isth training course · c v. platelet aggregation. par1 (100µm) adp (10µm) c v. collagen...
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ISTH Training CourseNew genes that cause thrombocytopenia
Dr Neil Morgan, University of Birmingham (UK)
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Disclosures for In compliance with COI policy, ISTH requires the following disclosures to the session audience:
Research Support/P.I. No relevant conflicts of interest to declare
Employee No relevant conflicts of interest to declare
Consultant No relevant conflicts of interest to declare
Major Stockholder No relevant conflicts of interest to declare
Speakers Bureau No relevant conflicts of interest to declare
Honoraria No relevant conflicts of interest to declare
Scientific Advisory Board No relevant conflicts of interest to declare
Presentation includes discussion of the following off-label use of a drug or medical device:<N/A>
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Example : exome sequencing only in dominant inherited disease
SLFN14 mutations underlie thrombocytopenia with excessive bleeding
and platelet secretion defects
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31 year old womanPlatelet count 100x109/lCutaneous bruisingProlonged bleeding (minor wounds and after tooth extraction)MenorrhagiaPostpartum hemorrhageSpontaneous muscle haematoma
Platelet transfusionAntifibrinolyticsUterine packing after postpartum haemorrhageRed blood cell transfusionsIron Therapy
Index Case : Family A IV:4
Bleeding disproportionate for platelet countSignificant family history with several similarly affected individuals
Clinical features of index case
IV:5IV:2IV:1
III:2 III:3
IV:4
II:1
? ?
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Multiple affected individuals
Reduced platelet count in multiple family members
Dominant inheritance patternII:1
? ?
III:2140x109/l
III:374x109/l
IV:2110x109/l
IV:4100x109/l
IV:5116x109/l
Normal platelet count: 150-450x109/l
Large family with inherited thrombocytopenia
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III:2140x109/l
III:374x109/l
IV:2110x109/l
IV:4100x109/l
IV:5116x109/l
II:1
WholeExomeSequencing
? ?
Whole Exome Sequencing – 3 affected patients
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Whole Exome Sequencing – 3 affected patients of family A
• novel = not in dbSNP 134, 1000 genomes project, in-house 600 exomes
Patient 1 Patient 2 Patient 3
Total variants 22,867 23,334 23,153
*Novel or Rare variants 124 137 128
Overlapping variants 8
minus synonymous variants 4
Segregation analysis (5 patients and 1 unaffected) 2
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III:2 III:3
IV:2 IV:4 IV:5
II:1
? ?
Patient GenotypesVariant III:3 IV:4 IV:2 III:2 IV:5 IV:1NEMF
p.H962Y C/T C/T C/T C/T C/T C/C
SLFN14p.V220D T/A T/A T/A T/A T/A T/T
IV:1
SangerSequencing
WholeExomeSequencing
• The candidates for disease causing variants were reduced to NEMF and SLFN14
• WES data from 100+ patients recruited to the GAPP study searched for SLFN14 / NEMF variants
• Identified 2 unrelated families with SLFN14 variants
Potential disease causing variants
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Family A Family B Family C
*
* *
protein p.V220D
nucleotide c.659 T>A
protein p.K219N
nucleotide c.657 A>T
protein p.K218E
nucleotide c.652 A>G
*
*
*
I
II
III
IV
1 2
1 2 3
1 2 3 4
1 2 3 4 5 6
I
II
III
I
II
1 2
1 2 3 4
1 2 3
1 2
1 2
??
?
A
-/- +/- +/- +/- +/- +/-
+/-+/-
+/-+/- +/-
+/-
+/--/-
-/-
3 unrelated families each with a heterozygous SLFN14 variant
Identification of SLFN14 candidate gene
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cv
cv
Platelet AggregationPAR1 (100µm) ADP (10µm)
cv
Collagen (3µg/ml) AA (1mM)
cv
Platelet ATP Secretion
PAR1 (100µm)
These patients show platelet dysfunction in addition to thrombocytopenia
Med
ian
CD
62-P
flu
ores
cenc
e In
tens
ity
(FL1
-H)
Flow Cytometry –Surface P-selectin expression
Family A – Patient IV:4
SLFN14 patient platelet phenotyping
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1 206 335 568 707 738 835 992ATPase-AAA-4
P-loop-NTPase
Family A – SLFN14 V220D Family B – SLFN14 K219N Family C – SLFN14 K218E
3 consecutive mutations identified in the AAA-4 ATPase domain of SLFN14 in affected individuals from 3 unrelated families
SLFN14 protein and mutations
Fletcher S…….Morgan NV J Clin Invest 2015; 125(9):3600-05
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Neumann et al., BBRC 2008, 370, 62-66.
The SLFN protein family
• “Slfn” box unique motif
• AAA domain (ATP/GTPbinding) – DNA helicases, chaperone molecules, transcription regulators(J. Wang, JSB 2004,148,259-267)
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The SLFN protein family
Neumann et al., BBRC 2008, 370, 62-66.
• “Slfn” box unique motif
• AAA domain (ATP/GTPbinding) – DNA helicases, chaperone molecules, transcription regulators(J. Wang, JSB 2004,148,259-267)
• “SWADL” domain (Ser-Trp-Ala-Asp-Leu)
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The Schlafen protein family• “Slfn” box unique motif• AAA domain (GTP/ATP binding)
• “SWADL” domain • (Ser-Trp-Ala-Asp-Leu)
• C-terminal extensions in • group III - homologous • to superfamily I of • DNA/RNA helicases
• Subgroups I and II localise to • cytoplasm • Subgroup III localise to nucleus
Putative roles in cell growth, haematopoietic cell differentiation, T cell development/maturationSLFN14 function unknown???
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10μm
SLFN14 patient platelets have significantly decreased SLFN14 protein levels
SLF
N14
pro
tein
exp
ress
ion
in
plat
elet
s (%
of c
ontro
l )
anti-SLFN14104kDa
anti GAPDH37kDa
SLFN14 protein expression in patient platelets
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10μm
δδ
δ
δδ
δ
δδ
δ
δ
δ
δδ
δ
δ
δ
δ
Control A Family B II:3 Family A IV:4
SLFN14 patient platelets have reduced dense granulesReduced dense granules may explain their defective secretory phenotype
Examination of δ-granules in SLFN14 patient platelets
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10μm
SLFN14 patient platelets displaying no difference in α-granules
Examination of α-granules in SLFN14 patient platelets
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10μm
• Identified 3 mutations in the gene SLFN14 in 12 patients with inherited thrombocytopenia from three unrelated families
• All mutations were consecutive and localised to a GTP/ATP binding domain
• All affected patient platelets displayed nearly identical aggregation and secretion defects
• Patient platelets displayed decreased endogenous SLFN14 protein relative to healthy control platelets
• A significant reduction of dense granules was observed in SLFN14 patient platelets
Key findings
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Proplatelet formation PlateletsMaturationEndomitosis
• Which stage does SLFN14 defects affect megakaryocytes?
• Need to investigate formation megakaryocytes in patients?
• Patients have few megakaryocytes in whole blood - bone marrow?
• Do patients have increased platelet clearance?
SLFN14 and unanswered questions?
Megakaryocytes
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AcknowledgementsDr Sarah FletcherMr Ben Johnson
Mr Abdullah KhanMiss Annabel Maclachlan
Dr Gillian LoweProf Steve Watson
Birmingham Platelet GroupGAPP study group
Prof Michael Simpson