ISTH Training CourseNew genes that cause thrombocytopenia

Dr Neil Morgan, University of Birmingham (UK)

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

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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

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

? ?

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

III:2140x109/l

III:374x109/l

IV:2110x109/l

IV:4100x109/l

IV:5116x109/l

II:1

WholeExomeSequencing

? ?

Whole Exome Sequencing – 3 affected patients

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

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

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

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

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

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)

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)

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???

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

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

10μm

SLFN14 patient platelets displaying no difference in α-granules

Examination of α-granules in SLFN14 patient platelets

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

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

AcknowledgementsDr Sarah FletcherMr Ben Johnson

Mr Abdullah KhanMiss Annabel Maclachlan

Dr Gillian LoweProf Steve Watson

Birmingham Platelet GroupGAPP study group

Prof Michael Simpson