1. Clinical Genetics M. Kent Froberg, MD 2009

2. Purpose This lecture is designed to illustrate two examples of the use of molecular genetics in the clinical laboratory Learn the difference between oncogenic and physiological gene rearrangements and how they are demonstrated by PCR & Southern blot Learn how Factor V Leiden leads to thrombosis and how to detect the mutation Learn the major genetic and environmental causes of thrombosis

3. CLONALITY Clonality of B-cell neoplasms can be demonstrated by molecular techniques Can replace demonstration of specific pathological alteration PCR and Southern Blot

4. TYPES OF REARRANGEMENTS Pathological  oncogenes Physiological  antigen receptor genes

5. ONCOGENE REARRANGEMENTS Translocations Not present in all lymphoid neoplasms Often detected by cytogenetics or FISH Need prior knowledge of type of tumor and genes rearranged Presently molecular probes not available for 2/3 of lymphomas

6. IG REARRANGEMENTS Physiological Since Ig rearrangement is normal for B lymphocytes, neoplasia of B-cell tumors show clonal Ig rearrangement Clonality = Malignancy (rare exceptions)

7. ANTIGEN RECEPTOR REARRANGEMENTS Immunoglobulin (Ig) and T-cell Receptor Ig rearrangements present in >90% of B-cell neoplasms Detected by Southern Blot or PCR Used for Dx, establish lineage, identify minimal residual disease, Rx

8. IG REARRANGEMENTS HIERARCHICAL Germline Ig specificity determined by somatic rearrangement of heavy & light chain genes Involve V (variable), D (diversity), and J (joining) gene segments D/J of heavy chain alleles, then V/DJ, followed by kappa light chain, then lambda Ig heavy chain selected first, so most commonly rearranged

9. Heavy chain rearrangement occurs first

10. Kappa light chain rearrangement follows, then lambda

11. Clonality Tissue from Pt (biopsy of enlarged lymph node) Extract DNA Southern blot using probe for conserved region of IgM heavy chain If polyclonal  smear (no discrete bands) besides germline If monoclonal see discrete bands (one germline & one clone)

17. Single bands indicate clonality

19. CONCLUSIONS Demonstration of Ig Clonal Rearrangement of B- cell lesions by PCR is becoming the standard for many molecular labs in Dx and lineage establishment for B-cell neoplasms This method is faster, more economical and more sensitive than other molecular techniques or cytogenetics

20. HEREDITARY THROMBOPHILIA Most Hypercoagulable States are Acquired –Deficiency of anticoagulant system or defective fibrinolysis –Post-operative, immobility, pregnancy, trauma, oral contraceptives, SLE, neoplasia –Need to eliminate acquired causes first

21. HEREDITARY THROMBOPHILIA Anticoagulant Deficiences –heterozygotes have 35-65% reduction in protein –have 3-7x risk for venous thrombosis –deficiencies may be quantitative or qualitative

24. HEREDITARY THROMBOPHILIA ANTICOAGULANT DEFICIENCIES Protein C, Protein S and Anti-thrombin III account for ~8% of hereditary thrombophilia 1st thrombotic event prior to 50 Anti-thrombin def  highest risk Need functional & antigenic assays

25. HEREDITARY THROMBOPHILIA PROTHROMBIN MUTATION Described 1996 G  A mutation at nucleotide 20210 Heterozygotes found in 2.3% of NL population 6.2% of pts with venous thrombosis Hence, 2.8-fold  risk Mechanism unknown Does  serum thrombin levels

26. HEREDITARY THROMBOPHILIA HYPERHOMOCYSTEINEMIA Described 1994 Acquired (def B 12, B 6, or folate) Hereditary: mutation in one of many enzymes Hereditary form accounts for ~20% of venous thrombosis that is genetic

27. HEREDITARY THROMBOPHILIA HYPERHOMOCYSTEINEMIA Also have  risk of atherosclerosis Mechanism: –endothelial dysfunction –interference with: antithrombin NO thrombomodulin

28. HEREDITARY THROMBOPHILIA FACTOR V LEIDEN Described 1993 as activated protein C resistance (APCR) Activated protein C cleaves factors V and VIII at specific arginine sites to inhibit coagulation Factor V Leiden is a point mutation at 506 in 95% of cases

29. HEREDITARY THROMBOPHILIA FACTOR V LEIDEN FV:Q 506 = arginine  glutamine mutation Hence factor V resistant to proteolysis by activated protein C Favors procoagulant state Carrier rate 5-7% of caucasians Accounts for >50% of hereditary venous thrombosis (~20% of unselected pts)

33. HEREDITARY THROMBOPHILIA FACTOR V LEIDEN Suspect when obvious etiologies of acquired thrombosis have been ruled out Pts with thrombosis < 50 Recurrent thrombosis one family member with thrombosis

35. HEREDITARY THROMBOPHILIA FACTOR V LEIDEN Diagnosis: Assay for APCR (activated protein C resistance) APCR: PTT performed + and - exogenous activated protein C Plus APC = prolonged PTT Normal: ratio +APC/-APC=2.0 APCR: ratio +APC/-APC<2.0

36. Normal Factor V Factor V Leiden

37. PTT ratio: +APC/-APC is > 2.5 with wild type Factor V Ratio is < 2.0 for heterozygous Factor V Leiden Ratio is lowest for homozygous Factor V Leiden Assay for Activated Protein C Resistance

39. Case 51 year old physician with acute SOB on climbing stairs  syncopal episode Taken to ED Perfusion Scan  multiple pulmonary emboli

41. Followup Pt had APCR PCR for Factor V Leiden  heterozygous 49 year old sister with Hx stroke 1 yr previously  Factor V Leiden heterozygote Son of pt also Factor V Leiden +

42. WT Heterozygote Homozygote PCR for Factor V Leiden Mutation Mutated band

43. HEREDITARY THROMBOPHILIA FACTOR V LEIDEN Confirmation: DNA analysis by PCR Factor V mutation eliminates a Mnl I restriction site Blood sample, isolate DNA, amplify mutation site by PCR, subject to Mnl I cleavage (cleaves at arginine site) Factor V Leiden is resistant to digestion at 506

46. Relative Risk of Thrombosis Thrombophilic Status RR Normal 1 OCP use 4 Protein C, S or ATIII def 5-7 Prothrombin 20210 3 Hyperhomcysteimemia 2-4 Factor V Leiden (heterozygous) 5-7 Factor V Leiden + OCP 30-35 Factor V Leiden (homozygous) 80

47. Rx of Hereditary Thrombophilia Anticoagulant therapy (heparin, warfarin) 3% risk of major hemorrhage/yr from Rx 1/5 fatal Avoid other risk factors (smoking, OCP) Genetic counseling