Case 5

Molecular Pathology

Case 5: Genetic Risk Factor Screening For Fatal Pulmonary Thromboembolism

  • Contributed by Jeffrey A. Porras, MD
    Department of Pathology, University of Oklahoma Health Sciences Center

  • Published on-line in November 1999

Patient History:

The patient was a Caucasian male who presented at 50 years-of-age with a  history of  chronic pulmonary embolism (PE) and pulmonary hypertension, first seen in 1996 for shortness of breath.  In 1997 an angiogram revealed "clots in his lungs" and obstruction of large pulmonary vessels to the right upper and left upper lobes of the lungs. Doppler scan of the legs showed no evidence of deep venous thrombosis (DVT).  At that time, pulmonary artery wedge pressure, revealed pulmonary hypertension.  The patient was referred to this institution for surgery evaluation.  A CT examination showed evidence of multiple pulmonary emboli and splenomegaly.  Pulmonary thromboendarterectomy was performed.  In the period following surgery the patient's respiratory status did not improve.  The patient developed progressive azotemia and metabolic acidosis.  The pulmonary hypertension did not resolve and pulmonary infiltrates developed. The patient died following cardiorespiratory arrest. 

Pathological Findings:


Macroscopic Findings
Cut surfaces of the lungs revealed patchy and segmental consolidation. Thrombi were present within segmental and sub-segmental pulmonary artery branches (Figure 1).

Figure 1. Gross morphology of the lung showing large thrombus in a pulmonary artery (center).  (Click image for larger view)

Figure2.  Section of lung showing thrombus within lumen of major artery with subsequent re-canalization (left of center).  (Click image for larger view)

Microscopic Findings

The lungs showed organizing bronchopneumonia, congestion, intra-alveolar hemorrhage, severe intimal fibroplasia with re-vascularization of old thrombi and fibrinoid necrosis. Figure 2 shows a major pulmonary artery in which the lumen has been altered by fibrous and granulation tissue following organization and re-canalization of older thromboemboli. These changes represent thromboembolic events occurring at various times.   

Molecular Pathology: 

Several common inherited causes of thrombophilia are now recognized.1 One is activated protein C resistance (aPC-R) and up to 90% of individuals with aPC-R are documented to have a single base mutation at nucleotide 1691 in the factor V gene (Factor V Leiden).  The other common inherited risk factor for thrombosis is a mutation at nucleotide 20210 of the prothrombin gene. 

Analysis of the patient for these two common inheritable causes of thrombosis involved separate polymerase chain reaction (PCR) amplification of specific sequences flanking the two mutation sites in the patient's DNA . The presence or absence of a mutation was then determined by restriction enzyme digestion (HindIII for prothrombin  and MnlI for factorV) and subsequent agarose gel electrophoresis of the amplified DNA. Specific banding patterns differentiated unaffected individuals from affected heterozygotes and homozygotes (Figures 3 and 4). Because DNA is used in these assays, even patients who have been anticoagulated can be tested. 

Figure 3: Prothrombin 20210 mutation analysis:Ethidium bromide stained agarose gel showing HindIII digests of PCR products from control (1) heterozygous (345bp and 322bp fragments) and (3) homozygous (322bp fragment) individuals and (2) patient who lacks the prothrombin mutation (345bp fragment). The mutation, when present for a given allele, creates a HindIII restriction site in the PCR product  - the 345bp PCR product forms 322 and 23bp (too small to see in gel) fragments.

Figure 4: Factor V Leiden mutation analysis: Ethidium bromide stained agarose gel showing MnlI digests of PCR products from  controls  (2) heterozygous (37, 67, 116, 153bp), and (3) homozygous (67, 153bp) for the factor V mutation and (1) the patient who lacks (37, 67, 116bp) the mutation. The mutation, when present for a given allele,destroys one of two MnlI restriction sites present in the PCR product, resulting in the formation of a new 153bp fragment (combination of 37 and 116bp fragments).

Final Diagnosis:  

Pulmonary thromboembolism with unknown underlying cause.


The fibrin-forming (coagulation) system is mediated by many coagulation proteins (factors) normally present in blood in an inactive state.  The process of blood coagulation involves activation of these factors through a series of biochemical reactions (coagulation cascade) that transform circulating soluble substances into insoluble fibrin clots.  Protein cofactors such as factor V, secreted by platelets, serve to assemble enzyme-cofactor complexes on the platelet surface, promoting factor X and prothrombin activation.  The product of these events is thrombin formation, which increases its own production many times by activating factor V and VIII.

Activation of coagulation simultaneously activates fibrinolysis and promotes coagulation inhibition.  One of the critical steps in this process is activation of protein C. During normal hemostasis, thrombin activates protein C by cleavage of a 12 amino-acid peptide from the amino terminus of the protein. The activated protein C, in turn, limits clot formation by proteolytic cleavage of factor Va and factor VIIIa, two major coagulation factors involved in thrombin formation.  

Two common genetic mutations have been recently described that affect normal coagulation hemostasis; the factor V Leiden (1691G-A) mutation2 and the G20210 mutation of the prothrombin gene.3

The human locus for factor V gene has been mapped to chromosome 1 (1q21-25).  A mis-sense mutation in the factor V gene substitutes guanine for adenine at nucleotide 1691 (1691G-A) which causes an amino-acid substitution of Gln (CAA) for Arg506 (CGA).  This substitution is known as factor V Q506 or Factor V Leiden.  Cleavage at Arg506 is necessary for inactivation of factor Va by activated protein C; and the introduction of a glutamine at position 506 prevents its inactivation, leading to a hypercoagulable state.

This Factor V Leiden mutation is found in approximately 3 to 5% of the general population and in up to 90% of individuals with activated protein C resistance (aPC-R). The relative risk of venous thrombosis associated with this mutation is 5 to10-fold for heterozygotes over individuals lacking the mutation, but this may rise to as high as a 30-fold increase in smokers, obese individuals, and women on oral contraceptives.  Homozygotes for the mutation have a 50- to 100-fold increase in relative risk. 

A second genetic risk factor for venous thrombosis involves the prothrombin gene, specifically a mutation at nucleotide 20210.  The prothrombin gene is localized on chromosome 11 (11p11-q12), and is organized into14 exons and 13 introns.  The 20210 mutation produces a G to A transition in the 3' untranslated region.  Carriers of this mutation have higher plasma levels of prothrombin and a 3-fold increase risk of venous thrombosis. The mutation is present in approx. 2 to 8% of patients with a first episode of DVT and is found in about 1% of apparently healthy control subjects. 

Studies have demonstrated the existence of a synergic interaction between the prothrombin and factor V Leiden mutations; thrombotic risk is increased 20 to 25 times above normal values and onset of thrombosis occurs much earlier in individuals with both mutations4.   

In a study of forty six necropsy cases done at this institution in which pulmonary thromboembolism (PE) was listed as the cause of death or secondarily associated with it, the factor V Leiden mutation was not found to be an independent risk factor associated with fatal PE.4   This study concluded that other etiologies must be responsible for non-fatal, chronic DVT/PE and fatal acute PE. 

In the present case, neither the factor V Leiden nor prothrombin 20210 mutations were present, indicating that other molecular mechanisms must underlie the thromboembolic disorder in this patient. These results appear to be consistent with the findings of the aforementioned5 and other studies6. Together they suggest that routine determination of factor V Leiden mutation is not indicated in patients with fatal PE; however, the value of determination of the prothrombin 20210 mutation in these cases remains to be elucidated in future studies.


  1. Coleman WB, Tsongalis, GJ 1997 Molecular Diagnostic, Humana Press.
  2. Bertina RM, et al.  Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature  1995; 369:64-67.
  3. Poort SR, et al.  A common genetic variation in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis.  Blood 1996; 88:3698-703.
  4. De Stephano V, et al. The risk of recurrent deep venous thrombosis among heterozygous carriers of both Factor V and the G20210A prothrombin mutation. NEJM 1999; 341:801-6.
  5. Dunn ST, Trong  S  Evaluation of role of factor V Leiden mutation in fatal pulmonary thromboembolism.  Thromb Res1998;91:7-14.
  6. Vandenbroucke, JP  Factor V Leiden and fatal pulmonary embolism.  Thromb Haemost 1998;79:511-6.