Transcript Advances in Genetic Testing for Inheritable Bleeding Disorders

Advances in Genetic Testing for
Inheritable Bleeding Disorders
Keith Gomez
Haemophilia and Thrombosis Centre
Royal Free Hospital
kd:ht
Katherine Dormandy Haemophilia
and Thrombosis Centre
Overview
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Status of genetic testing
Use in clinical practice
New technologies
Future challenges
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Clinical Uses of Genetic Diagnosis
• Prediction of phenotype
‒ Inhibitor formation
‒ Assay discrepancy
• Confirmation of severity
• Identification of carriers / relatives
• Pre-natal and pre-implantation genetic diagnosis
‒ Funded by the NHS for prevention of severe cases
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Testing Strategy for Mutation Detection
Haemophilia B
All Severities
Sequencing
Multiplex ligation-dependent
probe amplification
Single nucleotide variations
Large mutations
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Testing Strategy for Mutation Detection
Haemophilia A
Severe
Inversions
PCR
Southern
Non-severe
Large insertions
or deletions
MLPA
Southern
PCR: Polymerase chain reaction;
MLPA: Multiplex ligation-dependent probe amplification
Single nucleotide
variations
Sequencing
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National Haemophilia Database 2014
Coagulation Defect
Patients in Register
Treated
Total
Male
Female
Haemophilia A
5,686
5,686
-
54%
Haemophilia B
1,205
1,205
-
55%
Haemophilia A Carrier
1,377
-
1,377
5%
Haemophilia B Carrier
425
-
425
12%
Von Willebrand Disease
10,178
3,178
6,460
11%
Factor XI deficiency
2,459
1,051
1,408
3%
Factor VII deficency
964
461
503
6%
Other factor deficiencies
1,247
515
732
-
Platelet Dysfunction
1,768
570
1,198
5%
119
50
69
29%
26,581
13,696
12,885
21%
Glanzmann Thrombasthenia
Total Registered Patients
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Current Genetic Tests for Bleeding Disorders
Disorder
Genetic Testing
Haemophilia A and B
Readily available
von Willebrand Disease
Limited availability of partial
sequence
Other factor deficiencies
Limited availability
Platelet Disorders with gene identified
(e.g. Glanzmann’s)
Very limited availability
Platelet Disorders of no gene identified
No test
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Human Genome Project
• Sequence the entire human DNA
sequence
• Map and characterise all genes
• Started in 1990, completed in
2003
• 3,234 Mbp
• <2% contains 25,000 protein
coding genes
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Comparison of Genetic Testing Strategies
Method
Description
Advantages
Disadvantages
Single Gene
All tests for a single
gene
Identifies nearly all
mutations at a specific
locus
May need multiple
tests
Variable costs
Exome panel
(ThromboGenomics)
Coding regions from
selected genes
Rapid
Low cost
Fewer VUS
Only tests genes on
the panel
May miss gross
abnormalities
Whole Exome
Sequencing (BRIDGE)
Screens all exomes
(2%) in the genome
Covers entire human
coding sequencing
Discovers new genes
Laborious
More VUS
May miss gross
abnormalities
Whole Genome
Sequencing (BRIDGE)
>99% of human
sequence (coding and
non-coding)
Non-coding sequence
covered
Includes regulatory
elements
Discovers new genes
Very laborious
Many VUS
May miss gross
abnormalities
Exomes = coding sequence and flanking regions
VUS = Variant of uncertain significance
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BRIDGE or ThromboGenomics?
Patient with Inheritable Bleeding Disorder
Specialised Laboratory Assays
Specific gene suspected
No specific genetic defect
ThromboGenomics
~80 genes
BRIDGE BPD
Gene Discovery Platform
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ThromboGenomics
• Standardisation of symptoms
‒ Human Phenotype Ontology (HPO)
‒ Allows better genotype-phenotype correlation
• Exomes sequenced
‒ BRIDGE: whole genome
‒ ThromboGenomics: ~80 genes linked in OMIM to known
bleeding disorders
• Results reviewed in MDT
• Report sent to referring clinician
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Human Phenotype Ontology
Westbury et al. Genome Medicine 2015;7:36
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Human Phenotype Ontology
Cluster 18
Cluster 29
Westbury et al. Genome Medicine 2015;7:36
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ThromboGenomics vs Current Testing
ThromboGenomics
Current testing
One, single DNA based test
Multiple tests
Screening of 100+ genes
simultaneously
Screen one gene at a time
Single laboratory
Multiple laboratories each doing a
few genes
Affordable
Expensive for large genes
• ThromboGenomics screens 10 times as many genes as are currently
available on the NHS
• The ThromboGenomics platform will be regularly re-versioned to include
new genes discovered by BRIDGE-BPD and others
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ThromboGenomics – Genes
ITGA2B
Haemophilias + VWD
THPO
ITGB3
FGA
TBXA2R
FGB
FGG
F2
F5
F7
STXBP2
F8
ANO6
F9
VIPAS39
PLA2G4A
F10
VPS33B
F11
a granules
F13A1
F13B
SERPINE1
NBEAL2
VKORC1
GFI1B
vWF
SERPINF2
CYCS
LMAN1
COL3A1
MCFD2
GGCX
Receptors
Intracellular
Cytoskeletal signalling
GP6
P2RY12
GP1Bb
ACTN1
MYH9
FERMT3
SERPINC1
SERPIND1
THBD
PLAT
PROC
PROS1
HRG
MPL
GPV
9 HPS
LYST
NBEA
PLAU
ANKRD26
TBXAS1 (enzyme)
RASGRP2
GNE (enzyme)
Granule defects Transcription factors
Thrombotic
2+
Ca
ORAI1
GPIX
d granules
WAS
Rare Coagulation
Factor Defects
GP1Ba
Coagulation factors
STIM1 2+
Ca sensor
ER
FLI1
GATA1
RUNX1
HOXA11
RBM8A
ETV6
GNAS
MASTL
Membrane phospholipids
Thrombopoietin
Undefined
ThromboGenomics – Genes
ITGA2B
Glanzmann Thrombasthenia
THPO
ITGB3
FGA
TBXA2R
FGB
FGG
F2
F5
F7
STXBP2
F8
ANO6
F9
VIPAS39
PLA2G4A
F10
VPS33B
F11
a granules
F13A1
F13B
SERPINE1
NBEAL2
VKORC1
GFI1B
vWF
SERPINF2
CYCS
LMAN1
COL3A1
MCFD2
GGCX
Receptors
Intracellular
Cytoskeletal signalling
Bernard Soulier
GP1Ba
GP6
P2RY12
GP1Bb
ACTN1
MYH9
FERMT3
SERPINC1
SERPIND1
THBD
PLAT
PROC
PROS1
HRG
GPV
9 HPS
LYST
NBEA
PLAU
ANKRD26
TBXAS1 (enzyme)
RASGRP2
GNE (enzyme)
Granule defects Transcription factors
Thrombotic
2+
Ca
ORAI1
GPIX
d granules
WAS
MPL
Coagulation factors
STIM1 2+
Ca sensor
ER
FLI1
GATA1
RUNX1Hermansky-Pudlak,
HOXA11
Chediak-Higashi
RBM8A
ETV6 syndromes
GNAS
MASTL
Membrane phospholipids
Thrombopoietin
Undefined
ThromboGenomics – Genes
ITGA2B
Macrothrombocytopenia
GP1Ba
THPO
ITGB3
FGA
TBXA2R
FGB
FGG
F2
F5
F7
STXBP2
F8
ANO6
F9
VIPAS39
PLA2G4A
F10
VPS33B
F11
a granules
F13A1
F13B
SERPINE1
NBEAL2
VKORC1
GFI1B
vWF
SERPINF2
CYCS
LMAN1
COL3A1
MCFD2
GGCX
Receptors
Intracellular
Cytoskeletal signalling
GP6
P2RY12
GP1Bb
GPV
9 HPS
LYST
NBEA
ACTN1
MYH9
FERMT3
SERPINC1
SERPIND1
THBD
PLAT
PROC
PROS1
HRG
PLAU
ANKRD26
TBXAS1 (enzyme)
RASGRP2
GNE (enzyme)
Granule defects Transcription factors
Thrombotic
2+
Ca
ORAI1
GPIX
d granules
WAS
MPL
Coagulation factors
STIM1 2+
Ca sensor
ER
FLI1
GATA1
RUNX1
HOXA11
RBM8A
ETV6
GNAS
MASTL
Membrane phospholipids
Thrombopoietin
Undefined
https://haemgen.haem.cam.ac.uk/thrombogenomics
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Results Reporting
• Weekly web-conference
• Multi Disciplinary Team (MDT)
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Clinician responsible for the case
Clinical Geneticists
Bioinformaticians
Software developers
Project Scientific Coordinator
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Prediction of Mutation Effect
• 10 million variants in each person’s genome
‒ Can have 20-30 per gene
• Genotype – Phenotype correlation
• Previously described mutation
‒ Check database
• Novel mutation
‒ Found in normal controls or relatives (co-segregation)
‒ Sequence alignments
‒ Structure-Function analysis
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https://www.sapientia.co.uk/
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Interpretation of Variants
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Interpretation of Variants
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Pathogenicity Assignment
Description
Criteria
Clearly pathogenic
• High Impact Variants
• ≥4 previous reports in unrelated
pedigrees
• Molecular mechanism proven
Likely to be pathogenic
• <4 previous reports
• Co-segregation in pedigree with
phenotype
Variant of Unknown significance (VUS)
Unlikely to be pathogenic
• Present in normal population at
low frequency
Clearly not pathogenic
• Common in normals
• No obvious phenotype
• “Polymorphism”
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Pathogenicity Assignment
Description
Criteria
Clearly pathogenic
• High Impact Variants
• ≥4 previous reports in unrelated
pedigrees
• Molecular mechanism proven
Likely to be pathogenic
• <4 previous reports
• Co-segregation in pedigree with
phenotype
Variant of Unknown significance (VUS)
http://www.acgs.uk.com
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Genotype-Phenotype Correlation
Relevance to Phenotype
Examples
Full contribution to phenotype
• Heterozygous variant in condition
known to have dominant inheritance
• Homozygous variant in recessive
condition
Partial contribution to phenotype
• Heterozygous variant in recessive
condition
• Variant in related gene in same pathway
Uncertain contribution to phenotype
• Variant in gene with possible
relationship to main locus
None
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MDT Outcome
Multi Disciplinary Team (MDT)
Consultant Haematologists, Clinical Geneticists, Bioinformaticians,
Clinical Scientists, Project Scientific Coordinator,
Causative Variant
Yes
No
Confirmation by Sanger sequencing
Sample considered for NGS
Whole Genome Sequencing
Report to referring Consultant
BRIDGE BPD
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ThromboGenomics Results
203
samples
134
79
Variant known
No prior genetic
analysis
(35 different genes)
109
13
SNV
Indels
5
Exon(s)
deletions
7
74
5
Gross
Causal
to
BRIDGE
variants
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ThromboGenomics – Timeline
Accredited Service
2009.…..
2012
Available to all NHS
Hospitals
300 samples
processed
First sample processed
(Gene list version 1.0)
2013
Idea submitted to ISTH
by Willem Ouwehand
and Thomas Kunicki
2014
2015
2016
Gene list version 2.0
All UK Haemophilia Centres
TG Working Group
Genomics in Thrombosis and Haemostasis SSC
SSC = Scientific and Standardization Committee
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ThromboGenomics – Global Network
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Acknowledgements – TG
Genomics in T&H SSC Committee
Chair - Willem H Ouwehand, Cambridge UK
Co-chairs
Dan Bellissimo, Pittsburgh, USA
Paul Bray, Philadelphia, USA
Kathleen Freson, Leuven, Belgium
Anne Goodeve, Sheffield, UK
Michele Lambert, Philadelphia, USA
Pieter Reitsma, Leiden, The Netherland
Anthony Attwood
Louise Daugherty
Cedric Ghevaert
Jennifer Jolley
Myrto Kostadima
Karyn Megy
Sofia Papadia
Chris Penkett
Ilenia Simeoni
Jonathan Stephens
Ernest Turro
Matthias Ballmaier
Marco Cattaneo
Jose Guerrero
Dan Hampshire
Marian Hill
Marguerite Neerman- Arbez
CAMBRIDGE MEMBERS
Nancy Hogg
Paquita Nurden
Peter Smethurst
William Stevenson
Sarah Westbury
Tadbir Bariana
Peter Collins
Ron Kerr
Mike Laffan
Claire Lentaigne
Ri Liesner
Christopher Ludlam
Carolyn Millar
Andrew Mumford
Amit Nathwani
David Perry
Suthesh
Sivapalaratnam
CURATORS
CONTRIBUTORS
Funding