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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
������������� �� ���� ��������
Deficiencies- Gene Lesions
APCresistance -FV LeidenProthrombin GA20210
1994
1996
������������� �� ���� �������� �
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
(P = 0.0002)���� �! " # ���������
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� ������
! � ��"�� �#�����
�"�� ���� ��H1
H2
$ #��% � �
$ #��% � �
Gene A
Gene A
: polymorphisms in coding regions: promoter polymorphism
Protein A
���������������� �� ��������������
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
���������������� �� �������������� �� �� �� ��
�� � ��� �� �� � � � � � � ��� �� � � �� � ��� �� �� � � � � � � ��� �� � � � ��� � �� � �� � �� � ���� ��� � �� � �� � �� � ���
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
�' (��� �
�' ($�����*��� �
�' (������
+�� �������� ���' (����� ����' ����������������� ���������� ����� ����� ��������
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
" ������������ ������ ���' ��� ������
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
, ����������� ����(����, (���������������������������
" ���� �- �������
mapping ofchromosomal regions
fine mapping
characterization of candidate genes
� ������ !�"���� # ��
$ �%��&�����'�&�(��) * +,�++ -
$ �. ��'� +/�+0 -
$ ��*���1� +2 -
$ ������ 34 ++ �� -
“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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