Trombo-genomica

Rosa Santacroce

Genetica MedicaUniversità di Foggia

Malattie multifattoriali

Sono il risultato di più fattori sia ambientali chegenetici.

L’importanza relativa dei fattori genetici è variabile.

Esiste un’aggregazione familiare ma non segue ilcaratteristico pattern di segregazione delle malattie dasingolo gene.

Obesity

Immobility

Trauma orSurgery

Contraceptives

Fattori di rischio acquisiti per trombosi

Who has an increased risk?

Obesity

Immobility

Trauma orSurgery

Contraceptives

Genetic Predisposition

Fattori di rischio per trombosi

Coagulation

Factor V LeidenFactor II 20210A

AntithrombinProtein CProtein S

Gain of Function

Loss of Function

~35% of patients with thrombosis have one of these risk factors

Fattori di rischio genetici per trombosi

TEST PREDITTIVI

Conferma o esclude, in un soggetto

asintomatico, la propensione ad ammalarsi di una

malattia comune e/o complessa.

Questi test identificano il rischio relativo :l'incremento o la riduzione del rischio di ammalarsi di una determinata malattia nel corso della vita rispetto al

rischio medio di popolazione.

de HAAN et al. BLOOD 2012

A role for additional common genetic variants (SNPs)?

de HAAN et al. BLOOD 2012

A role for additional common genetic variants (SNPs)?

FV Leiden

FII A20210

ABO

La contemporanea esistenza all’interno di famiglie con trombosidi soggetti clinicamente non affetti ma genotipicamente portatoridei marcatori indagati rispecchia il lato multifattoriale dellatrombosi

la coesistenza di difetti multipli ne modula la variabilita’ clinica.

le alterazioni genetiche note giustificano soltanto il 60%dellecondizioni trombofiliche di tipo ereditario

altri polimorfismi o mutazioni rare non ancora individuati sonoprobabilmente responsabili dei casi ancora non chiari

Other Inherited Risk Factors

Hyperhomocysteinemia (?) Factor VIII ↑ (mixed) Dysfibrinogenemias Factor V mutations Very rare Factor II mutations Unique mutations

Pannello Trombofilia 13 mutazioni

I geni in considerazione sono quelli relativi al fattore V di Leiden, al fattore II della coagulazione (protrombina) ed il

gene MTHFR (Metilentetraidrofolatoreduttasi).Altri geni sono stati associati a stati trombotici, tra i quali:

Fattore XIII, Beta Fibrinogeno, PAI-1, HPA, HFE, APO E, ACE, AGT.

Pannello Trombofilia 4 mutazioni

Valutazione genetica del rischio di patologie cardiovascolari mediante analisi di mutazione dei geni del Fattore V di Leiden,

Fattore II (protrombina) e metilentetraidrofolatoreduttasi (MTHFR), varianti C677T e 1298 A/C

….dove stiamo andando…

….dove eravamo…

La NGS è anche chiamata high-throughput sequencing (sequenziamento

ad alta resa) perchè, a differenza del sequenziamento tradizionale col

metodo Sanger, consente di sequenziare moltissimi frammenti in parallelo.

Esistono diversi sistemi NGS, sviluppati da diverse compagnie.

Tutti questi sistemi, tuttavia, condividono almeno tre passi fondamentali: la

preparazione e immobilizzazione del DNA (cioè la preparazione della

cosiddetta sequencing library), la reazione di amplificazione e la reazione

di sequenziamento.

NEXT GENERATION SEQUENCING

Il principale vantaggio è la possibilità tecnica di produrre un

volume enorme di dati a costi estremamente più bassi ed in

tempi estremamente più rapidi

Il potenziale dell’NGS è simile ai primi tempi della PCR con il

limite principale dovuto all’immaginazione.

NEXT GENERATION SEQUENCING

“NEXT GENERATION SEQUENCING”….

AS054Whole exome sequencing in patients withinherited thrombocytopenia and excessivebleeding is an efficient way to identify geneticvariants in known and novel genesJohnson B

AS132Validation of a next generation DNAsequencing panel for haemostatic and plateletdisordersGoodeve A

AS196Next-generation DNA sequencingapproach to identify novel geneticrisk factors underlying cerebral veinThrombosis.Gorski MM

Conclusion: We have successfullyapplied NGS-based analysis of 740candidate hemostatic and pro-inflammatory genes in 469 ItalianCVT patients and controls,identifying a common indel variantrs8176719 in the ABOgene that hasbeen previously associated with deepvein thrombosis of the lower limbsand pulmonary embolism.

OR176Diagnosis of inherited bleeding, thrombotic and platelet diseasesby next generation sequencing.Simeoni I

Most monogenic inherited bleeding, thrombotic and platelet

disorders (BTPD) except Haemophilia and von Willebrand’s

diseases are extremely rare.

Reaching a conclusive laboratory diagnosis remains challenging

in the routine health care settings. Next generation sequencing

(NGS) will change their laboratory diagnosis bringing benefits

to patient care.

PO543-MONGenetic variability of KNG1and F11genes using next generation sequencingMartin-Fernandez L1

Background: Venous thromboembolism (VTE) is a complex disease with a

high heritability. However, only a portion of the genetic component is

accounted for by genetic risk factors. From the GWAS of factor XI (FXI)

performed in the GAIT-1 (Genetic Analysis of Idiopathic Thrombophilia)

project significant associations were identified at KNG1andF11loci.

Aims: We performed Next Generation Sequencing (NGS) of the KNG1and

F11 genes to identify genetic variants that might be involved in the risk of

thrombosis.

Bleeding symptoms or bleeding disorder? That is the question

Fasulo MR

Conclusion: Our results show that the majority of patients with

history of bleeding symptoms has not necessary a hemostatic

defect. In patients with a severe bleeding diathesis further

evaluation using next generation sequencing might help to

understand the role of novel genes encoding for unknown

proteins involved in haemostasis.

OR266Meta-analysis of 65,734 individuals identifies TSPAN15andSLC44A2as twonew susceptibility loci for venous thromboembolismMorange P-E

Conclusion: We identified 2 loci (TSPAN15andSLC44A2) that had novelassociations with VTE yet do not belong to canonical pathways leading tothrombosis and have not been associated with other cardiovascular endpoints.These findings may provide etiologic and mechanistic insights into VTEpathophysiology.

OR394Identification of new genetic risk factors for venous thrombosis by targetedsequencing: results of the MILES studyde Haan HG

Conclusion:Using a targeted sequencing strategy we were able to identify newgenetic risk factors for venous thrombosis. Replication and functional studiesare needed to confirm our findings and to unravel the biological mechanismsinvolved

PO653-MON

The use of next-generation sequencing to screen for disorders of

coagulation using a 24 gene panel

Bignell P

Aims: We sought to investigate the clinical utility of NGS for

understanding haemostatic disease by sequencing 24 coagulation genes

using a designed TruSeq Custom Amplicon (TSCA) panel.

Methods:Samples were run on the Illumina MiSeq platform

Sanger Sequencing

Determining the nucleotide sequence (A, T, C and G) of a DNA of a gene

Next generation sequencing (NGS)

Next generation sequencing (NGS) is often referred to as massively

parallel sequencing of clonally amplified or single DNA molecules

that are spatially separated in a flow cell.

NGS generates hundreds of megabases to gigabases of nucleotide-

sequence output in a single instrument run, depending on the

platform.

Next generation sequencing (NGS)

ADVANTAGES OF NGS

Revolutionary improvements in cost and speed of data generation

Provides exquisite resolution for many types of experiments

Next generation sequencing (NGS)

DIS-ADVANTAGES OF NGS

Shorter read lenght sequenced are produced

Bioinformatics-based analysis

File size traumatize IT infrastructures

Next generation sequencing (NGS)

Sanger vs Next Generation Sequencing

Sequencing workflow

Immagini.TIFF Intensità

ReadsFASTQ

Quality control: FastQC,

FastXtoolkit

Bowtie BWAMaq

GATKSAMtoolVarscan

AllineamentoSAMBAMBED

Identificazione delle varianti

.vcf

Filtraggio delle variantiVisualizzazione

NGS Data Analysis flowchart

Reads and coverage

Quella dei test genetici è forse l’area in più rapida espansione e ricca di

problematiche con un impatto pratico immediato sulla vita reale delle

persone.

L’informazione derivante dal test genetico èpermanente in quanto non si modificanell’arco di vita dell’individuo.

Il test genetico fornisce informazioni nonsolo sull’individuo testato ma anche sui suoifamiliari.

VTEThreshold

Number of risk factors for VT

Venous thrombosis is a multifactorial disease

GRAZIE PER L’ATTENZIONE!