CLINICIANS TEST FOR PROTHROMbotic

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1 CLINICAL REVIEW CLINICIAN S CORNER Predictive Value of Factor V Leiden and Prothrombin G20210A in Adults With Venous Thromboembolism and in Family Members of Those With a Mutation A Systematic Review Jodi B. Segal, MD, MPH Daniel J. Brotman, MD Alejandro J. Necochea, MD, MPH Ashkan Emadi, MD, PhD Lipika Samal, MD Lisa M. Wilson, ScM Matthew T. Crim, MSc, MA Eric B. Bass, MD, MPH CLINICIANS TEST FOR PROTHROMbotic genetic mutations including factor V Leiden (FVL) ( ) and G20210A ( ) when treating patients who have had or are at risk of venous thromboembolism (VTE). Factor V Leiden refers to a single base change in the factor V gene (G1691A) that eliminates 1 of its 3 activated protein C cleavage sites. Consequently, factor V is inactivated at a lower rate, leading to more thrombin generation. 1 A single FVL allele is present in about 5%, 2.2%, and 1.2% of white, Hispanic and African American populations in the United States. 2 The (factor II) mutation is the second most common inherited risk factor for VTE. This allele is present in 1.1% of non-hispanic whites and Mexican Americans and in 0.3% of African Americans. 3 The G20210A mutation is associated with an increase in levels by approximately 30% in heterozygotes and by 70% in ho- CME available online at and questions on p Context Testing for genetic risks for venous thromboembolism (VTE) is common, but the safety and utility of such testing need review. Objectives To define rates of recurrent VTE among adults with VTE with a factor V Leiden (FVL) or G20210A mutation compared with those without such mutations; to define rates of VTE among family members of adults with a FVL or G20210A mutation according to presence or absence of a mutation; and to assess whether testing adults with VTE for FVL or G20210A improves outcomes. Data s We searched MEDLINE, EMBASE, the Cochrane Library, the Cumulative Index to Nursing and Allied Health Literature, and PsycInfo through December Study Selection Studies were included if they assessed rates of VTE in individuals with a history of VTE who were tested for FVL or G20210A or in family members of individuals with these mutations. Studies assessing the harms and benefits associated with testing were also included. Data Extraction Two investigators abstracted data and assessed study quality. We pooled the odds of VTE associated with the mutations using random-effects models. We assessed the strength of the evidence using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) criteria. Results We reviewed 7777 titles and included 46 articles. Heterozygosity (odds ratio [OR], 1.56; 95% confidence interval [CI], ) and homozygosity (OR, 2.65; 95% CI, ) for FVL in probands are predictive of recurrent VTE compared with individuals without FVL. Heterozygosity for FVL predicts VTE in family members (OR, 3.5; 95% CI, ), as does homozygosity for FVL (OR, 18; 95% CI, ) compared with family members of adults without FVL. Heterozygosity for G20210A is not predictive of recurrent VTE in probands compared with individuals without G20210A (OR, 1.45; 95% CI, ). Evidence is insufficient regarding the predictive value of G20210A homozygosity for recurrent VTE and the risk of VTE in family members of individuals with G20210A. High-grade evidence supports that reduces recurrent VTE events in probands with either mutation. Low-grade evidence supports that this risk reduction is similar to that in individuals with a history of VTE and without mutations. Conclusions Patients with FVL are at increased risk of recurrent VTE compared with patients with VTE without this mutation. However, it is unknown whether testing for FVL or G20210A improves outcomes in adults with VTE or in family members of those with a mutation. JAMA. 2009;301(23): Author Affiliations: Department of Medicine, Johns Hopkins University School of Medicine (Drs Segal, Brotman, Emadi, Samal, and Bass, Ms Wilson, and Mr Crim), Johns Hopkins Evidence-Based Practice Center (Drs Segal and Bass and Ms Wilson), and General Preventive Medicine, Bloomberg School of Public Health (Dr Necochea), Baltimore, Maryland. Corresponding Author: Jodi B. Segal, MD, MPH, 1830 E Monument St, Room 8047, Baltimore, MD (jsegal@jhmi.edu). Clinical Review Section Editor: Mary McGrae McDermott, MD, Contributing Editor. We encourage authors to submit papers for consideration as a Clinical Review. Please contact Mary McGrae McDermott, MD, at mdm608@northwestern.edu JAMA, June 17, 2009 Vol 301, No. 23 (Reprinted) 2009 American Medical Association. All rights reserved.

2 mozygotes. In 2003, the US Food and Drug Administration approved the first DNA-based laboratory tests specifically for FVL and G20210A detection (Roche LightCycler Tag-IT Mutation Detection Method; Roche Diagnostics, Indianapolis, Indiana). Testing for these mutations is widely offered in the United States. The Evaluation of Genomic ApplicationsinPracticeandPrevention(EGAPP) initiative was developed by the Centers for Disease Control and Prevention to address the need for timely and objective evidence that allows health care providers and payers, policy makers, and consumers to identify genetic tests that are safe and useful. On their behalf, and as a part of an Agency for Healthcare Research and Quality supported Evidence- Based Practice Center, we conducted a broad review of testing for FVL and G20210A. 4 We systematically reviewed the published literature on the predictive value of these genetic tests for future VTE in 2 populations: individuals with a history of VTE (probands) and family members of individuals with VTE and 1 of the mutations. We also assessed whether testing adults with VTE for these mutations improves outcomes. METHODS Search Strategy We performed our search electronically, by hand, and through discussion with experts. We searched 5 databases, MEDLINE (1950 through December 2008), EMBASE (1974 through December 2008), the Cochrane Library (Issue 2, 2008), the Cumulative Index to Nursing and Allied Health Literature (CINAHL; 1982 through December 2008), and PsycInfo, to identify primary, published literature. Study Selection Two authors independently reviewed titles and abstracts to identify eligible articles. Abstractswereexcludedwhenboth investigators agreed they were not relevant, did not study adults, included no originaldata, orwerenotpublishedinenglish. Full articles underwent indepen- dentparallelreviewtodeterminetheirap- propriateness. Studies about the predictive value of the genetic tests were excluded if they did not report results separately for individuals with each mutation, did not objectively confirm VTE, studiedfewerthan10probandsor10family members of individuals with mutations, or did not prospectively study probands. studies of family memberswereacceptable.studiesofpregnantwomenwereexcluded. Studiesusing qualitative methods were excluded if less than 80% of the participants had either mutation or a history of VTE. Data Abstraction A primary reviewer completed all data abstraction forms. A second reviewer Figure 1. Literature Search Results Articles identified in preliminary search Identified by electronic database search 6096 MEDLINE 132 Cochrane Library 6744 EMBASE 711 CINAHL 28 PsycInfo 165 Identified by hand search 7777 Titles reviewed 2224 Abstracts reviewed 397 Articles reviewed 46 Articles included in meta-analysis b 40 Included in predictive value analysis 9 Included in effect of testing analysis with clinical expertise checked the results for completeness and accuracy. Reviewers were not masked to the articles authors, institutions, or journal. 5 We abstracted information on study characteristics, population characteristics, objective of the study, and results. When available, we noted whether the index VTEs were idiopathic events; ie, without identifiable precipitants. Information was entered into the SRS 4.0 database (TrialStat! Corp, Ottawa, Ontario, Canada). Study Quality Assessment Two investigators independently assessed articles internal validity based on study setting, inclusion/exclusion 6099 Duplicates excluded 5553 Excluded (did not apply to a key question) 1827 Excluded a 1461 Did not apply to a key question 2 Animal studies 577 No original data 10 In vitro testing with no human specimen 25 Studied only children 16 Not in English 130 Other 351 Excluded a 22 No original data 32 Not in English 326 Did not address study question 81 Other CINAHL indicates Cumulative Index to Allied Health and Nursing Literature; FVL, factor V Leiden; VTE, venous thromboembolism. a Number of excluded articles sums to more than total number because articles could be excluded for more than 1 reason at this level. b Forty articles addressed the predictive value of testing and 9 addressed the effect of testing; 3 of these articles addressed both American Medical Association. All rights reserved. (Reprinted) JAMA, June 17, 2009 Vol 301, No

3 Table 1. Characteristics of Studies Assessing Risk of Future Venous Thromboembolism Among Probands With a Mutation Ridker et al, Kearon et al, Lindmarker et al, Eichinger et al, Simioni et al, Høibraaten et al, Miles et al, Eichinger et al, Baglin et al, Palareti et al, Procare Group, Baarslag et al, Kyrle et al, Christiansen et al, Mansilha et al, Santamaria et al, Vossen et al, González-Porras et al, Study Aim Prospective study nested in RCT examining efficacy of prolonged Prospective study nested in RCT of recurrence rates by, with varying duration of Prospective study nested in RCT assessing resistance to activated protein C by hormonal therapies Prospective study to evaluate D-dimer testing to predict recurrence clinical outcomes of patients with upper extremity thrombosis Prospective study to evaluate the risk of recurrence in men vs women Prospective study nested in RCT of extended-duration Anticoagulation Duration No. of After Index Participants Event, mo Follow-up After Index Event, Mean, mo Age at Index Event, Mean, y Male, % Quality Deficits 77 NR a 68 NR 100 Incomplete description of covariates and no description of loss to follow-up 78 Minimum: 3 a Loss to follow-up 10% in 1 group (Median) 57 No major deficits Convenience sample; loss to follow-up 10% in 1 group Uncertain influence of funding source 121 NR NR Postmenopausal and 70 0 Incomplete description of sampling scheme; loss to follow-up 10% in 1 group; incomplete description of covariates; uncertain influence of funding source 218 NR 87 NR 100 Incomplete description of covariates a Incomplete description of sampling scheme; loss to follow-up 10% in 1 group NR NR NR Loss to follow-up 10% in 1 group; uncertain influence of funding sources 581 NR 16 (Median) Incomplete description of sampling scheme; uncertain influence of funding source 140 Minimum: Loss to follow-up 10% in 1 group; uncertain influence of funding source 28 Minimum: NR No description of loss to follow-up; uncertain influence of funding source 887 Mean (range): 9 (3-21) Incomplete description of sampling scheme; loss to follow-up 10% in 1 group 503 NR No major deficits Incomplete description of covariates; loss to follow-up 10% in 1 group Uncertain influence of funding source 100 NR Incomplete description of sampling scheme (Medians among subgroups) (Medians) NR No description of loss to follow-up; uncertain influence of funding source (continued) 2474 JAMA, June 17, 2009 Vol 301, No. 23 (Reprinted) 2009 American Medical Association. All rights reserved.

4 Table 1. Characteristics of Studies Assessing Risk of Future Venous Thromboembolism Among Probands With a Mutation (continued) Hron et al, Wåhlander et al, Prandoni et al, Strandberg et al, Kearon et al, Study Aim Prospective study examining whether family history predicts recurrence Prospective study nested in RCT to learn if prothrombotic defects affect efficacy of ximelagatran Prospective study evaluating many risk factors for recurrence Prospective study examining the association between factor V Leiden and circulating APC-PCI complexes Prospective study nested in RCT examining effect of intensity of Anticoagulation Duration No. of After Index Participants Event, mo Follow-up After Index Event, Mean, mo Age at Index Event, Mean, y Male, % Quality Deficits Incomplete description of sampling scheme; no description of loss to follow-up; uncertain influence of funding source 1133 Mean (range): 6 (5-7) Abbreviations: APC-PCI, activated protein C protein C inhibitor; NR, not reported; RCT, randomized controlled trial. a Index events were all idiopathic (unprovoked) events. 24 NR NR Incomplete description of covariates; no description of loss to follow-up; uncertain influence of funding source Uncertain influence of funding source NR NR NR Incomplete description of sampling scheme; no description of loss to follow-up 376 Minimum: 3 a Loss to follow-up 10% in 1 group criteria, key characteristics of the enrolled participants, losses to followup, and funding source. Our quality assessment of qualitative studies included items from the Joanna Briggs Institute Qualitative Assessment and Review Instrument. 6 A senior reviewer adjudicated the discrepancies in the quality assessment. Quality assessments are presented descriptively rather than quantitatively to highlight potential sources of bias. Data Synthesis We quantitatively pooled data from studies that assessed the predictive value of the tests when there were sufficient data and the studies were qualitatively homogeneous with respect to key variables. We calculated a pooled estimate of the odds ratio (OR) for VTE in probands and separately in family members. We used a random-effects model with the DerSimonian and Laird method for calculating between-study variance. 7 Fixed-effects models yielded very similar results. We assessed heterogeneity among the studies using a standard 2 test and a significance level of.10 and with an I 2 statistic. The I 2 statistic describes variability in effect estimates that is due to heterogeneity rather than chance. 8 A value greater than 50% suggests substantial variability. We used the Duval and Tweedie nonparametric trim-and-fill method of accounting for publication bias. 9 We also used the Egger test to evaluate the likelihood of missing studies, with P.05 for the bias estimate as an indicator of potentially missing studies 10 Finally, we sequentially removed each study from the calculation of the pooled estimates and recalculated the pooled ORs with a 95% confidence interval (CI). We used STATA (Intercooled, version 9.0, Stata Corp, College Station, Tex). We graded the quantity, quality, and consistency of the evidence by adapting a grading scheme recommended by the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) Working Group. 11 For each question, we assessed the strength of the study designs; the quality and consistency of the body of evidence, including limitations affecting individual study quality; certainty regarding the directness of the observed effects across studies; and the precision and strength of the findings. RESULTS The literature search identified 7777 unique titles, including 165 found by hand searching. We included 46 articles (FIGURE 1). Risk of Recurrent Thrombosis Among Probands With FVL and Prothrombin G20210A vs Individuals Without the Mutations Twenty-three articles addressed this question Study quality was moderate (TABLE 1). There was often insufficient description of modifiers of the relationship between the mutation and recurrent VTE, such as age and other thrombotic risk factors. However, in studies using multivariate models, statistical adjustment did not generally attenuate the mutation-specific effect size Funding sources were rarely described. Thirteen studies described rates of recurrent VTE in probands heterozygous for FVL compared with probands without mutations (TABLE 2). One presented data separately for 2 treatment groups (ximelagatran extended prophylaxis vs placebo). 22 One study tested whether FVL homozygosity is associated with a higher rate of 2009 American Medical Association. All rights reserved. (Reprinted) JAMA, June 17, 2009 Vol 301, No

5 VTE recurrence than FVL heterozygosity. 26 Overall, 979 heterozygous individuals experienced 161 recurrent VTE events and 3751 mutation-free individuals had 473 thrombotic events. The pooled OR for recurrent thrombosis was 1.56 (95% CI, ) for individuals heterozygous for FVL compared with patients without the mutation (FIGURE 2A). The I 2 value was 48%, but there was no significant evidence of publication bias. When each study was sequentially removed, the ORs changed little. In the 3 studies reporting annualized event rates in individuals heterozygous for FVL, event rates ranged from 2.8% to 7.5%. 16,19,26 Annualized event rates in individuals without FVL ranged from 1.1% to 3.1%. 16,19,23 When we included 3 studies that did not specify whether the probands were heterozygous or homozygous for FVL, the OR changed little (OR, 1.61; 95% CI, ). 16,19,27 Seven studies described rates of recurrent VTE in probands homozygous for FVL compared with those without mutations. 13,14,17,20,22-24 Overall, 49 homozygous individuals had 7 recurrent thrombotic events and 2333 mutation-free controls had 225 thrombotic events. The pooled OR was 2.65 (95% CI, ) (Figure 2B and Table 2). There was no evidence of heterogeneity (I 2 =0%). When each study was sequentially removed from analysis, the ORs changed little. Among 6 studies that included only patients with idiopathic index thromboses 16,18,23,24 or reported results separately for individuals with idiopathic index events, 15,28 the pooled OR for recurrence of VTE was 1.17 (95% CI, ) for individuals with FVL relative to individuals without. One of Table 2. Numbers and Rates of Recurrent Events in Probands With a History of Venous Thromboembolism According to Presence of a Genetic Mutation Heterozygous for Factor V Leiden No. of Events/Total Homozygous for Factor V Leiden Heterozygous for Prothrombin G20210A Comparison Double Group Heterozygous Ridker et al, b 7/63 4/14 NR NR Not studied 1.8; 7.5 Event Rate per Person-Year (95% CI) a Kearon et al, /55 c 2/19 1/1 NR 1/3 1.1 ( ) in comparison group Lindmarker et al, /338 c 19/118 4/11 NR 4/28 NR Eichinger et al, /450 c NR NR NR 3/42 NR Simioni et al, /186 18/38 NR 1/3 12/24 NR Høibraaten et al, /98 c 2/21 1/2 NR Not studied NR Miles et al, /178 5/26 NR 3/3 2/11 NR Eichinger et al, /204 17/83 NR NR Not studied NR Baglin et al, NR/410 NR/77 NR NR NR/20 NR Palareti et al, /469 15/68 0/2 0/4 3/38 NR Procare Group, NR 10/108 5/32 NR Not studied 5.8 ( ); 3.2 ( ) Baarslag et al, /24 1/3 d NR NR 0/1 NR Kyrle et al, NR/581 NR/245 d NR NR NR/61 NR Christiansen et al, /382 19/84 1/8 NR 4/29 NR Mansilha et al, NR/69 NR/18 NR NR NR/8 NR Santamaria et al, /138 5/15 d NR NR 3/14 NR Vossen et al, Placebo recipients NR 13/79 d NR 1/7 Not studied 3.5 ( ) for heterozygous group Anticoagulation recipients NR 0/13 d NR 0/1 Not studied 0 (0-0.08) for heterozygous group González-Porras et al, /126 5/29 NR NR 3/21 3.1; 2.8; 2.9 Hron et al, NR NR/245 d NR NR NR/61 NR Wåhlander et al, Placebo recipients 7/438 16/121 0/4 NR 2/27 NR Ximelagatran recipients 41/406 2/100 0/11 NR 0/26 NR Prandoni et al, /724 38/111 NR NR 11/45 NR Strandberg et al, /132 6/50 d NR NR Not studied NR Kearon et al, e 7/147 3/161 0/10 NR 0/58 1.1; 0.8 ( ) d ;0 Abbreviations: CI, confidence interval; NR, not reported. a Event rates correspond to columns with event data in the order in which data appear. b Data overlap with Miles et al 25 and are not included in pooling. c Comparison group may include individuals with other hypercoagulable conditions. d Mixed heterozygous and homozygous. e Anticoagulation recipients JAMA, June 17, 2009 Vol 301, No. 23 (Reprinted) 2009 American Medical Association. All rights reserved.

6 these 6 did not specify if the individuals were homozygous or heterozygous for FVL 28 ; the others studied heterozygous individuals. A single study described recurrence rates of VTE among individuals with vs without FVL who had a clearly provoked (nonidiopathic) VTE. Results of this study showed an OR of 6.5 (95% CI, ). 15 Three articles described rates of recurrent VTE in individuals with both the FVL and the G20210A mutation compared with mutationfree control patients. 15,17,25 Overall, 10 double-heterozygous individuals had 4 recurrent events and 833 mutationfree controls had 95 recurrent thromboses. The pooled OR was 4.81 (95% CI, ). In 1 study, all 3 double heterozygotes developed recurrent thrombosis. 25 Annual incidence rates were not reported in these studies. One additional study described recurrent VTE rates in double-heterozygous individuals but did not include a mutation-free comparison group. 29 Eighteen articles examined rates of recurrent VTE in probands with the G20210A mutation 12-15,17,18,21-25,27-33 (Table 2). Nine articles compared rates of recurrent VTE between probands heterozygous for G20210A and mutationfree probands. 14,15,17,19,21-25 The number of probands with a heterozygous G20210A mutation ranged from 3 to 58 (median, 26). The number of comparison probands ranged from 72 to 724 (median, 307). Among 281 probands heterozygous for the G20210A mutation, 38 recurrent thrombotic events occurred compared with 385 recurrent events among 3355 mutation-free probands. The pooled OR was 1.45 (95% CI, ) (Figure 2C). The I 2 for heterogeneity was 8% and there was no evidence of publication bias. In our sensitivity analysis, removal of 1 study with an OR of decreased the pooled OR to Annual recurrence rates were reported in 2 studies and were 0% and 2.9%. 19,24 One additional study reported a relative rate that did not differ significantly from unity but did not report numbers of recurrent VTE events. 32 When 4 studies were included that did not specify whether probands were heterozygous or homozygous, the combined OR was 1.23 (95% CI, ). 27 Only 2 articles quantified event rates in individuals with VTE who were homozygous for G20210A compared with mutation-free probands. 22,24 There were only 3 probands homozygous for Figure 2. Odds Ratios for Recurrent Venous Thromboembolism Among Probands A B C Probands heterozygous for factor V Leiden mutation Kearon et al, ( ) Lindmarker et al, ( ) Simioni et al, ( ) Høibraaten et al, ( ) Miles et al, ( ) Eichinger et al, ( ) Palareti et al, ( ) Christiansen et al, ( ) González-Porras et al, ( ) Wåhlander et al, a 1.36 ( ) Wåhlander et al, b 1.26 ( ) Prandoni et al, ( ) Kearon et al, ( ) Overall 1.56 ( ) Test for heterogeneity: I 2 = 48%; P =.03 Test for overall effect: P =.005 Probands homozygous for factor V Leiden mutation Kearon et al, ( ) Lindmarker et al, ( ) Høibraaten et al, ( ) Palareti et al, ( ) Christiansen et al, ( ) Wåhlander et al, a 0.98 ( ) Wåhlander et al, b 2.50 ( ) Kearon et al, ( ) Overall 2.65 ( ) Test for heterogeneity: I 2 = 0%; P =.62 Test for overall effect: P =.08 Probands heterozygous for G20210A mutation Kearon et al, ( ) Lindmarker et al, ( ) Simioni et al, ( ) Miles et al, ( ) Palareti et al, ( ) González-Porras et al, ( ) Wåhlander et al, a 0.66 ( ) Wåhlander et al, b 1.02 ( ) Prandoni et al, ( ) Kearon et al, ( ) Overall 1.45 ( ) Test for heterogeneity: I 2 = 7.91%; P =.37 Test for overall effect: P = Size of the data markers is proportional to study size. CI indicates confidence interval American Medical Association. All rights reserved. (Reprinted) JAMA, June 17, 2009 Vol 301, No

7 Table 3. Characteristics of Studies of VTE Rates Among Family Members of Probands With Mutations Samama et al, Middeldorp et al, Le Cam-Duchez et al, Simioni et al, Simioni et al, Castaman et al, Lensen et al, Martinelli et al, Simioni et al, Lensen et al, Middeldorp et al, Rintelen et al, Meinardi et al, Simioni et al, Design Prospective Prospective Prospective Vossen et al, Vossen et al, 47 Prospective 2005 Couturaud et al, 37 Prospective and 2006 retrospective s No. of Participants Duration of Observation, Mean (Range), y Male, % Quality Deficits Inclusion Criteria 125 NR NR 39 Incomplete description of sampling frame, inclusion/exclusion criteria, enrolled participants; unclear influence of funding source 467 Age 15 y; first-degree relatives living in the Netherlands; no terminal illness 370 Age 15 y; no protein C, protein S, or antithrombin III deficiency NR NR No major deficits NR NR Incomplete description of sampling frame and enrolled participants NR 50 Incomplete description of sampling frame 378 No malignancy; no protein C, protein S, or antithrombin III deficiency 417 NR 2.8 ( ) NR Incomplete description of sampling frame, inclusion/exclusion criteria, enrolled participants; unclear influence of funding source 40 No lupus or long-term anticoagulants; no antiphospholipid antibody; not pregnant; not on oral contraceptives or estrogen/progesterone 181 Age 15 y; first- and second-degree relatives; living in the Netherlands 1076 First- or second-degree relatives; no antithrombin, protein C, or protein S deficiency without FVL or G20210A; no overt autoimmune disease; no severe illness; no long-term NR NR Incomplete description of enrolled participants, unclear influence of funding source NR 50 No major deficits NR 44 Unclear influence of funding source 12 A single family NR 25 Incomplete description of sampling frame and enrolled participants 183 First-degree relatives NR NR Incomplete description of inclusion/exclusion criteria and enrolled participants 470 Age 15 y; no long-term 3.3 ( ) 50 No major deficits anticoagulant; first-degree relative; no history of VTE 40 No malignancy; no protein C, protein S, or antithrombin deficiency; no lupus anticoagulant 67 Family members who themselves had a previous VTE; age 15 y; living in the Netherlands; no terminal illness; first-degree relatives 561 Age 15 y; no previous VTE; no malignancy; no G20210A Abbreviations: FVL, factor V Leiden; NR, not reported; VTE, venous thromboembolism. NR NR Incomplete description of sampling frame, inclusion/exclusion criteria, enrolled participants; unclear influence of funding source NR 43 No major deficits 4 (0.3-6) 46 Incomplete description of sampling frame 1437 NR NR NR Incomplete description of sampling frame 1291 No long-term anticoagulant; no 5.7 ( ) NR Incomplete description of previous VTE sampling frame 552 Age 16 y; first-degree relatives Total years 41 No major deficits of observation; retrospectively and 1999 prospectively 2478 JAMA, June 17, 2009 Vol 301, No. 23 (Reprinted) 2009 American Medical Association. All rights reserved.

8 G20210A in these studies, and none developed recurrent thrombosis. Family Members With FVL and Prothrombin G20210A and Risk of Thrombosis vs Family Members Without the Mutations Seventeen articles assessed first venous thrombosis in family members of individuals with the FVL or G20210A mutations (TABLE 3). Study quality was mixed. Inclusion criteria other than that participants be family members of probands with VTE were not always stated. Three of the 5 prospective studies conducted clinical surveillance for VTE either annually or biannually Studies varied in their descriptions of the population from which participants were identified. Several had little description of eligibility criteria for family members Age and sex were inconsistently described. Nine studies described rates of VTE in relatives with heterozygosity for FVL compared with relatives without the mutation ,41-46 One of these included a single person homozygous for FVL among the 91 studied. 43 Two studies did not include a comparison group without a mutation. 35,46 Seven studies with comparison groups enrolled 1066 family members heterozygous for FVL and 940 family members without the FVL mutation (TABLE 4). The ORs across these studies were similar except for 1 small study in which there were no events among 8 heterozygous family members. 41 The pooled OR was 3.49 (95% CI, ) with little heterogeneity between studies (I 2 =0%) (FIGURE 3A). Removal of the study by Couturaud et al 37 increased the pooled OR to just over 4.0. In this study, the annualized event rate among family members heterozygous for FVL was 0.36% (95% CI, 0.24%-0.49%), lower than the rates in other studies. 36,43,45 It is unclear why VTE rates were lower in this highquality study. There was no evidence of publication bias. The inclusion of 5 studies in which relatives were not differentiated as to whether they were homozygous or heterozygous for FVL did not substantially change the OR for VTE (OR, 3.30; 95% CI, ) ,47-49 Six studies described results for 48 relatives who were homozygous for FVL compared with family members Table 4. Numbers and Rates of Events Among Family Members According to the Presence of a Genetic Mutation in the Family Members Heterozygous for Factor V Leiden No. of Events/Total Homozygous for Factor V Leiden Heterozygous for Prothrombin G20210A Comparison Double Group Heterozygous Samama et al, /41 b 11/84 c NR NR Not studied NR Middeldorp et al, /203 d 28/253 4/11 NR Not studied 0.1; 0.43 Le Cam-Duchez et al, /161 d 75/203 5/6 NR Not studied NR Simioni et al, /154 d 17/224 c NR NR Not studied 0.09; 0.28 Simioni et al, /201 d 3/216 c NR NR Not studied 0.34; 0.49 Castaman et al, /30 b NR NR NR 1/ ; 0.26 Lensen et al, /90 d 20/91 c NR NR Not studied 0.15; 0.56 Event Rate per Person-Year (95% CI) a Martinelli et al, /436 b 35/447 c NR 6/35 9/158 b 0.07 ( ) 0.19 ( ) 0.42 ( ) 0.13 ( ) Simioni et al, /1 b 0/5 0/1 0/5 Not studied NR Lensen et al, /82 d 9/101 c NR NR Not studied 0.11; 0.2 Middeldorp et al, /12 9/458 NR NR Not studied ( ) Rintelen et al, /12 b 0/8 1/2 3/10 1/8 NR Meinardi et al, NR 24/67 NR NR Not studied 2.3 Simioni et al, /248 d 8/299 0/14 NR Not studied 0.1 ( ) 0.67 ( ) 0 (0-0.37) Vossen et al, / /225 c NR NR Not studied 0.03 ( ) 0.15 ( ) Vossen et al, /1118 d 1/173 NR NR Not studied 0.1 (0-0.2) 0.1 (0-0.06) Couturaud et al, /225 d 35/298 7/14 2/9 0/ ( ) 0.36 ( ) 2.3 ( ) 0.53 (0-1.26) 0 (0-0.39) Abbreviations: CI, confidence interval; NR, not reported. a Event rates correspond to columns with event data in the order in which data appear. b No mutations. c No specification as to heterozygous or homozygous. d No factor V Leiden American Medical Association. All rights reserved. (Reprinted) JAMA, June 17, 2009 Vol 301, No

9 Figure 3. Odds Ratios for First Venous Thromboembolism Among Family Members A B Family members heterozygous for factor V Leiden mutation Middeldorp et al, ( ) Le Cam-Duchez et al, ( ) Lensen et al, ( ) Rintelen et al, ( ) Simioni et al, ( ) Couturaud et al, ( ) Overall 3.49 ( ) Test for heterogeneity: I 2 = 0%; P =.58 Test for overall effect: P <.001 Family members homozygous for factor V Leiden mutation Middeldorp et al, ( ) Le Cam-Duchez et al, ( ) Rintelen et al, ( ) Simioni et al, ( ) Couturaud et al, ( ) Overall ( ) Test for heterogeneity: I 2 = 0%; P =.93 Test for overall effect: P < Size of the data markers is proportional to study size. CI indicates confidence interval. Data not shown for Simioni et al, 42 which had no events in either group. studies (OR, 8.0; 95% CI, ). 50 This may be due to low event rates among family members without mutations (0.07% per year). This compares with somewhat higher rates in the mutation-free relatives in the other studies, ranging from 0% to 0.34% ,39,42,44,46-48 Analysis suggested potential publication bias, with a paucity of small studies reporting large effect sizes. Family members who were heterozygous for G20210A were compared with family members without the mutation in 3 studies 37,41,51 (Table 4). In 1 study, the age-adjusted relative event rate was 3.4 (95% CI, ) compared with family members without the mutation. 51 Remaining studies were small. 37,41 Pooling these small studies yielded an OR for events of 1.89 (95% CI, ), with an I 2 of 0%, suggesting that family members who are heterozygous for this mutation do not have an increased odds of thrombosis. A single study with a comparison group described 1 relative who was homozygous for G20210A. 41 This individual was identified among 44 family members from 4 families. None reported a venous thromboembolic event. Three studies described agespecific event rates. 43,45,49 One additional study described the age of the individuals at the time of their first thrombosis among family members with and without mutations. 44 Middeldorp et al 45 described age-specific relative event rates for family members who were homozygous or heterozygous for FVL mutation or who had both FVL and G20210A mutations compared with family members without mutations. The relative risk associated with a mutation was highest in the youngest patients (aged years; relative rate of approximately 15) and was lowest in the older patients (relative rate of approximately 2.5). However, other similarly designed studies did not demonstrate this age mutation interaction. 43,49 without the mutation. 36,37,41,42,44,45 One additional study had no comparison group 35 (Table 3). Annualized rates were described for only 3 studies The pooled OR was 18 (95% CI, ). The I 2 for heterogeneity was 0% (Figure 3B6). One small study was excluded from pooled analyses because it had only 1 individual in each group. 42 Removal of 1 study 44 decreased the pooled OR to 16. In this study, 5 of the 6 relatives who were homozygous for FVL had venous thromboembolic events. 44 The annualized rate was not described for the mutation-free comparison group. There was no evidence of publication bias. Four studies described venous thromboembolic events in 59 family members with double heterozygosity 37,41,42,50 compared with 674 family members without mutations. One study observed no events in either group 42 (Table 4). The pooled OR was 6.7 (95% CI, ), with an I 2 value of 0%. The OR for family members with double heterozygosity in 1 study was higher than in the remaining Impact of Testing and Resultant Management on VTE-Related Outcomes No studies directly addressed the effect of testing on outcomes. Four studies described VTE recurrence rates during among probands with FVL or G20210A. 22,24,29,52 This is relevant to our question if clinicians alter management based on a positive test result. Three of these studies consisted of individuals participating in randomized controlled trials. 22,24,52 The fourth was within a prospective study. 29 Two studies investigated the effect of warfarin on recurrence rates 24,52 and 1 the effect of ximelagatran, 22 and 1 did not specify the treatment 29 (TABLE 5). Ridker et al 52 assessed thromboembolism recurrence rates among individuals with FVL or G20210A treated with low-intensity warfarin or placebo. Of 77 patients with FVL or G20210A in the placebo group, 14 had recurrences (8.6 events per 100 person-years) compared with 3 of 66 assigned to the war JAMA, June 17, 2009 Vol 301, No. 23 (Reprinted) 2009 American Medical Association. All rights reserved.

10 farin group (2.2 events per 100 personyears). Low-intensity warfarin reduced the rate of recurrence among patients with thrombophilia by 75% (hazard ratio, 0.25; 95% CI, ). This risk reduction was not significantly different than the 58% reduction seen among patients without either mutation (hazard ratio, 0.42; 95% CI, ; P=.51 for interaction). Kearon et al 24 studied whether VTE recurrence rates differed among individuals with vs without thrombophilic defects receiving low-intensity (international normalized ratio goal, ) or conventional (international normalized ratio, goal, ) warfarin therapy. Among 171 patients with FVL, 3 had recurrences. None occurred in the 60 patients with G20210A. The recurrence rate for patients with FVL receiving lowor conventional-intensity warfarin therapy was 0.8% per year (95% CI, 0.2- Table 5. Effect of Anticoagulation on VTE Recurrence Among Individuals With Mutations Design Population Sample Size Outcome of Interest Key Conclusions Ridker et al, Randomized VTE recurrence with controlled trial; low-intensity subgroup of warfarin and patients within recurrence rate a larger trial among carriers (PREVENT) Vossen et al, Wåhlander et al, Kearon et al, Prospective Cohort analysis nested within randomized controlled trial; subgroup of patients within a larger trial (THRIVE III) Cohort analysis nested within randomized controlled trial; subgroup of patients within a larger trial (ELATE) FVL or G20210A (pooled); prior unprovoked VTE; had3moof uninterrupted warfarin; US-based study; age range, y; 53% male; mean follow-up 2.1 y (maximum, 4.3 y) FVL or combined FVL/ G20210A; prior VTE; European-based study; age range, y; 35%-48% male; followed up for 1710 person-years (mean, 5.6 y; range, 1-7 y) FVL or G20210A; prior VTE; had 6 mo of uninterrupted warfarin; European-based study; 18-mo study period FVL or G20210A; prior VTE; had 3 mo of uninterrupted warfarin; Canada-based study; mean (SD) age, 57 (15); 54%-58% male; mean follow-up 2.3 y 508 participants: 253 placebo (26.6% FVL carriers; 4.8% G2021A carriers); 255 low-intensity warfarin (22.0% FVL carriers; 4.7% G20210A carriers) 304 participants: 124 on long-term (13 FVL; 1 combined FVL/ G20210A); 180 not on long-term (79 FVL; 7 combined FVL/ G20210A) 1223 participants: 612 oral ximelagatran (111 FVL; 27 G20210A); 611 placebo (125 FVL; 27 G20210A) 661 participants (pooled): 337 low-intensity warfarin (91 FVL; 32 G20210A); 324 conventionalintensity warfarin (80 FVL; 28 G20210A) VTE recurrence with long-term and recurrence rate among carriers VTE recurrence with ximelagatran and recurrence rate among carriers VTE recurrence with low- or conventionalintensity warfarin and recurrence rate among carriers vs noncarriers Low-intensity warfarin reduced recurrence in noncarriers (hazard ratio, 0.42; 95% CI, ) and in carriers (hazard ratio, 0.25; 95% CI, ) Rate of recurrence for FVL carriers not on long-term was 3.5%; no events in the group on long-term Oral ximelagatran reduced recurrence in noncarriers as well as FVL carriers With anticoagulants, absolute rate of recurrence for FVL carriers was 0.8% per patient-year (95% CI, ) and was less than those without a defect (hazard ratio, 0.7; 95% CI, ) Quality Deficits Loss to follow-up not described No quality deficits noted Loss to follow-up not described; source of study funding represents potential bias Loss to follow-up not described Abbreviations: CI, confidence interval; ELATE, Extended Low-Intensity Anticoagulation for Unprovoked Thromboembolism; FVL, factor V Leiden; PREVENT, Prevention of Recurrent Venous Thromboembolism; THRIVE, Thrombin Inhibitor in Venous Thromboembolism; VTE, venous thromboembolism American Medical Association. All rights reserved. (Reprinted) JAMA, June 17, 2009 Vol 301, No

11 2.2). This rate was not statistically different from the rate among participants without mutations or coagulopathies on anticoagulants (hazard ratio, 0.7; 95% CI, ). Wåhlander et al 22 assessed the risk of VTE recurrence in probands receiving ximelagatran, an oral direct thrombin inhibitor (not presently available) vs placebo. Among 111 FVL carriers in the treatment group, 2 had recurrent VTE compared with 16 among 125 with mutations assigned to placebo. This difference in recurrence rates was statistically significant (hazard ratio 0.25; P value not reported). The relative reduction in recurrence with treatment was similar for individuals with vs without FVL (P=.92 for interaction). Results were similar for individuals with G20210A (P =.98 for interaction). Vossen et al 29 studied the recurrence rates of VTE among probands belonging to families with thrombophilia according to whether they were receiving long-term. Of 304 patients, 124 were receiving longterm and 180 were not. Fewer FVL carriers were in the longterm group than in the comparison group (13% and 44%, respectively). Of 79 patients with FVL who did not receive long-term, 13 had recurrences during 366 person-years (incidence rate, 3.5% per year; 95% CI, ). Among the 13 FVL carriers who received, none had recurrences during 43 person-years. There were important differences in the baseline characteristics and duration of follow-up between the 2 groups and few details about the regimens. Table 6. Studies of the Effect of FVL and Prothrombin G20210A Testing on Outcomes Other Than VTE Design Population Sample Size Outcome of Interest Key Conclusions Bank et al, Saukko et al, 55, /2007 Kaptein et al, Heshka et al, Qualitative, structured interview Qualitative, structured interview Cross-sectional survey Cross-sectional survey Asymptomatic first-degree relatives of probands; tested for FVL; Netherlands-based study; age y; 53% male Probands or relatives referred for FVL testing; UK-based study; 12% male Probands and relatives recruited from EPCOT study; tested for FVL and antithrombin, protein C, and protein S deficiency; multicountry European study; age y; 46% male Asymptomatic first-degree relatives of probands; not on long-term ; tested for FVL or G20210A; Canada-based study; age y; 38% male 17 Participants (65% response rate) 42 Participants (43% response rate) 174 Participants (78% response rate) 70 Participants: 44 carriers (86% response rate) and 26 noncarriers (55% response rate) Overall experience with testing; effects of testing on daily life Understanding of testing process and implications of results on daily life VTE risk perception and disease-related worry Perception of VTE risk and changes in behavior following FVL or G20210A testing Abbreviations: EPCOT, European Prospective Cohort on Thrombophilia; FVL, factor V Leiden; VTE, venous thromboembolism. For most, testing not stressful; more concern about relatives health than their own; no changes in daily life for most participants Most did not consider FVL testing different than other tests ordered by clinician; less understanding among those of lower socioeconomic status; few changed daily routine as result of test Increased risk perception and worry among those with prior VTE Little difference in recognition of VTE risk factors between carriers and noncarriers; behavior changes in response to testing were uncommon Quality Assessment Did not mention ethical approval; sampling bias (participant self-selection); survey methods not described in detail Sampling bias (participant self-selection) Sampling bias (participant self-selection) Sampling bias (participant self-selection) 2482 JAMA, June 17, 2009 Vol 301, No. 23 (Reprinted) 2009 American Medical Association. All rights reserved.

12 Effect of Testing and Results on Other Outcomes Four studies addressed how probands and family members knowledge, behaviors and health care experiences were affected by their being tested for FVL or G20210A (TABLE 6) Heshka et al 53 surveyed the perception of VTE risk and changes in behavior following testing for FVL or G20210A among first-degree relatives of probands. Kaptein et al 54 investigated whether the type of thrombophilic mutation and history of VTE affect perception of risk and worry among probands or their relatives with FVL compared with individuals with other thrombophilic mutations. Bank et al 57 conducted a qualitative study of asymptomatic relatives of probands with FVL to assess their experience with testing and how results affected their daily lives. Saukko et al 55,56 assessed the level of understanding of the testing process and the implications of the results among probands and relatives referred for FVL. Testing had minimal impact on knowledge or behavior and was associated with no more than modest distress. COMMENT Moderate evidence supports that both homozygosity and heterozygosity for FVL are predictive of recurrent VTE among individuals who have had a prior VTE. Homozygosity for G20210A is a rare genotype and its association with recurrent venous thrombosis in probands is little known. Moderate evidence supports that heterozygosity for G20210A in probands is not predictive of recurrent VTE. Evidence is insufficient about double heterozygosity in probands. High-grade evidence supports that homozygosity for FVL in family members predicts a higher rate of incident VTE. Moderate evidence supports that heterozygosity for FVL predicts a higher rate of incident VTE. Evidence is insufficient about risk of VTE among family members who are homozygous or heterozygous for G20210A. Low-grade evidence supports that double heterozygosity among relatives is associated with higher risk of VTE. There is no direct evidence that testing for these mutations, and the resultant management, reduces VTE relatedoutcomes in individuals who have had VTE or in the probands family members who have been tested. We identified studies demonstrating that treatment reduces recurrent events in patients with FVL or G20210A; however, the magnitude of this relative reduction is comparable with that seen in individuals without mutations. This suggests that other nongenetic factors may be as important as the presence or absence of the FVL or G20210A mutation in determining the risk of recurrence and the absolute magnitude of benefit conferred by. This may be especially so in individuals with idiopathic VTE. We conclude that the incremental value of testing individuals with VTE for these mutations is uncertain. The literature does not conclusively show that testing individuals with VTE or their family members for FVL or G20210A confers other harms or benefits. If testing is done in conjunction with education, it may increase knowledge about risk factors for VTE. This review has limitations, including the use of only English-language articles. Furthermore, we included observational studies knowing that confounding is a possibility, particularly if there were factors that might have been associated with both the exposure () and the outcome (thrombosis). We were limited to the covariates reported in each study, and the studies did not often present patient-level data that would allow testing for potential confounders. For the family studies, the expected confounder would be genes that are coinherited with the mutation of interest. For both the proband studies and family studies, there might be nongenetic factors such as more aggressive surveillance for events that might bias the estimate of the association between the mutation and outcome. In the studies that did report group-level data (eg, mean duration of or mean age at diagnosis by ), we did not see any consistent evidence that these factors affected the outcomes, but we cannot definitively exclude confounding. Studies are needed to measure how practice actually changes in response to results from FVL or G20210A testing and whether that improves patient outcomes. Future studies should focus on whether management decisions based on testing results affect the recurrence rates (as well as complication rates) in carriers of each of these mutations. Ideally, future trials would randomize patients with thrombosis or family members of individuals with mutations to a test group or a no-test group, and individuals would be managed by their physicians based on the results of the testing, perhaps with evidence-based guidance. Studies of clinical validity should include event rates over time (and relative rates of recurrence between specified groups) rather than just the number of events. Studies should consistently differentiate between heterozygosity and homozygosity. By examining specific subsets of patients, it may be possible to clarify whether there are any interactions between and clinical variables in predicting recurrence. The data presented in the studies were insufficient to definitively exclude the effect of confounders on the relationship between the mutations and the outcome. Uncertainty remains about the magnitude of risk for family members with mutations given the very wide CIs surrounding the ORs and the small number of studies the reported event rates (rather than counts). The studies that we included were exclusively studies of European populations. Future research would be appropriate in white populations outside of Europe or in other populations with appreciable frequencies of mutations American Medical Association. All rights reserved. (Reprinted) JAMA, June 17, 2009 Vol 301, No