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Il Laboratorio di Emostasi e Trombosi:AttualitàBologna 19 Giugno 2010

Biologia Molecolare e…

�Terapia sostitutiva

�Terapia genica

�Terapia ad RNA

��DiagnosticaDiagnostica

Biologia Molecolare e Diagnostica delle trombofilie

Fattori dellacoagulazione

Inibitori dellacoagulazione

Gene Lesions Deficiencies

antithrombin 1965

protein C 1981

protein SHCII

19841996

thrombomodulin 2002

ZPI 2004

������������� �� ���� ��������

�� 131 different mutations131 different mutations

�� 203 patients203 patients

8 8 homozygohomozygotestes or compoundor compound heterozyheterozygotesgotes195195 heterozygoheterozygotestes

�� 7 7 mutations responsible for mutations responsible for protein dysfunction protein dysfunction (type II PS deficiency)(type II PS deficiency)

• Rare and Heterogeneous mutationswith variable penetrance

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Deficiencies- Gene Lesions

APCresistance -FV LeidenProthrombin GA20210

1994

1996

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Functional polymorphisms

Frequent mutations>1% in the general population

Biologia Molecolare della trombofiliaBiologia Molecolare della trombofilia

FVFV LeidenLeiden

PS DeficiencyPS Deficiency

PC DeficiencyPC Deficiency

FII A20120GFII A20120G

AT DeficiencyAT Deficiency

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1,08

1,23

0

0,2

0,4

0,6

0,8

1

1,2

1,4

GG(n = 20)

GA(n = 52)

U/ml

Prothrombinactivity

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H2

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Gene A

Gene A

: polymorphisms in coding regions: promoter polymorphism

Protein A

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5380

G/A

(173

6 Val/

Met)

EX 25EX 25

6755

A/G

(219

4 Asp

/Gly)

GGly96

EX 13EX 13 EX 16EX 16

2298

T/C

(708

Ile)

2325

C/T (7

17Asn

)

2379

A/G

(735

Glu)

2391

G/A

(739

Ser

)

2663

G/A

(830

Arg/L

ys)

2684

G/A

(837

Arg/H

is)

3943

C/A

(125

7 Leu/Ile

)

4070

A/G

(129

9 His/

Arg)

EX 4EX 4 EX 8EX 8

495 G

/A (1

07 A

la)

642 G

/T (1

56 S

er)

1328

T/C

(385

Met

/Thr)

GAla78

GSer82

CThr100

CIle

100

TAsn100

GGlu100

ASer100

ALys100

AHis100

CLeu100

GVal100

G (R2)Arg100

The HR2 FV haplotype

100%100% conservedconserved 94%94%n.c.n.c.

Bernardi et al. 1997, Castoldi et al. 2000

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Bernardi et al 1997

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CoinheritanceCoinheritance of the HR2of the HR2 Haplotype Confers an Increased Haplotype Confers an Increased RiskRisk ofof Venous Thromboembolism to CarriersVenous Thromboembolism to Carriers ofof FVLeidenFVLeiden

Faioni et al 1999

FVLeiden

FVLeiden +FVHR2

1 2 3 6 84 5 7 109

1

1

2

2

3

3

84 75 6 9

FV R506QPT 20210 G/AFV HR2FV Y1702C

$�����% &�

$������' ()$

�' ()$

�' ()�' % &� �' ()�' % &�

Castoldi et al Blood 2000

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�' ($�����*��� �

�' (������

+�� �������� ���' (����� ����' ����������������� ���������� ����� ����� ��������

Simioni et al Blood 2005

Met2120Thr

His1299Arg (4070 G/A)

Asp79HisAsp2194Gly

Heavy chain (105 kDa)

Connecting region

Light chain (71/74 kDa)

A2A1 A3 C2C1

B

Ca2+

Several FV intragenic polymorphisms contribute to the

modulation of FV levels

Scanavini D et al ATVB

0.0

0.5

1.0

1.5

2.0

2.5

3.0F

VIII

:C (I

Um

l)

CC CG+GG CC CG+GG

0

ABO groupnon-0

-25 C/G LRP genotypes

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Marchetti et al

�Clear evidence for genetic determinantsof coagulation factor levels within coagulation factor genes

FVII FV FVIII FX FIX FXI

Open question

�� Do geneDo gene polymorphisms modulating coagulation polymorphisms modulating coagulation factor levelsfactor levels modulate FVmodulate FV Leiden penetranceLeiden penetrance??

ESENZIONI (RDG020) PER MALATTIE RARE RELATIVE ALLE ALTERAZIONI TROMBOFILICHE

• a) omozigosi per la mutazione fattore V Leiden (R506Q)

• b) omozigosi per la mutazione G20210A della protrombina

• c) difetti combinati in forma eterozigote delle due mutazioni suddette

• d) deficit di antitrombina

• e) difetti combinati di una delle condizioni suddette insieme a deficit congenito di Proteina C o di Proteina S

Il panel di esami per trombofilia eredofamiliarecompresi nell’esenzione R99

• 1. mutazione FV Leiden• 2. mutazione della protrombina 20210A• 3. difetto di Antitrombina (metodo funzionale e

immunologico)• 4. difetto di proteina C (metodo cromogenico e

immunologico)• 5. difetto di proteina S (determinazione proteina S libera e

funzionale)

Per i test funzionali il difetto deve essere confermato in due occasioni distinte.

Coronary Artery Disease

“AN ELEVATED LEVEL OF FACTOR VII COAGULANT ACTIVITY IS A RISK FACTOR, ESPECIALLY OF FATAL

EVENTS, IN ISCHAEMIC HEART DISEASE” (Meade, 1986)

“AN ELEVATED LEVEL OF FACTOR VII COAGULANT ACTIVITY IS A RISK FACTOR, ESPECIALLY OF FATAL

EVENTS, IN ISCHAEMIC HEART DISEASE” (Meade, 1986)

FXaFVIIaFVIIa

Ca2+

TF FX

Tissue Factor (extrinsic) PathwayCoagulation initiation

“Intra-Vascular” Tissue Factor (TF)

Microparticle shedding from humanDendritic Cells delivers TF

D E F

10 µµµµm10 µµµµm

G

10 µµµµm

H

10 µµµµm

I

10 µµµµm

10 µµµµm10 µµµµm 10 µµµµm10 µµµµm

Tissue FactorTissue Factor andand MicroparticlesMicroparticles• MPs are quantitatively important carriers for

the full length TF form• A large fraction of TF, and its procoagulant

potential, are “deliverable” after physiological or pathological stimuli

Baroni et al 2006

Coagulationand

Inflammation

IL-6

Protein S

Fibrinogen

FactorVIII

Tissue Factor

Combined Effect of Hemostatic Gene Polymorphisms and the Risk of Myocardial Infarction in

Patients with Advanced Coronary Atherosclerosis

Martinelli et al 2008

<5 risk alleles vs �5 OR 2.0 CI 1.3- 3.2 <3 risk alleles vs >7 OR 7.3 CI 2.0-26.4

High-throughput Studies

Bezemer et al JAMA 2008

“Functional” SNPs and Deep Vein Thrombosisassociation studies

Bezemer et al JAMA 2008

“Functional” SNPs and Deep Vein Thrombosis

Biologically plausible candidates but conferring modest risk for thrombosis

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mapping ofchromosomal regions

fine mapping

characterization of candidate genes

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$ �%��&�����'�&�(��) * +,�++ -

$ �. ��'� +/�+0 -

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“unknown candidates”

Quantitative Trait Loci (QTL) for thrombosis- Genome wide scan

Genome-wide association of early-onset MI with SNPs

Myocardial Infarction Genetics Consortium NATURE GENETICS 2009

Genome-wide association of early-onset myocardial infarction: OR of validated SNPs

Myocardial Infarction Genetics Consortium NATURE GENETICS 2009

Genome-wide association of early-onset myocardial infarction:

Lipid metabolismCoronaryCalcification?

HeartRhythm?

Myocardial Infarction Genetics Consortium NATURE GENETICS 2009

Genome-wide association of early-onset MI:allelic dosage score of validated SNPs

Biologia molecolare e diagnostica delle malattie emorragiche ereditarie

Fattori dellacoagulazione

Inibitori dellacoagulazione

Hemophilia A

Margaglione,Castaman et al 2008

Biologia Molecolare e Diagnosi Prenatale

Tagariello, Belvini et al 2008

60 centers involved worldwide

FVII gene mutations

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