Clinical Genetics

M. Kent Froberg, MD

2009

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

CLONALITY

• Clonality of B-cell neoplasms can be demonstrated by molecular techniques

• Can replace demonstration of specific pathological alteration

• PCR and Southern Blot

TYPES OF REARRANGEMENTS

• Pathological oncogenes

• Physiological antigen receptor genes

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

IG REARRANGEMENTS

• Physiological

• Since Ig rearrangement is normal for B lymphocytes, neoplasia of B-cell tumors show clonal Ig rearrangement

• Clonality = Malignancy (rare exceptions)

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

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

Heavy chain

rearrangement occurs

first

Kappa light chain

rearrangement

follows, then lambda

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)

Single bands indicate clonality

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

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

HEREDITARY THROMBOPHILIA

• Anticoagulant Deficiences

– heterozygotes have 35-65% reduction in protein

– have 3-7x risk for venous thrombosis

– deficiencies may be quantitative or qualitative

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

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

HEREDITARY THROMBOPHILIA HYPERHOMOCYSTEINEMIA

• Described 1994

• Acquired (def B12, B6, or folate)

• Hereditary: mutation in one of many enzymes

• Hereditary form accounts for ~20% of venous

thrombosis that is genetic

HEREDITARY THROMBOPHILIA HYPERHOMOCYSTEINEMIA

• Also have risk of atherosclerosis

• Mechanism:

– endothelial dysfunction

– interference with:

• antithrombin

• NO

• thrombomodulin

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

HEREDITARY THROMBOPHILIA FACTOR V LEIDEN

• FV:Q506 = 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)

HEREDITARY THROMBOPHILIA FACTOR V LEIDEN

• Suspect when obvious etiologies of acquired thrombosis have been ruled out

• Pts with thrombosis < 50

• Recurrent thrombosis

• < 50 and > one family member with thrombosis

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

Normal Factor V

Factor V Leiden

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

Case

• 51 year old physician with acute SOB on climbing stairs syncopal episode

• Taken to ED

• Perfusion Scan multiple pulmonary emboli

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 +

WT Heterozygote Homozygote

PCR for Factor V Leiden Mutation

Mutated band

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

Relative Risk of Thrombosis

Thrombophilic Status RRNormal 1OCP use 4Protein C, S or ATIII def 5-7Prothrombin 20210 3Hyperhomcysteimemia 2-4Factor V Leiden (heterozygous) 5-7Factor V Leiden + OCP 30-35Factor V Leiden (homozygous) 80

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