Basi molecolari delle piastrinopenie ereditarie

IV Corso Nazionale di aggiornamento

PIASTRINE 2013

Milano, 14-16 novembre 2013

Università degli Studi di Milano (Milano)

Anna Savoia

IRCCS Burlo Garofolo

University of Trieste, Trieste, Italy

Inherited thrombocytopenias

• Rare disorders

• Reduced platelet number (<150x109/L)

• Mainly primary haemostasis defects

• Bleeding tendency not always correlates with platelet count

• Often misdiagnosed (unnecessary therapies)

• Genetically heterogeneous though often with similar phenotypes

trab

ecu

lar

bo

ne

stem cell niche Vascular niche

blo od flow HSC self renewal immature Mk mature Mk vessel cytoskeleton-driven migration TPO/MPL activation pathways GATA1 RUNX1 FLI1 ANKRD26 NBEAL2 cytoskeleton-regulated PPF

Signaling pathway

CTRUS

TAR

CAMT

Severe defects in megakaryocyte production

CTRUS

TAR

CAMT

Bone marrow transplantation

Hypomegakaryocytic thrombocytopenia improving

during life

Bilateral radial aplasia Radio-ulnar synostosis

Alterations of gene expression levels

associated with defective megakaryocyte maturation

Transcription control of genes during mk-poiesis GATA1

FLI1 RUNX1

FLI1 Large deletion on 11q23-ter: contiguous gene syndrome

Paris-Trousseau thrombocytopenia/Jacobsen syndrome

Macrotrombocytopenia Increased mk number

Malformations

GATA1 related diseases

Increased number of small megakaryocytes Large platelets with defective a-granules content

Defective erythropoiesis Mild b-thalassemia

Propositus

Carrier

Balduini et al. Thromb Haemost 91:129, 2004

R216Q D218G D218Y G208S V205M DNA face FOG1

a

-granule defects: gray platelet syndrome

• Thrombocytopenia • Large platelets

• Pale platelets for absence of a-granules • Myelofibrosis

• Splenomegaly

• High serum vitamin B12

NBEAL2

BEACH _{ARM WD40}

Protein-protein interactions Membrane dynamics

RUNX1 Familial platelet disorder/predisposition to acute myelogenous leukemia

Thrombocytopenia

Normal size platelet

Predisposition to leukemia

ANKRD26 related disease ANKRD26

Unknown function

• Platelet normal size (MPV: 9.3 ± 1.6 fL) 0 5 10 15 20 25 30 35 40 0 1 2 3 4 WHO No p atien ts •Moderate thrombocytopenia (47.5 ± 28.3x 109/L)

Noris et al, Blood 117:6673-6680, 2011

Features of ANKRD26RD

• Mild bleeding tendency

(petechiae, ecchymosis, gum bleeding, epistaxis, menorrhagia) • a-granule deficiency P a tie nt TSP1 actin c on tr ol

Features of ANKRD2RD

• Higher TPO concentration

7.0 times higher than in controls 2.9 times than in ITP patients

Maturation defects

Noris et al, Blood 117:6673-6680, 2011

Pt

• Presence of PaCSs

(Particulate Cytoplasmic Structures)

Structures associated with cancers

Predisposition to leukemia

Noris et al. Blood 117:6673, 2011 Noris et al. Blood 122:1987, 2013

Leukemia MDS CML ANKRD26RD /100.000 123 56 34 General population /100.000

(National Cancer Institute)

Thon and Italiano. Seminars in Hematology 47:220, 2010

• Microtubule network: TUBB1

• Cytoskeleton organization: MYH9, ACNT1, FLNA, WASP

• Apoptosis: CYCS

GPIIIa myosin IIA actin filaments signalling to cytoskeleton GPIbα GPIbβ GPIX GPV cell membrane GPIIb

Role of cytoskeleton in inherited thrombocytopenias

A

R

P2/

3

WASP

Congenital features

• Myosin aggregates in neutrophils

Acquired features • Hearing loss

• Nephropathy/End-stage failure • Cataracts

• Elevated liver enzymes

Moderate thrombocytopenia Large platelets

No or modest bleeding tendency

Kunishimaet al. Am J Hum Genet 92:1, 2013

Thrombocytopenia due to ACTN1 mutations

α-actinin Actin filaments R1 R2 R3 R4 R4 R3 R2 R1 CAM CAM Missense mutations

Disease Transm Gene Features Wiskott-Aldrich syndrome (WAS) X-L WAS (Xp11) Severe immunodeficiency. Small platelets X-linked thrombocytopenia (XLT)

Possible mild immunodeficiency.

Small platelets

Von Willebrand factor Thrombin ….. Extracellular matrix GPIIIa myosin IIA actin filaments signalling to cytoskeleton GPIbα GPIbβ GPIX GPV cell membrane GPIIb

GPIb-IX-V complex: Bernard-Soulier syndrome

A R P2/ 3 WASP (Wiskot-AldrichSyndromeProtein)

Bernard-Soulier syndrome

Biallelic form Monoallelic form

Genetics Autosomal recessive Autosomal dominant

Bleeding Severe/mild Mild

Thrombocytopenia Variable degree Mild

GPIb-IX-V

expression Usually absent

50%

(carrier of p.Ala156Val) Ristocetin induced

platelet agglutination

Absent Normal or marginally

0 10 20 30 40 50 60 0 1 2 3 4 WHO No p atien ts

Features of patients with Ala156Val

(103 patients from 42 unrelated families)

Noris et al, Haematologica 97:82:2012

• Platelet macrocytosis

(MPV, 15 fL; 25th_-75th_{: 14.3-16.5)}

• Moderate thrombocytopenia

(Imp: 89 x 109_{/L; 25}th_-75th_{: 63-109)}

(Man: 114 x 109_{/L; 25}th_-75th_{: 89-141)}

• Mild bleeding tendency

• Autosomal dominant with complete penetrance (plt)

GPIIIa myosin IIA actin filaments signalling to cytoskeleton GPIbα GPIbβ GPIX GPV cell membrane GPIIb _Fibrinogen /3 WASP

(Wiskot-Aldrich Syndrome Protein)

a_IIbb3/fibrinogen interaction regulates proplatelet formation Glanzmann thrombasthenia (GT)

Autosomal recessive disease Severe platelet aggregation defect Normal platelet number and morphology

Gain of function mutations

• Constitutive mild a_IIbb3 activation • Macrothrombocytopenia

• Abnormal proplatelet formation Increased on fibrinogen

Increased proplatelet swellings • Only mutation not responsible for GT

GPIIIa GPIIb

Ghevaertet al. Blood 111:3407, 2008 Greseleet al. Haematologica 94:663, 2009 Kashiwagiet al. MolGenetGenMed1:77, 2013

*

*

Defective interaction with cytoskeleton

Diagnostic algorithm for inherited thrombocytopenias

History and examination Syndromic

Non-syndromic

Platelet size evaluation

Small platelets Normal-sized platelets Large/Giant platelets Specific tests Specific tests Specific tests Balduini et al. Haematologica88:582, 2003

Balduini et al. Semin Thromb Hemost 2013;39:161

GRUPPO DI STUDIO DELLE PIASTRINE

Other features help in differential

Application of diagnostic algorithm

Case series of 210 consecutive families

Biallelic BSS GATA1RD GPS ITGB3 CAMT Unknown ANKRD26RD Monoallelic BSS MYH9RD Normal MPV MPV

Suspected inherited thrombocytopenia

screening Gene identification

Molecular diagnosis

Yes

No

ACKNOWLEDGMENTS

IRCCS Burlo Garofolo Università di Trieste Daniela De Rocco Chiara Gnan Roberta Bottega Elena Nicchia Michela Faleschini Chiara Greco Mariateresa Di Stazio

IRCCS San Matteo Università di Pavia

Alessandro Pecci Patrizia Noris Carlo Balduini

International Consortiun Bernard Soulier Syndrome