Fatal pulmonary embolism: A study of genetic and acquired factors

Molecular Diagnosis Vol. 5 No. 1 2000

Fatal Pulmonary Embolism: A Study of Genetic and Acquired Factors KATHLEEN J. SLOVACEK, MD, AUDREY F. HARRIS, BA, CLSp (MB), JOHN F. GREENE, JR, MD, ARUNDHATI RAO, MD, PhD Temple, Texas

Background: hwestigators speculate that hereditary thrombotic disease coupled with acquired factors account for a large number of pulmonary thromboemboli. Clinical correlation between genetic and acquired factors with fatal pulmonary thromboemboli has not been extensively studied. Methods: Archival autopsy material was obtained from patients who died of or with puhnonary emboli for whom confirmed autopsy results were available. Polymerase chain reaction-restriction fragment length polymorphism analysis was performed for factor V Leiden and factor II/20210A allele. Retrospective chart review was performed to determine the presence or absence of acquired factors that can predispose to pulmonary thromboemboli. Results: Two of 36 patients (5.5%) were heterozygous for factor V Leiden. No patients had detected abnormalities for factor I1/20210A allele. Eight patients (22.2%) had a malignancy present, one of whom was heterozygous for factor V Leiden. Fourteen patients (38.8%) had recent major surgery or were immobilized. Conclusions: The incidence of factor V Leiden and factor II/20210A allele in patients with fatal pulmonary thromboemboli is not greater than published results of the incidence of these factors in the general population. Recognized acquired factors such as malignancy, recent surgery, and immobilization are fi'equently present in these patients. Our results suggest that genetic profiling of thrombotic disease will not replace clinical vigilance in reducing the risk for death from pulmonary thromboemboli. Key words: PCR-restriction fragment length polymorphism, factor V Leiden, factor II/20210A, thromboemboli.

Pulmonary emboli cause significant preventable mortality in patients across the United States and the world. Recent articles have evaluated risk factors that predispose patients to venous thrombosis From the Department of Anatomic Pathology, Scott and White Memorial Hospital and Clinic, Scott, Sherwood and Brindle)' Foundation, Texas A&M Universio' Health Science Centel, College of Medicine, Temple, TX. Reprint requests: Kathleen J. Slovacek, MD, Department of Anatomic Pathology, 2401 South 31st St, Temple, TX 76508. Email: [email protected] Copyright © 2000 by Clnuvhill Livingstone ® 1084-8592/00/0501-0010510.00/0

and subsequent pulmonary emboli [1]. These factors are commonly divided into genetic and acquired factors. Some genetic factors include antithrombin deficiency, dysfibrinogenemia, factor II/ 20210A allele, factor V Leiden, protein C deficiency, and protein S deficiency [1,2]. Our ability to detect the homozygous or heterozygous state of these inherited factors has greatly improved with the advent of new molecular techniques in the laboratory. Acquired factors predisposing to venous thrombosis include but are not limited to age, hormonal replacement therapy, immobilization, malig-

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Molecular Diagnosis Vol. 5 No. 1 March 2000

nancy, major surgery, orthopedic surgery, and oral contraceptive use, as well as antiphospholipid syndrome [ 1]. The interplay of genetic and acquired factors in the manifestation of fatal pulmonary emboli has not been extensively evaluated. Some investigators have studied the association of factor V Leiden with the development of pulmonary emboli. One group found that the prevalence of factor V Leiden was high in a population of patients with nonfatal pulmonary embolism and deep vein thrombosis [3]. Other investigators evaluated patients with fatal pulmonary embolism and found that the factor V Leiden mutation did not appear to influence the development of emboli in the face of severe illness [4]. This investigation is designed to examine the hypothesis that the incidence of hereditary thrombotic disease is greater in patients with fatal pulmonary thromboemboli and also to evaluate the frequency of acquired factors in this patient population.

Methods Autopsy records from January 1994 through June 1998 were reviewed (330 autopsies). From this initial population, 36 patients were found to have pulmonary emboli diagnosed at autopsy by the pathologist. In 29 cases, the pathologist determined that the pulmonary embolus(i) caused the patient's immediate demise. In seven cases, pulmonary emboli were listed at autopsy as a diagnostic finding contributing to the patient's death. All 36 patients, as determined by autopsy record review, were included in this study.

DNA Extraction For each of the 36 cases, DNA was extracted from formalin-fixed, paraffin-embedded spleen or lymph node sections. DNA from autopsy blocks was slightly more difficult to amplify than standard surgical blocks; therefore, standard paraffin extraction methods were combined with the GlassMAX DNA Isolation Spin Cartridge System (Life Technologies/Gibco BRL, Gaithersburg, MD)[5]. For DNA preparation and extraction, four to five tissue sections of 8 Ixm were collected in a sterile 1.5-mL microcentrifuge tube. Sections were dewaxed by two steps of soaking in 1 mL xylenes (commercially available mixture of ortho-, meta-, and paraisomers with some ethyl benzene) at 65°C,

vortexed, spun down, and the xylenes removed. Sections were washed in 1 mL 100% ethanol, spun down, and the ethanol removed, then 1 mL 70% ethanol, spun down, the ethanol removed, and the samples air dried. Samples were then incubated overnight in 200 IxL of a solution containing 50 mM Tris-C1, pH 8.5:1 mM EDTA; 0.5% Tween 20 (polyoxyethylene(20)sorbitan monolaurate; Mallinckrodt OR, Paris, KY); 200 Ixg/mL proteinase K; and 0.1% sodium dodecyl sulfate at 65°C. The following day, 100 ixL of a 1:1 Chelex 100 slurry (Chelex ® 100 resin; Bio-Rad Laboratories, Hercules, CA) was added, and samples were boiled for 10 minutes. After samples were centrifuged for 5 minutes at maximum speed, the supernatant was purified using the GlassMAX Spin Cartridge System according to the manufacturer's instructions. First, a chaotrope, sodium iodide (provided in the kit), was added to the sample at a ratio of 4.5 volumes binding solution per volume DNA solution. This DNA/sodium iodide solution was added to the GlassMAX Spin Cartridge, and the DNA binds to a silica-based membrane. After centrifugation, three washes with a cold buffer (provided in the kit) removed impurities and the sodium iodide. The spin cartridge was transferred to a clean recovery tube, and DNA was eluted in 65°C Tris-EDTA buffer (1.0 M Tris-HC1, 0.1 M EDTA).

Factor V Leiden: Polymerase Chain ReactionmRestriction Fragment Length Polymorphism Analysis The region surrounding nucleotide 1,691 of exon 10 in the factor V gene was amplified to screen for the Leiden mutation (G---~A substitution) that results in the loss of a recognition site for the restriction endonuclease MnlI [6]. Each 100 IxL polymerase chain reaction (PCR) contained a final concentration of: 1X Extaq PCR buffer (Panvera, Madison, WI), 0.2 mM each dNTP, 0.6 i,zM primer PR-6697 ( 5 ' - T G C C C A G T G C T T A A C A A G A C C A - 3 ' ) , 0.6 ixM primer PR-990 (5'-TGTTATCACACTGGTGCTAA-3'), 2.5 U ExTaq (Panvera), and 6.0 I.zL DNA extract. Cycling conditions on a Perkin Elmer (Norwalk, CT) 9600 were: initial denaturation of 94°C for 5 minutes, followed by 35 cycles of 94°C for 1 minute, 58°C for 1 minute, 72°C for 1 minute, and a final extension of 72°C for 7 minutes. After thermocycling, a 25.0-1xL aliquot PCR product was digested overnight with 24 U MnlI enzyme, 1X

Factors in Fatal Pulmonary Embolism

1

200 b p ~

2

3

4

5

..

163 bp

67 bp.

Fig. 1. Factor V Leiden mutation analysis. Amplificationof a 267-bp product with subsequent digestion by MnlI results in three possible outcomes.Homozygouswild-typemutantsshow fragments of 163, 67. and 37 bp; heterozygousmutant shows fragmentsof 200, 163, 67, and 37 bp; and homozygousmutant shows only 200- and 67-bp fragments.Lane 1, marker;lane 2, heterozygous amplicon; lane 3, heterozygousafter digestion; lane 4, normal amplicon; lane 5, normal after digestion. Amersham (Arlington Hts, IL) M buffer (containing 100 mM Tris-HC1, pH 7.5; 100 mM MgC12; 10 mM dithiothreitol; 500 mM NaCI) in a 200-txL volume at 37°C. DNA was precipitated with 100% ethanol, washed with 70% ethanol, dried, and resuspended in sample loading buffer. Undigested and digested PCR products were run side by side on 10% native polyacrylamide gels at 150 V for 1.5 hours, with Marker V (Boehringer Mannheim/Roche Molecular Biomedicals, Indianapolis, IN). Amplification of a 267-bp product with subsequent digestion by MnlI results in three possible outcomes. Homozygous wild-type shows fragments of 163, 67, and 37 bp; heterozygous shows fragments of 200, 163, 67, and 37 bp; and homozygous mutant shows only 200and 67-bp fragments (Fig. 1).

Factor 11/20210A Allele: PCR-Restriction Fragment Length Polymorphism Analysis A region from exon 14 and the 3'-untranslated region of the prothrombin gene was amplified. A mutagenic primer produ_ces a new HindIII recognit i o n site in samples with the G--->A substitution at

•

Slovacek et al.

55

position 20210 [7]. Each 100 I-~LPCR reaction contained a final concentration of 1X ExTaq buffer, 0.2 mM each dNTP, 0.6 txM primer pr93-787 (5'TCTAGAAACAGTrGCCTGGC-3'), 0.6 t-~M mutagenic primer pr95-315 (5'-ATAGCACTGGGAGCATTGAA*GC-3'), 2.5 U ExTaq, and 6.0 txL DNA extract. Cycling conditions on a Perkin Elmer 9600 were: initial denaturation at 94°C for 5 minutes, followed by 35 cycles of 94°C for 1 minute, 58°C for 1 minute, 72°C for 1 minute, and a final extension of 72°C for 7 minutes. After thermocycling, a 35.0-~L aliquot P e R product was digested overnight with 75 U HindlII enzyme, 1X Amersham M Buffer (containing 100 mM TrisHC1, pH 7.5; 100 mM MgCI2; 10 mM dithiothreitol; 500 mM NaC1) in a 200-1-~L volume at 37°C. DNA was precipitated with 100% ethanol, washed with 70% ethanol, dried, and resuspended in sample loading buffer. Undigested and digested PCR products were run side by side on 6% native polyacrylamide gels at 150 V for 1.5 hours, with Marker V. Amplification of a 345-bp product with subsequent digestion by HindlII results in three possible outcomes. Homozygous wild-type will not cut and only shows a fragment of 345 bp; heterozygous shows fragments of 345, 322, and 23 bp; and homozygous mutant shows only 322- and 23-bp fragments (Fig. 2). Homozygous mutant and heterozygous controls for the 20210A allele of the prothrombin gene were generously provided by Kimball Genetics, Inc (Denver, CO).

Review for Acquired Factors The clinical histories of the 36 patients in the study were reviewed. Initially, acquired factors considered in each case included age, pregnant or postpartum state, oral contraceptive use or hormonal replacement therapy, tamoxifen use, autoimmune disease, malignancy, immobilization, recent major surgery (within 3 weeks of death), recent orthopedic surgery (within 3 weeks of death), and myeloproliferative disorders.

Results Thirty-six patients were included in the study set of autopsied patients. Twenty-nine patients (80.5%) were diagnosed with a pulmonary embolus, as the primary cause of death by the attending pathologist, In other words, the pulmonary embolus(i)yeas deemed to be the immediate cause of the patient's

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Molecular:Diagnosis Vol. 5 No. 1 March 2000

1 234

345bp

56 7 89

~-~.~ ~=~J~==J~==~~ k~--JL _,

~! " , ~ 322bp

:. ,~

Fig. 2. Factor II/202~0A allele mutation analysis. Amplification of a 345-bp product with subsequentdigestion by HindlII results in three possible outcomes.Hom0zygouswild-typewill not cut and only shows a fragment of 345 bp; heterozygous shows fragments of 345, 322, and 23 bp; and homozygous mutant shows only 322-and 23-bp fragments. Lane 1, marker; lane 2, normal amplieon; lane 3, normal after digestion; lane 4, :heterozygous amplicon; lane 5; heterozygous after digestion; latte 6~ homozygous rnutant amplicon; lane 7, hom0zygousmutant after digestion; lane 8, water (contamination control); lane 9, marker.

death. The remaining seven patients had pulmonary emboli present, and these were believed to be a c ontri,buting factor in the patient's death, according to the attending pathologist. Twenty-three patients with fatal pulmonary emboli were women, and 13 patients were men. Twenty-nine patients were white, four patients were black, and the remaimng three patients were of other ethnic origins. The average age of the patients in this study was 67.6 years, with an average age for women of 68 years and an average age for men of 67 years. .Only two patients (5.5%) had abnormalities in factor V. Both patients were heterozygous for the mutation, and both were wl~i.te. One patient did not have an identifiable acquh, ed risk factor present, whereas the other patient had a coexisting malignancy (Table 1). NO patient had detectable abnormali,ties ir~ ~ e factor II/20210A allele. Of the acquired factors evaluated, no pregnant or postpartum~ patient.s were present. T ~ e e patients were on hormonal replacement therapy. One patient had documented systemic 1,upus erythematosus and an antiphospl~olipid syndrome. The most common acquired factors eontribufi,ng to venous thrombosis and/or pulmonary embohas in l~his study population were malignancy (eight patients; 22.2%), recent

major surgery (four patients; 11.1%) or orthopedic surgery (four patients; 1 1.1%), and immobilization (six patients; 16.6%).

Discussion Based on study results, it is apparent that pulmonary embolus remains a relatively common cause of death. From 330 autopsies performed over 54 months at our institution, pulmonary embolism was listed as the primary cause of death in 29 cases (9% of autopsies performed). In seven additional cases, pulmonary emboli contributed to death. Recent studies have attempted to identify predisposing risk factors for venous thrombosis and subsequent pulmonary embolus [1]. Investigators hope to delineate those factors that should be considered and heighten the clinician's suspicion and/or awareness of the problem. Ideally, genetic and acquired factors will be identified, and future patients may be risk su'atified. The results of this study did not support the hypothesis that patients dying of pulmonary thromboembolism have a greater incidence of heritable thrombotic disease than the general population. In various studies, the prevalence of factor V Leiden mutation is from 3% to 7% [1,2]. The prevalence of the mutation is also variable within certain ethnic groups and is greater in whites [7]. The incidence of the factor V Leiden mutation in our population of patients dying of pulmonary embolism was within this normal range (5:.5% of patients in our study). Both patients who were heterozygous for the mutation were white. In both instances, pulmonary embolism was believed to be the cause of death, not only a contributing factor. We also examined another heritable condition, factor IU20210A allele, which is a mutation in the prothrombin gene previously noted to be associated with venous thrombosis [8], The prevalence of factor II/202IOA is 2.3% [8]. In the 36 patients included in this study, none had detectable abnormalities in the factor II/20210A allele. However, with a study size o f only 36 patients, We cannot exclude the possibiSty that this gene defect could be associated with pulmonary thromboembolism, but it is not Iikely to have a large role in the incidence of this disease. Other low-incidence genetic defects have been identified and linked to venous thrombosis and pu,l-

Factors in Fatal Pulmonary Embolism •

Slovacek et al.

57

Table 1. S u m m a r y of Patient Data for Chart Review and Mutation Analysis Patient No.

Age (y)

Sex

I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

69 80 42 65 57 79 71 71 57 75 82 67 79 76 82 60 47 50 63 71 74 39 67 62 65 65 87 54 71 66 89 70 87 55 71 70

F F F F F M M F F M F F M F M M M F F M F M F M F M F F M F F M F F F F

Malignancy Present

Immobilization

Recent Surgery

Factor V Leiden

Factor II/2021A

Yes

Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Heterozygous Normal Normal

Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal

Yes Yes Yes

Yes Yes Yes Yes Yes

Yes Yes Yes

Yes

Heterozygous

Yes

Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal

Yes Yes Yes Yes Yes

Yes

monary embolism, such as dysfibrinogenemia, protein C deficiency, and protein S deficiency, as well as antithrombin deficiency [1,2]. We did not examine these genetic defects in this study because our study group was relatively small. However, investigation is still needed in this field. It is still not absolutely clear whether the presence of a given genetic mutation will increase a patient's risk for fatal or nonfatal thromboembolic disease. Our study did not include a control population of patients without thromboembolic disease, and results were compared with published data from the general population. Two patients in our study were heterozygous for the factor V Leiden mutation. One patient had no other identifiable acquired risk factors and presented to the emergency room a week after hospital discharge. Immobilization in this recently

Yes Yes

Allele

hospitalized patient was not a factor. In this particular patient, the role of the genetic defect may have been more important, or it could have been acting in concert with some as yet unidentified acquired factor. The other patient with factor V Leiden mutation had a coexisting malignancy. In this patient, the combination of the two factors could have increased the risk for thromboembolism. This, however, is only speculation. Much larger studies will be neeessary to accurately and definitively define an interplay of genetic and acquired factors in fatal pulmonary embolism. Review of our patients' records showed that malignancy, recent major and/or orthopedic surgery, and immobilization were the most common associated acquired factors. Of interest was a single patient with systemic lupus erythematosus and docu-

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Molecular Diagnosis Vol. 5 No. 1 March 2000

mented antiphospholipid syndrome. Another patient had polycythemia vera, known to be associated with an increased risk for pulmonary embolus. The high frequency of recognized acquired factors associated with fatal pulmonary embolism in our patients indicates that a genetic profile of thrombotic disease will not replace clinical vigilance in patients with these conditions. Received July 8, 1999. Received in revised form November I. 1999. Accepted November 12. 1999.

References 1, Rosendaal FR: Risk factors for venous thrombosis: Prevalence, risk and interaction. Semin Hematol 1997;34:171-187 2, De Stefano V, Finazzi G, Mannucci PM: Inherited thrombophilia: Pathogenesis, clinical syndromes, and management. Blood 1996;87:3531-3544 3. Martinelli I, Cattaneo M, Panzeri D, Mannucci PM:

Low prevalence of factor V:Q506 in 41 patients with isolated pulmonary embolism. Thromb Haemost 1997;77:440-443 4. Vandenbroucke JP, Bertina RM, Holmes ZR, et al.: Factor V Leiden and fatal pulmonary embolism. Thromb Haemost 1998:79:511-516 5. Pinto AP, Villa LL: A spin cartridge system for DNA extraction fi'om paraffin wax embedded tissues. J Clin Pathol Mol Pathol 1998;51:48-49 6. Voelkerding KV, Huber S, Strobl F, et at.: Resistance to activated protein C: Comparison of three different PCR methods for detection of FV R506Q. Mol Diagn 1996;1:297-304 7. Gregg JP, Yamane AJ, Grody WW: Prevalence of the factor V-Leiden mutation in tour distinct American ethnic populations. Am J Med Genet 1997;73: 334-336 8. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM: A common genetic variation in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996;88:3698-3703