiden and Prothrombin Mutations Friedline et al 9

6 Figure 3. Case. Agarose gel electrophoresis for detection of the factor V Leiden by polymerase chain reaction and MnlI restriction fragment length polymorphism Lanes: Uncut 373 indicates uncut D amplicon (67 base pair [bp] band); X74, molecular size marker; 373, wild-type control (63-bp band); F3, heterozygous control (- bp and 63-bp bands); F67 F638, patients Ds; and H O, negative (no-d) control. The patient s D in lane F638 shows homozygosity (-bp band). Figure 4. Case. Agarose gel electrophoresis for detection of the prothrombin mutation by polymerase chain reaction and HindIII restriction fragment length polymorphism. Lanes: HET/U indicates uncut D amplicon (345 base pair [bp] band); M, molecular size marker; HET/ C, heterozygous control cut (345-bp and 3-bp bands); WT, wild-type control (345-bp band); H O, negative (no-d) control; and F638 F643, patients Ds. The patient s D in lane F638 shows heterozygosity (345-bp and 3-bp bands). nificant increase in the risk for recurrent DVT among patients with only the FVL mutation (carriers with no other genetic or acquired thrombotic risk factors) compared with patients without the genetic defect. However, they did find an increased risk of recurrent DVT in carriers of both the FVL and prothrombin GA mutations. In the LETS study, the prothrombin gene mutation was demonstrated in 6.% of symptomatic patients. We found a slightly lower frequency of 4.5% in our study group and a slight female preponderance (male-female ratio of :.6). All 8 of our cases were heterozygous for the mutation, a finding highly likely in a genetic defect with a relatively low overall prevalence. On the basis of a prevalence of %, Wulf et al 5 calculated the expected prevalence of homozygosity for the prothrombin mutation at in. Of our 7 patients with available histories, 9 (5.9%) had recurrent disease, and 8 of 6 (5%) with documented thrombosis had their first thrombotic episode before age 45 years (range, 43 years). Seven of the 8 early-onset cases (87.5%) had recurrent disease and 6 of the 8 (75%) had additional risk factors. These results are comparable to those we have obtained from the patients with FVL mutations. While identifying cases of compound genetic defects involving the FVL and prothrombin GA mutations does not allow us to make any statistical comparison with patients with only a single genetic defect, the cases themselves seem to support the hypothesis of amplified risk. Ehrenforth et al 6 and Zoller et al 7 found a threefold increase in the thrombotic risk in patients with combined heterozygosity in their studies. Increased risk, as estimated by statistical models, was also suggested by Alhenc-Gelas et al 9 and Salomon et al. 3 Although there have been no reported values for thrombotic risks in combined homozygous cases, it is conceivable that this risk would be much greater than the risk associated with homozygosity for a single type of mutation. It would then appear that of our patients who is homozygous for the FVL mutation and heterozygous for the prothrombin GA mutation has a substantially greater risk compared with a doubly heterozygous patient, possibly explaining in part the fact that this patient had episodes of thrombosis by the time of analysis. The other compound case, a patient who was doubly heterozygous, had a first thrombotic episode at age 3 years and had already had episodes of thrombosis at age 36 years. While De Stefano et al found increased recurrence rates only in patients with both genetic defects, Margaglione et al 3 demonstrated increased recurrence rates in FVL mutation carriers (6.%) and in prothrombin mutation carriers (.%). In addition, they found significantly higher rates of recurrence in subjects carrying both FVL and prothrombin gene mutations in their study, reporting a recurrence rate of 36.4%. Pregnancy and the use of oral contraceptives pose added thrombotic risks to women of childbearing age. Thromboembolic complications are major causes of morbidity and mortality during pregnancy and the puerperium. We found that oral contraceptive use and pregnancy-related events (ie, pregnancy, cesarean section, and the postpartum state) were common precipitating or additional risk factors for our female patients under 45 years of age. The reported risk for developing VTE among pregnant women is 5 times higher than the risk in nonpregnant women. 3 In a recently published work, Gerhardt et al 3 studied 9 women with history of VTE during pregnancy and the puerperium and found a prevalence of 43.7% for the FVL mutation, 6.9% for the prothrombin GA mutation, and 9.3% for combined FVL and prothrombin mutations. Among carriers of both defects, the calculated risk of thrombosis was 4.6%, which is disproportionately higher than that among women with only mutation (FVL mutation,.% prothrombin mutation,.5%). In a study of Arch Pathol Lab Med Vol 5, January Factor V Leiden and Prothrombin Mutations Friedline et al

7 FVL mutation carriers designed to investigate the multigenic nature of VTE, Tosetto et al 3 observed a particularly high risk of developing VTE in a fraction of their subjects; this fraction consisted of women carrying both FVL and prothrombin gene mutations and using oral contraceptives. We have presented data and observations that further support the contention that VTE is a multifactorial disease, the result of a complex interplay of or more risk factors that may be inherited or acquired. Although statistically limited due to a small number of cases, this study revealed cases of combined FVL and prothrombin gene mutations that exemplify the nature of VTE. The co-inheritance of these genetic defects appears to be associated with a risk of thrombosis and recurrence that is higher than the risk associated with single mutations. Whereas individuals with single genetic mutations may be largely asymptomatic (and thus remain undiagnosed), the presence of acquired or transient risk factors may precipitate a thrombotic episode. As most of these thrombotic episodes may be preventable, it is important to consider possible screening procedures to identify those at high risk. Individuals with family histories of thrombosis are particularly at high risk, not only for early occurrence of thrombosis, but also possibly for the presence of a second genetic defect, as some reports have shown. Identifying high-risk patients is also important in therapeutic decision making. We have come a long way in our efforts to understand thromboembolic disease and a growing number of studies are being undertaken. However, due to the small number of cases in most of these studies, larger studies are needed to address the issue of precise assessment of risks in combined genetic defects. 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