Genetic Background of Venous Thromboembolism

Haifeng M. Wu, M.D.

Associate Professor

Departments of Pathology and Internal Medicine

Director of Clinical Coagulation Laboratory

Ohio State University

Columbus, OH USAHaifen Wu, MD discloses no significant financial interests or other relationships with commercial interests. Presentation will not include discussion of commercial products or services and will not include unapproved or off-label usage of a commercial product or device.

The following planning committee members have no significant financial interests or relationships with commercial interests to disclose, their educational unit does not have a financial interest or affiliation with an organization that may receive direct benefit from the subject of the proposed CME activity, and they will not be personally compensated for their role in the planning or execution of this proposed CME activity by an organization other than The Ohio State University: Amy Ehrlich, MA and Henry Zheng, PhD, MBA

Types of clotting disorders

• Stroke• Heart attack• Peripheral vascular disorder• Venous thromboembolism

– Deep vein thrombosis (DVT)– Pulmonary embolism (PE)

Risk factors for clotting disorders

• Factor V Leiden (G1691A), (SNP)• Prothrombin G20210A (SNP)• MTHFR C677T (SNP)• Protein C deficiency, functional assay• Protein S deficiency, functional assay• Antithrombin deficiency, functional assay

• Antiphospholipid antibodies• Malignancy• Age• Immobilization• Surgery• Pregnancy• Estrogen-containing oral

contraception or hormone replacement therapy

• Inflammation

Clotting disorders

Acquired and clinical factors

Genetic risk factors

Venous thromboembolism

• Deep vein thrombosis (DVT) and pulmonary embolism (PE) are caused by abnormal clot formation in veins and are associated with significant morbidity and mortality.

• The most common clinical risk factors for DVT and PE include advanced age, immobilization, pregnancy, hormonal imbalances, and cancer.

Virchow’s Triad

Rudolf Ludwig Karl Virchow (1821 – 1902). One of his major attributions is a theory delineating the pathogenesis ofvenous thrombosis in 1856.

FactorsContributing

toVenous Thrombosis

FactorsContributing

toVenous Thrombosis

Altered Blood Flow(Stasis)

Blood Coagulability

Vessel WallDamage

Venous thromboembolism, continued

• However, in many cases, venous clotting disorders occur at a younger age or at an unusual site, without apparent contributing clinical factors. These cases of clotting events are often caused by underlying genetic predisposition(s), also called thrombophilia. Thrombophilia refers to hereditary conditions that put patients at risk of thromboembolism

Hereditary conditions associated with DVT and PE

• Rare DNA mutations that result in a deficiency of proteins that are naturally present in blood to prevent excessive clot formation – Types

• Antithrombin deficiency• Protein C deficiency• Protein S deficiency

– Features• Low Prevalence (<0.5%)• Due to many possible DNA mutations of the

gene• Higher expression of disease in the affected

population • Earlier onset of disease• Thromboses at unusual sites

Hereditary conditions associates with DVT and PE,

continued• Most of the hereditary factors for clotting disorders

is due to certain SNPs with relatively high frequencies in the general population. – Types

• Factor V Leiden (G1691A)• Prothrombin G20210A

– Features• These prothrombotic SNPs are found in almost

half of all cases of idiopathic thromboembolism• high gene frequency• low disease expression

Protein C/thrombomodulin pathwayis a critical mechanism to control clotting

disorders

Factor V Leiden• Background– As early as 1994, factor V Leiden SNP (G1691A)

was among the first SNPs to be linked to a disease phenotype, venous thrombosis in this case.

– The mutation in Factor V Leiden results in a change of one amino acid from Arginine to Glutamine at the cleavage site of activated protein C (APC)

– This in turn prevents effective inactivation of factor V by APC, leading to a tendency for abnormal clot formation.

– Prevalence of factor V Leiden is about 5% in Europeans but lower in Asian and African populations.

– Dramatically increases the risk of thrombosis in conjunction with pregnancy or estrogen containing oral contraceptives (odd ratio of ~30x).

Factor V Leiden, continued• In a study of 4,047 men and women by Ridker

et al, – carrier frequencies for Factor V Leiden

(heterozygosity) were: • 5.3% of Caucasians • 2.2% of Hispanic Americans • 1.25% of Native Americans • 1.2% of African Americans • 0.45% of Asian Americans

– In the Leiden Thrombophilia Study, the relative risk for thromboembolism was: • Increased 7 fold for heterozygotes • Increased 80 fold for homozygotes • The risk is further increased by the presence of another

risk factor for thrombosis – Oral contraceptives – Pregnancy – Surgery

Mechanism of APC Resistance of Factor V

Leiden – ineffective inactivation of Factor V

Normal Factor V cleavage by APC

Cleavage of Factor V Leiden by APC

Laboratory Diagnosis Factor V Leiden

– Functional assay• Factor V Leiden is also called

APC resistance • When APC is added to the

plasma of affected individuals, APTT clotting time is resistant to the effect of APC, while normal individuals exhibit a dose-dependent prolongation of APTT corresponding to the amount of APC added.

– DNA based test

APC resistance

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Control

Prothrombin Gene G20210A• Discovered by Poort et al. in 1996 • A polymorphism (G20210A) in the 3’ untranslated region

of the prothrombin gene – Mutation leads to :

• increased plasma levels of prothrombin • may be an underlying mechanism for an increased

risk of venous thrombosis – Mutation leads to higher incidence of

thromboembolism • The prevalence of this SNP is approximately 2% in

the general population – 3% of southern Europeans – less commonly seen in Asian and African

populations • 10 - 20% of patients with thrombotic events

Laboratory methods for detection of SNPs, Factor V Leiden or prothrombin

G20210A• A typical genotyping approach is to first increase the

number of SNPs that will be analyzed. – For example, polymerase chain reaction (PCR)

amplification of a desired SNP-containing region is performed initially to introduce specificity and increase the number of allele-specific molecules.

• Afterwards, amplified DNA fragments containing a specific SNP are measured by a device based on – Mass: Mass spectrometry (MS) is a widely used method

for the mass determination of various biomolecules including oligonucleotides

– Allele Specific hybridization or ligation– DNA sequencing– Restriction fragment length polymorphisms analyzed by

agarose gel

Relative risk

• Patients who are carriers of factor V Leiden have a 5–7 fold increased risk of venous thrombosis

• Patients with prothrombin G20210A have about a 2-3 fold increased risk for venous thrombosis

Combined Risk factors

Martinelli I. Pharmacogenetics 2003; 13:589-594

Patients on OC who are homozygous for Factor V Leiden have >20-fold increased risk of VTE

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Clinical usefulness of genotyping test for clotting disorders

• Since 1996, genotyping studies for detection of SNPs have been used routinely for evaluation of thrombotic disorders and for clinical management of patients. – Under current medical practice, genetic testing for factor

V Leiden (G1691A) and prothrombin G20210A have been routinely performed for patients at a higher risk for thrombotic disorders, along with functional assays to detect deficiencies in protein C, protein S, and antithrombin. Risk assessment.

– These patients at higher risk include those who presented with unexplained or ‘idiopathic’ thromboembolism, patients with thromboembolism that is unusually extensive or in an unusual location (e.g., portal vein thrombosis), or patients with a striking family history of venous thromboembolism

Clinical usefulness of genotyping test for clotting disorders, continued

• When testing for a SNP is positive, it is recommended that healthcare providers counsel patients as to the enhanced risk of thrombosis to themselves and family members, the importance of early recognition of signs and symptoms of venous thromboembolism, and the risks and benefits of thromboprophylaxis.

Life modifications

• For the patients who are carriers of factor V Leiden (G1691A) or prothrombin G20210A, it is important to provide counseling about the contribution of each genetic factor to the overall risk of thrombosis. More importantly, genetic testing allows for identifying patients with more than one risk factor. Such individuals usually have a much greater incidence of venous thrombosis than the sum of the individual risks. For example, in women who both carry a factor V Leiden mutation and use oral estrogen, venous thrombosis risk is substantially increased (RR is about 30), compared with nonusers of estrogen.

Limitation of the genotyping tests• Genotyping tests for factor V Leiden and prothrombin

G20210A are not screening tests for the general population. This is because prevalence of these SNPs is high in the general population, but the relative risk for clotting disorders is relatively low for each of these SNPs alone. When genotyping tests are performed without a proper clinical indication, a positive test result often causes unnecessary psychological stress for the patient and family members.

• With regard to the utility of the genetic tests to correctly identify the patients with regard to risk factors for recurrent thrombotic events, there has been a lack of clinical data supporting this clinical application. Recently, some experts have recommended not performing genetic tests for patients with clotting disorders. Many other investigators, however, believe that clinical usefulness of genotyping tests does exist. For instance, thrombophilia testing may help young females with strong family histories of clotting disorders in regards to the use of oral contraceptives or planning for raising a family.