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Thrombocytopenia: an Australasian Perspective ISTH Advanced Training Course 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: <N/A> ISTH Advanced Training Course Dubai, UAE - 2 - Outline . Introduction . Traditional phenotypic testing approach . Genetic testing – rationale . Our experience using a candidate gene panel . Observations from inherited platelet disorders caused by transcription factor mutation. 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 mutations. 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 genes 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 Cytoskeleton 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 protein 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
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