Lessons from Genome-Wide Association Studies in Venous Thrombosis

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Raul A. DeLa Cadena, M.D. Temple University School of Medicine

Philadelphia, Pennsylvania “La Mujer” David Alfaro Siqueiros 1964-1971

GWAS

GWAS and Venous Thrombosis

• The revolution started with: – Human Genome – HapMap projects

Lander ES, et al.: Nature, 2001 Venter JC, et al.: Science, 2001

Risk Factors known prior to GWAS Era

• Until the end of the 20th Century: Association and linkage studies.

• Compared to the number of studies performed, successes in VT were relatively modest despite notable discoveries.

• SERPINC1, PROC, and PROS1.. – Association with VT over 25 years.

• F5, F2, ABO y FGG – More frequent SNPs and associated with VT.

Risk Factors Known prior to GWAS Era

F5 Gene

• Rs6025 • Factor V Leyden • R506Q • Resistence activated Proteina C • Allele Q506 has a frequency of 5% in

Caucassians with an increased risk of about 3 times compared to heterozygous carriers

Bertina RM, et al.: Nature 1994 Seligson U, et al.: N Engl J Med 2001

F2 Gene

• Rs1799963-A • 20210 • G20210A • Elevated plasma levels of FII • Allele G20210A associated with an increased

risk of about 2.5 times when compared with heterozygous carriers

Poort SR, et al.: Blood 1996 Seligson U, et al.: N Engl J Med 2001

Gene FGG

• Rs2066865.

• Frequency of approximately 25%.

• Modulating the levels of a form of fibrinogen in plasma by affecting a polyadenation site.

Tregouet DA, et al.: Blood, 2009

ABO Blood Groups

• A1 • B • Associated with VT apparently by increased

levels of plasma FVIII y vWF due to decrease clearance but not clearly determined today.

• Associated frequency of about 30%

Wu O, et al.: J Thromb Haemost 2008

Contributions due to the GWAS Era

New Risk Factors from the GWAS Era

• First study conducted by Bezemer et al. – 200,000 SNPs. – DNA analysis performed in group and individually. – Two novel susceptibility loci identified: GPVI y F11

• Second study concentrated in approximately 300,000 SNPs. – No new susceptibility loci for VT identified, but strong

associations were observed at two known loci, the FV and the ABO genes.

Bezemer ID, et al.: JAMA 2008

Tregouet DA, et al.: Blood, 2009

GP6 • Allele rs1613662-G • Confirmed by three other studies. • Substitution A/G, amino acid 219, exon 4,

gene coding for GP6: isoforms GPVIa y GPVIb. • GPVI for collagen in platelets

Bezemer ID, et al.: JAMA 2008 Snoep JD, et al.: J Thromb Haemost, 2010

F11

• Alleles rs2289252 y rs2036914 • Additive effect regulating the plasma levels of

FXI.

Bezemer ID, et al.: JAMA 2008 Meijers JC, et al.: N Engl J Med, 2000

New Susceptibility Locus on Chromosome 6p24.1

• Allele rs169713 • Allele lies about 100kb downstream of the

HIVEP1 gene. It belongs to a family of genes participating in transcriptional regulation of a variety of inflammatory genes.

Morange PE, et al.: Am J Hum Genet, 2010

GWAS contribution in the understanding of shorthened aPTT

• Short aPTT, a laboratory tool, due to is association with VT.

• A study with 1477 normal subjects with the identification of 3 SNPs – Rs27431672 (F12) – Rs9898 (HRG) – Rs710446 (KNG1)

Tripodi A, et al.: Blood, 2004 Houlihan LM, et al.: Am J Hum Genet, 2010

F12, HRG, KNG1 Association con Thrombosis

• Study with 1,542 patients with VT and 1,110

normal controls. • The rs710446 (KNG1) was found associated

with a risk for thrombosis. • Confirmed with another study with 596

patients and 590 normal controls. • Rs710446 (KNG1), consists of a single amino

acid substitution (Ile581Thr) in KNG1 (HK).

Morange PE, et al.: Blood, 2011

HK

Domain 3

Domain 2

Domain 1

Domain 4 Domain 5

Domain 6

This image cannot currently be displayed. PK TTPa

Additional Information Associated with VT as a result of GWAS

Additional Information from GWAS

• Some GWAS) studies were conducted within the context of the pathophysiology of VT and instead of VT risk assessment.

• One of them evaluated SNPs to study the variation of Protein C in plasma.

• Conducted with 8,000 participants identified 5 genes. – PROCR which codes for the Proteina C receptor identified rs867186 explaining

about 10% of Protein C variability in plasma. It is associated with an increased susceptibility of the receptor to proteolysis and thus associated with an increased risk to VT.

• Additional genes were identified, namely EDEM2, BAZIB, y GCKR requiring careful attention for their association with VT. – GCKR gene plays a major role in the levels of reactive C protein, in favor of the

known association between inflammation and coagulation.

Dehghan A, et al.: Circulation, 2011

INTRINSIC PATWAY

EXTRINSIC PATHWAY

FXII

FXI

FIX

Contact System Kallikrein-Kinin System

FV FII

FVIII FVIIIa Trombin

TFPI

AT-III

Protein C rs6025 F5 rs1799963F2

SERPINC1

rs2289252 y rs2036914 F11

Rs710446 KNG1

aPTT

Trombin Fibrinogen

vWF

Fibrin Generation

FXIII

α2-anti-plasmin

rs1799963 FGG

rs2154299 rs12194855

F13A1

ABO B, A1

Platelets

Platelet Microparticules

GPIb

GPIIb/IIIa

VIIIa

FIXa Ca

FX

FVa FXa

Ca

FIIa

FII

Fibrinogen

PF4

TSP1

FXIIIa

rs2154299 rs12194855

F13A1

rs1799963 FGG

ABO B, A1

Normal Endothelium

NO PGI2 ADPase

Heparan + ATIII

Inhibition FXa + Trombin

tPA uPA

Plasmin Plasminogen

Trombomodulin

Protein C Protein Ca Protein S

Trombin

FVa FVIIIa

Platelet Inhibition

PAI

PROC

PROS1

What to do now?

GWAS

Case Report

Saygi S., et al.: Echocardiography 2011;28:E64-E67

A 46-year old man admitted to ER with: 1. Chest pain 2. Diaphoresis

History, mild smoker. 1. Anxious 2. Hypotensive (70/50 mm Hg

3. EKG:

• Bradycardia • Prolongation QRS complex • Total AV block • ST elevation

Troponin I 15.1 ng/ml (reference range <0.01 ng/ml) Diagnosis of acute myocardial infarction complicated with cardiogenic shock and total AV block was made.

Case Report

Coronary Angiogram

Normal right and circumflex coronary artery and a noncritical plaque in Left anterior descending artery. During coronary angiography chest pain was resolved abruptly and Electrocardiographic changes dissapeared. Patient transferred to ICU. After administration of heparin and aspirin Transthoracic echocardiography was performed. • Suspicion of a mass placed in noncoronary sinus of Valsalva Transesophageal echocardiography was performed.

Coronary Angiogram - Normal

•A round mass (25 x 10mm) filling the noncoronary sinus of Valsalva.

•The mass was highly mobile and prolapsing into right coronary artery ostium intermittently.

Transesophageal Echocardiography

Platelet count - Normal AT-III - Normal Protein S - Normal Protein C - Normal Factor V Leiden - Normal Prothrombin - Normal Factor XII - Normal Methyltetrahydrofolate (MTHFR) - Normal

SPECIALIZED COAGULATION LABORATORY

Surgical Procedure

Emergency surgery, a solid round mass 25mm diameter occupying the Noncoronary sinus of Valsalva was seen and removed. Mass penetrated into the right coronary artery ostium.

Normal Endothelium

NO PGI2 ADPase

Heparan + ATIII

Inhibition FXa + Trombina

tPA uPA

Plasmin Plasminogen

Trombomodulina

Proteina C Proteina Ca Proteina S

Trombina

FVa FVIIIa

Inibicion Plaquetas

PAI

PROC

PROS1

Homozygote Rs1799768

PAI-1 G4/G5

What to do now?

Homozygote Rs1799768

PAI-1 G4/G5

Conclusions

• GWAS, require a large number of participants, for instance, to identify 10 SNPs associated with cardiovascular disease, over 140,000 subjects needed to be included.

• Noteworthy to mention is that a gene housing a rare mutation associated with VT may house SNPs with a moderate risk of thrombosis.

• For instance by the use of the deep gene analysis method like the one use for F9 a rare mutation was found (R338L) associated with elevated numbers of FIXa and thrombotic events in young adults which is expressed in other family members and known as FIX Padua.

Schunkert H, et al.: Nat Genet 2011 Simioni P, et al.: N Engl J Med, 2009

Lessons from Genome-Wide Association Studies in Venous Thrombosis

THANK YOU FOR YOUR ATTENTION

Raul A. DeLa Cadena, M.D. Temple University School of Medicine

Philadelphia, Pennsylvania “La Mujer” David Alfaro Siqueiros 1964-1971