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Keywords:

  • venous thromboembolism;
  • factor V Leiden;
  • hypercoagulability;
  • thrombophilia;
  • activated protein C resistance

Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Factor V Leiden (FVL) leads to a sevenfold increased risk of venous thrombosis and is present in 50% of individuals from families referred because of unexplained familial thrombophilia. We assessed the association of FVL with venous thromboembolism (VTE) in 12 thrombophilic families of symptomatic probands with FVL in a retrospective follow-up study. We screened 182 first- and second-degree relatives of the 12 unrelated propositi for the FVL mutation and the occurrence of VTE. The incidence rate of VTE in carriers of FVL (0·56%/year) was about six times the incidence for the Dutch population (0·1%/year). The incidence rate in non-carriers also appeared to be higher (0·15% per year). At the age of 50 years, the probability of not being affected by VTE was reduced to 75% for carriers and to 93% for non-carriers (P = 0·009). Identification of carriers of FV Leiden may be worthwhile in young symptomatic individuals and their relatives with a strong positive family history of venous thromboembolism or a history of recurrent venous thrombosis who may be at risk (e.g. pregnancy, use of oral contraceptives). After adjustment for prothrombin G20210A (present in two families), even higher thrombotic incidence rates were found in carriers and non-carriers of FVL. This makes the presence of other unknown prothrombotic risk factors more probable in these families.

Venous thromboembolism is a major cause of morbidity with an incidence of about one per 1000 per year ( Nordström et al, 1992 ; National Medical Registration of the Foundation Information Centre for Health, 1996). Predisposing factors can be either genetic or environmental. Environmental risk factors include recent surgery, immobilization, oral contraceptives, pregnancy and puerperium ( Nachman & Silverstein, 1993). Until 1993, a specific genetic defect was identified in a maximum of 10–15% of affected subjects ( Allaart & Briët, 1994). These defects included deficiencies of the main inhibitors of the clotting system: protein C, protein S and anti-thrombin ( Hirsh et al, 1986 ). Familial resistance to activated protein C (APC-resistance), first described in 1990 ( Dahlbäck et al, 1990 ), is the most frequent genetic risk factor for thrombosis ( Griffin et al, 1993 ; Koster et al, 1993 ; Svensson & Dahlbäck, 1994 ; Dahlbäck, 1995; Lane et al, 1996 ). In the large majority of cases, APC resistance is associated with a single point mutation (1691 G to A substitution) in the factor V gene that predicts the synthesis of a factor V molecule (Factor V Leiden) that is not properly inactivated by activated protein C (APC) ( Bertina et al, 1994 ; Greengard et al, 1994 ; Voorberg et al, 1994 ). In Caucasians, this mutation is present in about 5% of healthy individuals ( Rees et al, 1995 ; Zivelin et al, 1997 ). Factor V Leiden leads to a sevenfold increased risk of venous thrombosis ( Rosendaal et al, 1995 ). It is present in 20% of unselected, consecutive patients with deep vein thrombosis and in 50% of individuals from families referred because of unexplained familial thrombophilia ( Bertina et al, 1994 ; Zöller et al, 1994 ; Lane et al, 1996 ).

We demonstrated an earlier age of onset in a series of selected patients from thrombophilic families with factor V Leiden than in a panel of unselected patients with a first venous thrombosis who turned out to be carriers of the factor V Leiden mutation ( Lensen et al, 1996 ). This suggests a higher thrombotic tendency in members from selected families than in consecutively diagnosed patients, even if both carry the same or a similar molecular defect. Important to clinicians is the question of what prophylactic measures are advisable for patients and their relatives in these selected thrombophilic families with factor V Leiden. Before this question can be answered, the risk of thrombosis in these individuals needs to be assessed. Therefore, we studied 12 thrombophilic families with the factor V Leiden mutation in which, next to the proband, at least two persons had experienced thrombosis.

PATIENTS and METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients and families The 12 probands originate from a larger panel of 28 patients who were referred to our centre for diagnostic work-up for venous thrombophilia, i.e. patients with a positive family history of venous thrombosis (at least two symptomatic relatives in addition to the proband) and who did not have deficiencies of protein C, protein S or anti-thrombin. These 28 patients were screened for the presence of the factor V Leiden mutation which was detected in 12 patients. We invited the siblings, parents and children of these 12 probands, as well as uncles and aunts of the affected parental side, and, if these were carriers, their children (first cousins of the proband) to participate. Family members under 15 years of age were excluded for practical purposes. Of the 12 probands, 182 first- and second-degree family members (93%) participated in the study and 12 did not, three members because they lived abroad and nine for reasons unknown. In all cases of couples with factor V Leiden-positive children, we verified from which parent the mutant allele was transmitted by testing the other parent. In 30 of these cases, none of the other parents who had married into these pedigrees, carried the factor V Leiden allele.

The 12 probands and their 182 family members were seen by the same physician for venepuncture and interview; risk factor status was assessed by the interviewer before carrier status was determined (except for the proband). A standardized history was taken of the occurrence of deep venous thrombosis (excluding superficial thrombophlebitis) and pulmonary embolism, including the age at each episode, the method of diagnosis, treatment and predisposing circumstances. The history included occurrence of risk factors in the past: surgery, hospital admission, immobilization (period exceeding two weeks), plaster casts, malignancies, pregnancies, postpartum periods and use of oral contraceptives, hormonal replacement therapy or oral anticoagulants. When a patient reported to have experienced a thrombotic event, clinical information was gathered for confirmation from the physician responsible for the treatment of the patient. We counted as venous thrombotic events only those that had been diagnosed by physicians (80% of deep venous thrombotic events were objectively diagnosed, i.e. by ultrasound, venography, pulmonary angiography or lung ventilation perfusion scan; 20% were not, mainly because of events occurring before objective diagnostic techniques were available).

Laboratory methods Blood samples were collected from the antecubital vein into 0·106 mmol/l trisodium citrate. Plasma was prepared by centrifugation for 10 min at 2000 g at room temperature and stored at −70°C in 1·5 ml volumes. The mutation of the factor V gene was detected with the use of amplification and restriction-enzyme digestion. Normalized APC-sensitivity ratios were measured as described previously ( de Ronde & Bertina, 1994). APC resistance was defined as normalized APC-sensitivity ratio of < 0·84; carriers of factor V Leiden usually have a normalized APC-sensitivity ratio of < 0·71 ( Bertina et al, 1994 ). We measured normalized APC-sensitivity ratios in all relatives who were not treated with oral anticoagulants (n = 166) and found a clear association of factor V Leiden and a normalized APC-sensitivity ratio of < 0·71. We screened all individuals for deficiencies of protein C, protein S, anti-thrombin and the 20210 G to A prothrombin variant. These laboratory measurements were carried out by one laboratory technician who had no knowledge of the medical history.

Statistics We analysed the lifetime risk of thrombosis by standard life-table techniques (Kaplan–Meier method). To compare the two curves we used the logrank test, resulting in a chi-square distribution with one degree of freedom. This analysis of lifetime risk of thrombosis based on retrospective data is valid under the assumption that there is no excess mortality in these families. Absence of excess mortality for factor V Leiden has been reported by Mari et al (1996) and Hille et al (1997) , and is analogous to other reports on the absence of excess mortality in thrombophilic families with deficiencies of anti-thrombin or protein C ( Rosendaal et al, 1991 ; van Boven et al, 1994 ; Allaart et al, 1995 ). We have assessed occurrences of superficial thrombophlebitis (diagnosed by a physician), but we performed further analyses without this type of venous thrombosis (because of difficulties in the subjective diagnosis and for reasons of comparability with other published studies). When necessary, we also excluded second-degree relatives for comparability with other studies.

We compared the incidence rates of first venous thrombotic events in relatives with factor V Leiden (carriers) and without factor V Leiden (non-carriers) of the 12 probands. We calculated these incidence rates by counting patient-years of observation (follow-up time) and dividing the number of events in each group by the total number of patient-years of all the individuals in the group. Follow-up for symptomatic individuals started at birth and ended at the date of the first venous thrombosis. Follow-up for asymptomatic individuals started at birth and ended at the date of the interview. For all participants, follow-up was complete. Incidence rates of recurrent venous thrombotic events were computed as the number of recurrences divided by the total of follow-up time in symptomatic relatives between the first event and the date of the recurrence or the date of the interview, whichever came first.

We used Poisson regression analysis to study the role of the putative risk factors: age (seven 10-year age groups ranging from age 10–19 years to 70–79 years), sex, heterozygosity for factor V Leiden, surgery (exposition window: 1 year per operation), immobilization longer than 2 weeks (including hospital admissions and plaster casts; exposition window: 1 year), obesity (body mass index > 25 kg/m2), smoking, pregnancy/post partum period (exposition window set at 1 year) and use of oral contraceptives (only for time periods lasting at least 1 month). As the prothrombin G20210A variant was present in two families, we adjusted for this coagulation defect in order to assess more precisely the thrombogenicity of factor V Leiden.

Results

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Clinical data

We studied 182 relatives of 12 probands with factor V Leiden. Of these 182 family members, 91 were heterozygous for the factor V Leiden mutation (44 men and 47 women; median age 39 years). One relative was homozygous (62-year-old man) and 90 relatives were non-carriers (46 men and 44 women, median age 36 years).

Twenty-five (14%) of the 182 relatives were symptomatic. Of the 92 factor V Leiden carriers (probands excluded), 20 (22%) had experienced one or more venous thrombotic events in their lifetime (first event was a pulmonary embolism in four carriers and a venous thrombosis in a lower extremity in 16 carriers). Five of the 90 non-carriers (6%) experienced a venous thrombotic event (first event was a pulmonary embolism in three non-carriers and a deep venous thrombosis in a lower extremity in two non-carriers). Among carriers, the incidence of venous thrombosis was 0·56%/year (20 events on 3585 person-years), which was 3·7-fold (95% CI 1·4–10·0) higher than the incidence of 0·15%/year among non-carriers (five events on 3354 person-years). First event was a superficial thrombophlebitis in nine carriers and in one non-carrier.

We calculated the thrombosis-free survival by Kaplan–Meier analysis ( Fig 1). At the age of 50 years, the probability of being free of deep venous thromboembolism was reduced to 75% for heterozygotes; for non-carriers, 93% were still free of thrombosis at this age (P = 0·009). These significant differences could not be explained by different frequencies of other risk factors in the two groups. Overall relative risk of carriers vs. non-carriers, as calculated by the Cox proportional hazards model, was 3·4 (95% CI 1·3–9·2). Interestingly, the symptomatic fraction of first-degree family members with factor V Leiden was significantly higher than the symptomatic fraction of second-degree family members with factor V Leiden. Among 36 first-degree relatives carrying factor V Leiden, 17 (47%) had experienced venous thrombosis, as compared with three (5%) among 56 second-degree relatives with factor V Leiden. This is visualized in Fig 2, which shows the thrombosis-free curves for the first-degree relatives only.

image

Figure 1. Venous thrombosis-free survival curves in 92 carriers (lower line, probands excluded) and 90 non-carriers (upper line). The difference in curves was significant (logrank test: P = 0·009).

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image

Figure 2. Venous thrombosis-free survival curves in 36 carriers (lower line, probands excluded) and 31 non-carriers (upper line) after exclusion of second-degree relatives. The difference in curves was significant (logrank test: P = 0·002).

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The incidence of first venous thrombosis in the 92 carriers of factor V Leiden (probands excluded) was 0·2%/year up to age 24 years (vs. 0·05%/year in non-carriers). Between 25 years and 45 years of age the incidence was 1·1%/year (vs. 0·3%/year in non-carriers) and over 45 years of age the incidence was 1·2%/year (vs. 0·3%/year in non-carriers) ( Table I).

Table I.  Incidence rates of deep venous thromboembolism per year in carriers and non-carriers per age category in thrombophilic families with factor V Leiden.
AgeFactor V Leiden +Factor V Leiden –
Person-years (n) Events (n) Incidence (95% CI) *Person-years (n) Events (n) Incidence (95% CI)
  • * 

    Incidence rates are in percentage per years.

< 24230550·2 (0–0·4)215910·05 (0–0·1)
24–45959111·1 (0·5–1·8)87430·3 (0–0·7)
> 4532141·2 (0–2·5)32110·3 (0–0·9)
Total3585200·56 (0·3–0·8)335450·15 (0–0·3)

There had been 18 recurrent events in ten of the 20 symptomatic carriers; none of them was taking oral anticoagulants at the time of the second event. One symptomatic non-carrier experienced four recurrent events despite treatment with oral anticoagulants. The incidence of first recurrence was 3·7%/year in carriers, which was 3·7 times higher (95% CI: 0·5–29·9) than the incidence of recurrence in non-carriers (1·0%/year).

The median age at first thrombosis for the 20 symptomatic relatives with the factor V Leiden mutation was 30 years (range, 16–75 years). This median age of onset was equal in both sexes. The median age of onset for the five non-carriers was 28 years (range, 23–54 years).

Effect of other risk factors

In none of the families was deficiency of protein C, protein S, or anti-thrombin found. However, two of the 12 propositi (16·7%) were heterozygous carriers of the recently described 20210 G to A prothrombin variant, i.e. they had a second defect in combination with factor V Leiden ( Poort et al, 1996 ). After exclusion of these two families with the prothrombin 20210A variant in addition to factor V Leiden, the thrombotic incidence rate in factor V Leiden carriers of the remaining ten families was 0·58%/year and was 0·17%/year in non-carriers (relative risk 3·4, 95% CI 1·2–10·1). Cox regression analysis revealed for the 12 families to have a risk ratio of 3·5 (95% CI 1·3–9·3) for factor V Leiden and a risk ratio of 2·2 (95% CI 0·5–9·2) for the prothrombin 20210A variant (multivariate analysis).

The distribution of environmental risk factors in carriers and non-carriers was very similar with the exception of pregnancies: the 47 female carriers experienced 100 pregnancies, while the 44 female non-carriers experienced 50 pregnancies. Precipitating risk factors during or shortly before the first event were present in 15 (75%) of the 20 carriers and in three (60%) of the five non-carriers with thrombosis ( Table II). Four of the 20 carriers with thrombosis experienced their first episode post partum. Other risk factors in carriers were surgery, use of oral contraceptives, pregnancy and immobilization; in only five carriers (25%) was the first episode apparently spontaneous. Risk factors in the five non-carriers with thrombosis were surgery and pregnancy; two non-carriers experienced a spontaneous thrombotic event. All carriers experienced, in total, 244 episodes of surgery (n = 84), immobilization (n = 60) or pregnancy (including puerperium; n = 100) and 12 of these episodes were complicated by a venous thrombotic event (4·9%), which was the case in 1·6% of the non-carriers (185 episodes were complicated by three thrombotic events). Eleven of the 18 recurrent events (61%) in carriers were apparently spontaneous.

Table II.  Potential risk factors associated with the first venous thrombosis.
Potential risk factorSymptomatic carriers (n = 20) Symptomatic non-carriers (n = 5)
  1. Probands excluded. There were five spontaneous venous thrombotic events among carriers and two among non-carriers.

Puerperium41
Pregnancy30
Surgery32
Oral contraceptives30
Immobilization20
Total15 (75%)3 (60%)

In Table III, rate ratios are presented when the main risk factors were entered in a Poisson regression model. In this multivariate-adjusted model, the strongest environmental risk factor was pregnancy and puerperium (rate ratio 37·7), followed by surgery (rate ratio 14·1), immobilization (rate ratio 8·9) and use of oral contraceptives (rate ratio 4·1). We found a highly increased risk (interaction under a multiplicative model) when factor V Leiden was combined with pregnancy and puerperium, and only a moderate increased risk (interaction under an additive model) when factor V Leiden was combined with surgery. As the thrombotic risk of surgery was also high in non-carriers, the joint risk estimates are high for all combinations of risk factors with factor V Leiden, with annual rates of 2–7%.

Table III.  Adjusted rate ratios (95% CI) for the main potential risk factors on venous thrombotic events for the 182 relatives.
Potential risk factorRate ratio (95% CI)
  1. The rate ratios are the risks of thrombosis in the presence of the risk factor as compared to its absence, adjusted for the other factors in the model. All variables are coded as yes/no, age is coded in 10 year intervals.

Factor V Leiden3·0 (1·1–8·2)
Age1·6 (1·3–2·1)
Pregnancy & puerperium37·7 (13·9–101·7)
Surgery14·1 (4·8–41·8)
Contraceptive pill use4·1 (1·1–15·1)
Immobilization8·9 (1·9–41·1)

Discussion

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We performed a study on 182 members of 12 referred families with thrombophilia. At the age of 50 years, 25% of carriers had experienced at least one venous thrombotic event (vs. 7% in non-carriers). The incidence rate of deep vein thrombosis (superficial thrombophlebitis excluded) in carriers is six times higher than the reported incidence rate for the general Dutch population (about 0·11%/year) ( Nordström et al, 1992 ; Briët et al, 1994 ; National Medical Registration of the Foundation Information Centre for Health Care, 1996). This incidence rate of 0·11%/year for the general population is probably an overestimation as recurrences are included. Interestingly, in the families we studied the incidence among non-carriers (0·17%/year, adjusted for the prothrombin G20210A allele) also exceeded this population figure. This increased incidence of venous thrombosis in non-carriers is probably caused by the selection of families that are very prone to thrombosis, in which the presence of more than one defect may be suspected ( Lane et al, 1996 ; Lensen et al, 1996 ; Rosendaal, 1997). We also think that the significant higher thrombotic incidence found in first-degree relatives as compared with second-degree relatives (which is characteristic in polygenic inheritance) gives support to the hypothesized presence of more co-existing genetic defects and justifies further research work into this matter.

Previously, in a population-based case-control study on venous thrombosis (Leiden Thrombophilia Study, LETS; Rosendaal et al, 1995 ) among 471 unselected consecutive patients with a first, objectively confirmed deep vein thrombosis (all younger than 70 years) and 471 unrelated age- and sex-matched controls, we found an odds ratio for heterozygosity of 7·3. The lower risk ratio of 3·7 for heterozygosity we found in thrombophilic families with factor V Leiden may be the result of the high incidence of venous thrombosis in non-carriers and the difference in study design.

Considering Tables I and II, we think that the minimally higher thrombotic risk found in carriers older than 45 years as compared with the 25–45 year group is caused by the occurrence of many thrombotic events during pregnancy and puerperium or the use of oral contraceptives in the 25–45 years group, demonstrating their important role as a thrombogenic risk factors in this age group.

Recently, Middeldorp et al (1998) presented their results of a family study on factor V Leiden. They investigated first-degree family members of consecutive patients with venous thromboembolism, as opposed to referred high-risk families in our study. They and Zöller et al (1994) found that 97% of carriers were still free of thrombosis at age 30 years. We found that 28% of carriers in thrombophilic families had experienced thrombosis at this age ( Fig 2). The incidence rate for first-degree heterozygous family members younger than 61 years of age was 0·9%/year, more than two times the incidence rate found by Middeldorp et al (1998) (0·4%/year; in both calculations, follow-up started at the age of 15 years). These comparisons emphasize that the estimated risk of thrombosis for carriers of factor V Leiden depends on the inclusion criteria and is much higher in selected and referred families than in relatives of consecutive unselected patients. This is also illustrated by comparing our data with data from a study recently performed by Simioni et al (1999) . They included relatives of unselected patients and found an annual thrombotic incidence of 0·28% in carriers and 0·09% in non-carriers (rate ratio 2·8, 95% CI 1·1–8·6) (in first-degree relatives we found 1·7% in carriers and 0·3% in non-carriers, resulting in a doubled rate ratio of 5·7, 95% CI 2·0–26·8). We recently found thrombotic incidences in first-degree relatives of 47 unselected consecutive patients with a first venous thrombosis that were five times lower in carriers and three times lower in non-carriers than the thrombotic incidences we found in the thrombophilic families. Only one of these 47 families (2%) met the inclusion criteria used in the current study (more than one symptomatic relative besides the proband) and 8% of the first-degree relatives had experienced a deep vein thrombosis (as Middeldorp et al, 1998 found in their study population; Lensen et al 2000 ) vs. 47% in the current study. This illustrates the role of a strong family history as a probable risk factor for venous thrombosis.

The inclusion of thrombotic events that were not objectively diagnosed (20%) could have led to an overestimation of the thrombotic incidence. As the possibility of misclassification concerns only this fraction of all events, and as the medical history in these patients strongly indicated a deep venous thrombosis, this cannot have affected our figures materially (after exclusion of the 20% who were not objectively diagnosed, we found similar risk estimates with wider confidence intervals).

Two years and 6 years after the first event, 10% (two out of 20) and 25% (five out of 20) of the symptomatic carriers had experienced a second event (superficial thrombophlebitis excluded). These high recurrence risks in factor V Leiden carriers are similar to the results of previous studies from the United States and Italy ( Ridker et al, 1995 ; Simioni et al, 1997 ; Prandoni et al, 1998 ). The protective role of oral anticoagulants is well illustrated in these 20 symptomatic carriers. None of the eight carriers who received oral anticoagulant treatment experienced a recurrence, while ten of the 12 carriers who received no oral anticoagulant treatment experienced a recurrence.

Remarkably, two (16·7%) of the propositi had a combined defect, i.e. factor V Leiden and the prothrombin G20210A variant. This mutation has a population prevalence of about 2% and, therefore, it seems probable that the combination of defects led to these patients becoming index patients ( Poort et al, 1996 ). The elevated incidence in non-carriers could not be explained by the presence of the prothrombin G20210A variant: none of the relatives carrying only the prothrombin G20210A variant had experienced a venous thrombosis and the thrombotic incidence rate in non-carriers was even higher after exclusion of the two families with the prothrombin G20210A variant.

We compared our data with data from a study performed by Allaart et al (1993) on similar selected thrombophilic families with heterozygous protein C deficiency (who were also known to our centre) for thrombophilia work-up. Using these data, we found that the median of thrombotic incidence and recurrence rates were very similar for both defects, which does not lend support to different thrombotic risks for protein C deficiency and factor V Leiden.

In conclusion, in clinical practice special attention should be paid to young symptomatic individuals and their relatives with a strong positive family history of venous thromboembolism or a history of recurrent venous thrombosis who are at risk, especially women who would like to use oral contraceptives or who intend to become pregnant. Identification of carriers of factor V Leiden may be worthwhile in these persons in order to discourage contraceptive pill use among carriers and to protect carriers during pregnancies against venous thrombosis. However, data that such a policy would be beneficial are lacking. In addition, our data are based on a study among selected families with thrombophilia and should not be applied to screening of other asymptomatic individuals (i.e. prior to prescribing oral contraceptives). Finally, our data provide no grounds to treat patients with factor V Leiden differently from patients with heterozygous protein C deficiency. However, considering the fact that the prevalence of factor V Leiden is at least tenfold higher than the prevalence of all other known genetic deficiencies, further prospective studies will be needed to evaluate clinical policy.

Acknowledgments

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This study was supported by a grant from the Netherlands Heart Association (grant no. 95 026).

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  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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