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Clotting Disorders

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Clotting Disorders

  1. 1. Genetic Background of Venous Thromboembolism<br />Haifeng M. Wu, M.D.<br />Associate Professor<br />Departments of Pathology and Internal Medicine<br />Director of Clinical Coagulation Laboratory<br />Ohio State University <br />Columbus, OH USA<br />Haifen 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.<br />
  2. 2. Types of clotting disorders <br />Stroke<br />Heart attack<br />Peripheral vascular disorder<br />Venous thromboembolism<br />Deep vein thrombosis (DVT)<br />Pulmonary embolism (PE)<br />
  3. 3. Risk factors for clotting disorders<br /><ul><li>Factor V Leiden (G1691A), (SNP)
  4. 4. Prothrombin G20210A (SNP)
  5. 5. MTHFR C677T (SNP)
  6. 6. Protein C deficiency, functional assay
  7. 7. Protein S deficiency, functional assay
  8. 8. Antithrombin deficiency, functional assay</li></ul>Genetic risk factors <br />Clotting disorders<br /><ul><li>Antiphospholipid antibodies
  9. 9. Malignancy
  10. 10. Age
  11. 11. Immobilization
  12. 12. Surgery
  13. 13. Pregnancy
  14. 14. Estrogen-containing oral contraception or hormone replacement therapy
  15. 15. Inflammation</li></ul>Acquired and <br />clinical factors<br />
  16. 16. Venous thromboembolism<br /><ul><li>Deep vein thrombosis (DVT) and pulmonary embolism (PE) are caused by abnormal clot formation in veins and are associated with significant morbidity and mortality.
  17. 17. The most common clinical risk factors for DVT and PE include advanced age, immobilization, pregnancy, hormonal imbalances, and cancer. </li></li></ul><li>Virchow’s Triad<br />FactorsContributingtoVenous Thrombosis<br />Vessel WallDamage<br />Altered Blood Flow(Stasis)<br />Blood Coagulability<br />Rudolf Ludwig Karl Virchow (1821 – 1902). One of his major attributions is a theory delineating the pathogenesis of<br />venous thrombosis in 1856.<br />
  18. 18. Venous thromboembolism, continued<br /><ul><li>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 </li></li></ul><li>Hereditary conditions associated with DVT and PE<br /><ul><li>Rare DNA mutations that result in a deficiency of proteins that are naturally present in blood to prevent excessive clot formation
  19. 19. Types
  20. 20. Antithrombin deficiency
  21. 21. Protein C deficiency
  22. 22. Protein S deficiency
  23. 23. Features
  24. 24. Low Prevalence (<0.5%)
  25. 25. Due to many possible DNA mutations of the gene
  26. 26. Higher expression of disease in the affected population
  27. 27. Earlier onset of disease
  28. 28. Thromboses at unusual sites</li></li></ul><li>Hereditary conditions associates with DVT and PE, continued<br /><ul><li>Most of the hereditary factors for clotting disorders is due to certain SNPs with relatively high frequencies in the general population.
  29. 29. Types
  30. 30. Factor V Leiden (G1691A)
  31. 31. Prothrombin G20210A
  32. 32. Features
  33. 33. These prothrombotic SNPs are found in almost half of all cases of idiopathic thromboembolism
  34. 34. high gene frequency
  35. 35. low disease expression</li></li></ul><li>Protein C/thrombomodulin pathwayis a critical mechanism to control clotting disorders<br />
  36. 36. Factor V Leiden<br /><ul><li>Background
  37. 37. 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.
  38. 38. 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)
  39. 39. This in turn prevents effective inactivation of factor V by APC, leading to a tendency for abnormal clot formation.
  40. 40. Prevalence of factor V Leiden is about 5% in Europeans but lower in Asian and African populations.
  41. 41. Dramatically increases the risk of thrombosis in conjunction with pregnancy or estrogen containing oral contraceptives (odd ratio of ~30x).</li></li></ul><li>Factor V Leiden, continued<br /><ul><li>In a study of 4,047 men and women by Ridker et al,
  42. 42. carrier frequencies for Factor V Leiden (heterozygosity) were:
  43. 43. 5.3% of Caucasians
  44. 44. 2.2% of Hispanic Americans
  45. 45. 1.25% of Native Americans
  46. 46. 1.2% of African Americans
  47. 47. 0.45% of Asian Americans
  48. 48. In the Leiden Thrombophilia Study, the relative risk for thromboembolism was:
  49. 49. Increased 7 fold for heterozygotes
  50. 50. Increased 80 fold for homozygotes
  51. 51. The risk is further increased by the presence of another risk factor for thrombosis
  52. 52. Oral contraceptives
  53. 53. Pregnancy
  54. 54. Surgery</li></li></ul><li>Mechanism of APC Resistance of Factor V Leiden – ineffective inactivation of Factor V<br />Normal Factor V cleavage by APC<br />Cleavage of Factor V Leiden by APC<br />
  55. 55. Laboratory Diagnosis <br />Factor V Leiden<br /><ul><li>Functional assay
  56. 56. Factor V Leiden is also called APC resistance
  57. 57. 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.
  58. 58. DNA based test</li></li></ul><li>Prothrombin Gene G20210A<br /><ul><li>Discovered by Poort et al. in 1996
  59. 59. A polymorphism (G20210A) in the 3’ untranslated region of the prothrombin gene
  60. 60. Mutation leads to :
  61. 61. increased plasma levels of prothrombin
  62. 62. may be an underlying mechanism for an increased risk of venous thrombosis
  63. 63. Mutation leads to higher incidence of thromboembolism
  64. 64. The prevalence of this SNP is approximately 2% in the general population
  65. 65. 3% of southern Europeans
  66. 66. less commonly seen in Asian and African populations
  67. 67. 10 - 20% of patients with thrombotic events</li></li></ul><li>Laboratory methods for detection of SNPs, Factor V Leiden or prothrombinG20210A<br /><ul><li>A typical genotyping approach is to first increase the number of SNPs that will be analyzed.
  68. 68. 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.
  69. 69. Afterwards, amplified DNA fragments containing a specific SNP are measured by a device based on
  70. 70. Mass: Mass spectrometry (MS) is a widely used method for the mass determination of various biomolecules including oligonucleotides
  71. 71. Allele Specific hybridization or ligation
  72. 72. DNA sequencing
  73. 73. Restriction fragment length polymorphisms analyzed by agarose gel </li></li></ul><li>Relative risk<br /><ul><li>Patients who are carriers of factor V Leiden have a 5–7 fold increased risk of venous thrombosis
  74. 74. Patients with prothrombin G20210A have about a 2-3 fold increased risk for venous thrombosis </li></li></ul><li>Combined Risk factors <br />Heterozygotes<br />Patients on OC who are homozygous for Factor V Leiden have<br />>20-fold increased risk of VTE<br />Martinelli I. Pharmacogenetics 2003; 13:589-594<br />
  75. 75. Clinical usefulness of genotyping test for clotting disorders<br /><ul><li>Since 1996, genotyping studies for detection of SNPs have been used routinely for evaluation of thrombotic disorders and for clinical management of patients.
  76. 76. 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.
  77. 77. 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 </li></li></ul><li>Clinical usefulness of genotyping test for clotting disorders, continued<br /><ul><li>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. </li></li></ul><li>Life modifications<br /><ul><li>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.</li></li></ul><li>Limitation of the genotyping tests<br /><ul><li>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.
  78. 78. 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.</li>

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