Post on 26-Mar-2020
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!