Genetic Testing in Inherited Thrombocytopenia: An Australasian Perspective ISTH Advanced Training Course

Presented By: Dr David Rabbolini 7th September 2016

ISTH Advanced Training Course Dubai, UAE Disclosures for David Rabbolini 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 No relevant conflicts of interest to declare Board

Presentation includes discussion of the following off-label use of a drug or medical device:

ISTH Advanced Training Course Dubai, UAE - 2 - Outline

. Introduction

. Traditional phenotypic testing approach

. Genetic testing – rationale

. Our experience using a candidate panel

. Observations from inherited platelet disorders caused by transcription factor .

. Concluding remarks

ISTH Advanced Training Course Dubai, UAE - 3 - Inherited platelet disorders

. Uncommon conditions . True prevalence is likely underestimated

. Under recognised . Many lack a preceding family history . MYH9-RDs – 20-30% de novo .

. Variable bleeding tendencies.

. Not all present in childhood.

Savoia A., et al., Journal of Thrombosis and ISTHHaemostasis Advanced Training, 2010. Course Balduini CL., et al., Journal of Thrombosis and HaemostasisDubai, UAE , 2013. Diagnosis is of importance . Prevent potentially futile and harmful treatments . Many inherited thrombocytopenias are diagnosed as ITP ~20%

. Predisposition to other illnesses . RUNX1 (FPD/AML) – Acute myelid leukaemia. . ETV6 and EVI1 – Solid organ and haematological malignancies. . MYH9-RDs – renal failure, cataracts, sensorineural deafness.

. Provide appropriate perioperative strategies

. Genetic counseling

Novelli EM., et al., Seminars in Thrombosis andISTH HaemostasisAdvanced Training ,Course 2008. Balduini CL., et al., British Journal of Haematology, 2011.Dubai, UAE Phenotypic approach to diagnosis

Macrothrombocytopenia Inclusions No inclusions

Light transmission aggregometry Non-specific -MultiImmunofluorescence- step - Sometimes not pattern possible (thrombocytopenia) - Availability Clusters of NMMIIA Flow cytometry β1 -Tubulin -RT α -Actinin- RT Monoallelic BSS MYH9-RD VWDIIB/ ITGA2B/ TCPT/ JBS PT-VWD ITGB3- RT Filamin A-RT

ISTH Advanced Training Course Rabbolini DJ., et al, Semin. Thrombosis and Hemostasis,Dubai, UAE 2014. Phenotypic approach to diagnosis

Anisopoikilocytosis Gray platelets

LTA: LTA: LTA: Non-specific ADP, AA Variable: ADP, Non-specific and collagen collagen, thrombin ⬇ ⬇

Complex to perform EM

GATA1-RT GFI1B-RT GPS

ISTH Advanced Training Course Rabbolini DJ., et al,, Semin. Thrombosis and Hemostasis,Dubai, UAE 2014. The diagnostic utility of candidate gene array in cases of uncharacterised macrothrombocytopenia

. Effective analysis of all the candidate by Sanger sequencing was not feasible.

. Good gene coverage and representation.

. Focussed approach minimises the problems with unexpected findings and developing downstream pipelines for analysis

. Fast

. Relatively cheap

ISTH Advanced Training Course Sikkema-Raddatz B, Human Dubai,Mutation, UAE 2013 Our approach at the Northern Blood Research Centre

Initial list of Literature genes (n=173)

Updated macrothrombocytopenia Candidate with list (n=19) subsequent + runs (n=32)

Exclusion Panel design NGS, genes with distinct (Illumina analysis and clinical Design reporting phenotypes studio) variants

ISTH Advanced Training Course Dubai, UAE Candidate gene identification and gene panel design

. Panel (1)  19 genes

ITGA2 GP1BA ITGB1 CD36 GP6 ITGA2B MPL ITGB3 GPIV TBXA2R GP1BB

P2RY12

GP9

GFI1B FLI1 α granules GATA1 ACTN1 RUNX1 ETS1 MYH9 BCL2L1 F2R TUBB1 NBEAL2

Receptors Transcription factors Granule defects Undefined Intracellular

ISTH Advanced Training Course Dubai, UAE Candidate gene identification and gene panel design

Panel (2)  27 genes

ITGA2 GP1BA ITGB1 CD36 GP6 ITGA2B MPL ITGB3 GPIV TBXA2R GP1BB

P2RY12

GP9

GFI1B FLI1, ETS1 α granules GATA1 and GATA2 RUNX1 and ZFPM1 ANKRD26 F2R BCL2L1 ACTN1 BCL2L1 NBEAL2 MEIS1 MYH9 PRKACG NFE2 TUBB1 RASGRP2 TPM4 TUBA4A Receptors Cytoskeleton Transcription factors Granule defects Undefined Intracellular ISTH Advanced Training Course Dubai, UAE Candidate gene identification and gene panel design Panel (3)  32 genes

ITGA2 GP1BA ITGB1 CD36 GP6 ITGA2B MPL ITGB3 GPIV TBXA2R GP1BB PTGS1 P2RY12

GP9

GFI1B FLI1, ETS1 ANKRD26 α granules GATA1 and GATA2 RUNX1 and ZFPM1 BCL2L1 F2R SLFN14 BCL2L1 ACTN1 PRKACG CHST14 NBEAL2 MEIS1 MYH9 RASGRP2 NFE2 VIPAS39 TUBB1 FYB TPM4 RGS18 VPS33B TUBA4A RGS2 ETV6 Receptors Cytoskeleton Transcription factors Granule defects Undefined Intracellular ISTH Advanced Training Course Dubai, UAE Illumina MiSeq platform and workflow

gDNA

. Assembled sequences were aligned to the reference genome (GRCh37/hg19) DNA enrichment . Variant calls were generated using ANNOVAR software. Library preparation . Genomic datasets were viewed using the Integrative Genomics Browser (IGV) NGS run . Sanger sequencing

Data analysis ISTH Advanced Training Course Dubai, UAE Illumina MiSeq platform and workflow - 2

. Data was analysed using genome browsers (UCSC). Results were cross checked against databases . NHLBI-extended sequencing project. . 1000 genomes project. . Database of single-nucleotide polymorphisms (dbSNP)

. Bioinformatic tools (SIFT, PolyPhen-2 and Mutation Taster) were used to predict effects on structure in the cases of variants lacking published literature MYH9, heterozygous, c.2104C>T (Arg702Cys), exon17, rs80338826, Pathogenic

ISTH Advanced Training Course Dubai, UAE Cumulative candidate gene panel results

. Number of individuals tested n=140

. Pathogenic variants 33 individuals (23.6%)

. Variants of uncertain significance 60 individuals (42.9%)

. Nil pathogenic 33.6%

ISTH Advanced Training Course Dubai, UAE Transcription factors act in a combinatorial manner priming and activating lineage restricted genes

ISTH Advanced Training Course Pimkin M., et al, Genome Dubai,Research, UAE 2014. Transcription factors in inherited Thrombocytopenia: Observation # 1

. Mutations in transcription factors RUNX1, GATA1, GFI1B, FLI1 and ETV6 share common features including a variable bleeding history often associated with abnormal but nonspecific changes in platelet morphology and platelet function testing

ISTH Advanced Training Course Dubai, UAE Transcription factors affect multiple target genes – This may cause a complex platelet phenotype

RUNX1

CBF complex CBFα CBFβ

N RHD TAD C

MYH10 MPL MYL9 MYH9 CBFα TUBB1 &2 PKC- theta IIbIIIa

Cohen MM Jr., et al., Am J Med Genet A, 2009. Heller PG., et al., Blood, 2005. Sun L., et al., Blood, 2004. Bluteau D., et al., Blood. ISTH Advanced Training Course Antony-Debre DNAI et al., Blood, 2012. Dubai, UAE RUNX1 platelet Phenotype Patients with mild to moderate bleeding usually present from childhood (variable).

(A) LTA . Reduced response to several platelet agonists (ADP, epinephrine, AA, collagen, TRAP).

Flow cytometry . Deficiency of platelet dense granules with reduced uptake and release of mepacrine.

Thrombocytopenia with normal sized platelets Ho CY, et al., Blood, 1996. Dowton SB, et al., Blood, 1995. Song WJ, et al., Nature genetics, 1999. Buijs A, et al., Blood, 2001. Beri-dexheimer M, et al., Eur J Hum Genet, 2008.ISTH Advanced Training Course Gerrard JM, et al., Leuk Lymphoma, 1992. Dubai, UAE FLI1 acts at the promoters of multiple platelet specific genes- mutation disrupts their function

GP6 GP9 ITGA2B

35 8 16 30 ** 12 ** 25 6 20 ** 8 4 15 4 10 2 5 0

Fold in change luciferase activity 0 0 Empty R324W Empty WT R324W

GPVI GPIbIX GPIIb 120% 120% 120% 100% 100% 100% 80% 80% 80% ** * 60% * 60% 60% ** 40% * 40% 40% 20% 20% 20% Protein content Protein content 0% 0% 0%

. Arg324 dose not directly bind DNA. . DNA binding is altered via the disturbed interaction with the N-terminal autoinhibitory Domain. . The change alters the transition of the ISTH Advanced Training Course Protein between a folded and unfolded state. Stevenson WS., et Dubai,al., Blood, UAE 2015. FLI1 platelet phenotype - R324W Individuals presented with moderate to severe bleeding. Most significant post-operatively

LTA

1 µg/ml Collagen 10 µM ADP 0.5 mg/ml AA 110 µM Epinephrine 1.5 mg/ml Ristocetin

. Impaired aggregation to ADP and epinephrine.

. No aggregation to collagen

. Normal aggregation to AA, TRAP (not shown) and agglutination to Ristocetin. . . Macrothrombocytopenia Normal platelet mepacrine uptake and release. . Platelets with giant fused . α-granules

ISTH Advanced Training Course Stevenson WS., et Dubai,al., Blood, UAE 2015. Transcription factors in inherited thrombocytopenia: Observation # 2

. The phenotype of the underlying platelet disorder is often variable despite mutations in the same transcription factor suggesting that the site of mutation and the protein domain that is perturbed is an important determinant of the clinical syndrome

ISTH Advanced Training Course Dubai, UAE The site of mutation and the protein domain that is perturbed is an important determinant of the clinical syndrome GATA1 Transactivation domain Zinc finger domain 83 204 228 258 282 414 N N-f C-f C

X V205M, G208R, G208S, D218G, D218Y XLTDA GATA1 -Zf X DNA R216Q XLTT

Balduini CL, et al., Thrombosis andISTH HaemostasisAdvanced Training ,Course 2004. Ciovacco WA, et al., Gene, 2008. Dubai, UAE GATA1 genotype- phenotype relationships

X V205M, G208R, G208S, D218G, D218Y XLTDA GATA1 -Zf

. GATA1 – X-linked thrombocytopenia Symptoms: Moderate to severe bleeding from infancy. Blood film: Moderate to severe macrothrombocytopenia Pale platelets – reduced alpha-granules Other: Red cell anisopoikilocytosis and anaemia Bilateral cryptoorchidism (V205M and G208R) Key observation: The greater the destabilisation of GATA1: FOG1 interaction  the more severe the

phenotype. ISTH Advanced Training Course Ciovacco WA, et Dubai,al., Gene, UAE 2008. GATA1 genotype- phenotype relationships

GATA1 -Zf X R216Q XLTT

. GATA1 X-linked thrombocytopenia with thalassaemia Symptoms: mild to moderate bleeding (from childhood) Blood film: Mild to moderate macrothrombocytopenia Pale platelets with reduced alpha-granules Other: Unabalanced α:β haemoglobin chain production resembling mild β- thalassaemia. Mild haemolytic anaemia Splenomegaly

Balduini CL, et al., Thrombosis andISTH HaemostasisAdvanced Training ,Course 2004. Ciovacco WA, et al., Gene, 2008. Dubai, UAE GFI1B genotype- phenotype observations

I (A) (B) 78 B

II 121 249 83 1 2 883 4 6 249

III C168F x2 185 121 91 (C) 1 2 3 2294 2505 215 H294fsX307 185 121 91 229 250 215 GFI1B 1 2 3 4 5 6 (C) DNA binding

1-20 21-163 164-330 amino acids

Stevenson WS, JTH, 2013. Monteferrario D, NEJM, 2014.ISTH Advanced Training Course Chen, Science, 2014. Dubai, UAE GFI1B genotype- phenotype observations

C168F H294fsX307

GFI1B C168F patients have:

Thrombocytopenia (78-121 x109/L)

But

No red cell anisopoikilocytosis

Normal platelet granule contents

No defect on aggregometry

ISTH Advanced Training Course Dubai, UAE Transcription factors in inherited thrombocytopenia: Observation # 3

. Transcription factor mutations may be associated with other physical abnormalities including an increased risk of acute leukaemia as well as solid-organ malignancies

ISTH Advanced Training Course Dubai, UAE RUNX1 - Familial platelet disorder with propensity to develop AML (FPD/AML)

. Propensity to develop AML/ MDS is (A) (B) dependent on the action of the ?

variant (Dominant negative > Myelofibrosis Haploinsufficient) ~40% ? ? ? “ITP” AML MI AMML . Median age to progression ~ 33 RUNX1 years old.

RUNX1 RUNX1 . Detection: RUNX1, c.C884G (p.S295X). RUNX1, stop/gain, c.766C>T . Referral to a specialist team (p.Gln256X). (clinician and genetic counsellor) . FBC, Bone marrow biopsy, HLA typing . Biannual haematological follow up.

ISTH Advanced Training Course Dubai, UAE Ets variant 6 (ETV6)

. Transcriptional repressor that is important in haematopoiesis . ETV6 modulates the activity of other transcription factors (FLI1) and other partners required for megakaryocyte maturation and platelet production. . ETV6 is commonly translocated in childhood ALL (ETV6-RUNX1) and somatic mutations are described in B-ALL, MDS and T cell leukaemias.

N PNT Central ETS C

Protein:Protein DNA binding interactions Promotes DNA binding, Essential domain for repressive function

Noetzli L., et al., Nature Genet, 2015. Zhang MY., et al., Nature Genet, 2015. ISTH Advanced Training Course Topka S., et al., Plos genetics, 2015. Dubai, UAE Ets variant 6 (ETV6)

Blood film: Moderate thrombocytopenia. LTA: Reduced responses to platelet agonists ADP and AA. Flow cytometry: Normal platelet glycoprotein expression. Other: Red cell macrocytosis Predisposition to solid tumours and haematological malignancy, particularly ALL. • 9 out of 17 (3 leukaemias) – Washington group • 3 out of 10 with leukaemia – Colorado group P214L R369Q, R399C, R418G, 385_418del

N PNT Central ETS C

Protein:Protein DNA binding interactions Promotes DNA binding, Essential domain for repressive function

Noetzli L., et al., Nature Genet, 2015.

Zhang MY., et al., Nature Genet, 2015. ISTH Advanced Training Course Topka S., et al., Plos genetics, 2015. Dubai, UAE Transcription factor mutations account for a significant portion of Inherited thrombocytopenia

Disease Inheritance Gene Inherited Defective MEG maturation thrombocytopenia CAMT AR MPL classified according to TAR AR RBM8A mechanism of defective CTRUS AD HOXA11 platelet production Defective MEG maturation FPD/AML AD CBFA2 (RUNX1) TCPT/ JBS AD/ AR FLI1 GATA1-RT XL GATA1 GFI1B-RT AD GFI1B ANKRD26-RT AD ANKRD26 GPS AR NBEAL2 Defective proplatelet/ plt formation MYH9-RD AD MYH9 ACTN1-RT AD ACTN1 FLNA-RT XL FLNA WAS XL WAS BSS AR GP1BA, GP1BB, GP9 Monoallelic ITGA2B/ITGB3 AD ITGA2B, ITGB3 TUBB1 AD TUBB1

CYCS-RT AD CYCS ISTH Advanced Training Course Pecci A et al., British Journal of HaematologyDubai, UAE , 2014. Diagnostic considerations – The role of genetic testing

. Consider transcription factor mutations causing thrombocyopenia: . Act in a combinatorial manner interacting with each other and other factors . Affect multiple genes important in megakaryopoiesis and platelet production . Platelet phenotype is often complex and non-specific

. Consider platelet phenotypic tests: . Descriminate single pathways or structural characteristics

Bleeding Hx Bleeding Hx Blood film Blood film Platelet aggs. RUNX1 Platelet aggs. Non-specific Flow cytometry Flow cytometry EM EM

Prioritization of genetic testing ISTH Advanced Training Course Dubai, UAE Correlation of genetic results with phenotypic data is essential

Heavy chain Genotype–phenotype MYH9 relationship

Light chains Mutations of the motor domain:

. More Severe thrombocytopenia . Higher risk for the development of nephritis and deafness

Motor domain Tail domain Mutations in the tail region N 2 11 17 31 33 41 C . Less severe thrombocytopenia and systemic complications.

. Total: 21 Variants in 77 individuals . Pathogenic: 7 variants in 16 individuals

Walck-Shannon E., et al., Nat Rev Mol Cell Bio, 2014 Althaus K., et al., Semin. Thrombosis and Hemostasis, 2009.ISTH Advanced Training Course Pecci A., et al., Human Mutation, 2008. Dubai, UAE Lessons learnt by correlating NGS and phenotypic data – MYH9-RDs

. Only 1 individual had a diagnosis of MYH- RD prior to NGS, and NGS confirmed a pathogenic variant in this individual.

. In all other cases a diagnosis of MYH-RD was not suspected by referring clinicians.

. MYH9-RDs: . Thrombocytopenia with giant platelets . Neutrophil inclusions are not always appreciated on blood films. Reliance on their detection may cause missed diagnoses. . IF and/or NGS are satisfactory Top panel: MYH9, c.5770_5779del (p. G1924)fs diagnostic tests. Bottom panel: MYH9, c.G283A (p.A95T) . NGS provides diagnostic and prognostic information and should be considered earlier in diagnostic algorithms. ISTH Advanced Training Course Dubai, UAE - 35 - Conclusions

. Phenotypic testing remains the standard approach to diagnose inherited platelet functional and number disorders. These are multi-step, complex and poorly standardised.

. A genetic based approach using NGS may be an effective diagnostic tool to examine inherited thrombocytopenia. Quality of sequence, coverage and sound analytical and post-analytical pipelines are required.

. Candidate gene panels require optimisation with iterative runs and appraisal of target genes.

. Targeted genotypic screening has the potential to be developed into a regional service. ISTH Advanced Training Course Dubai, UAE Acknowledgements:

. The Northern Blood Centre Research Team . Prof. Christopher Ward . A/Prof. William Stevenson . Dr. Marie-Christine Morel Kopp . Dr. Giles Best . Walter Chen . Sara Gabrielli . Lucinda Beutler Royal North Shore Hospital . All Referring clinicians and participating patients

ISTH Advanced Training Course Dubai, UAE - 37 - ISTH Advanced Training Course Dubai, UAE - 38 - ISTH Advanced Training Course Dubai, UAE - 39 - ISTH Advanced Training Course Dubai, UAE - 40 - Ets variant 6 (ETV6)

. Mutations cause: . Aberrant cellular localisation of mutant and endogenous ETV6. . Decreased transcriptional repression . Altered megakaryocyte maturation.

. Incomplete penetrance of malignancies indicates that other factors are required for oncogenesis. . Little is currently known about what other oncogenic mediators may act in cooperation with mutated ETV6.

ISTH Advanced Training Course Kirkpatrick G., et al., OncotargetDubai, UAE , 2015.