1. Thrombocytopenia

2. Platelet Counts Vary with – Age (slight decrease in mean with increased age) – Sex (Slightly lower mean in men)

3. Consequences of Misdiagnosis of Inherited Thrombocytopenia Wrong therapy Wrong prognosis Missed opportunity for genetic counseling

4. Inherited Thrombocytopenias with Adulthood Presentation Italian Experience Mean age of diagnosis – 35 yrsMYH9-related disease – 33 yrsmonoallelic Bernard-Soulier syndrome – 30 yrsANKRD26-related thrombocytopenia Baluini CL et al. JTH. 2013;11:1006-1019

5. Inherited Thrombocytopenias with Adulthood Presentation – MYH9-related disease – monoallelic Bernard-Soulier syndrome – ANKRD26-related thrombocytopenia – Familial platelet disorder with predisposition to AML (RUNX1) – ITGA2B/ITGB3-associated thrombocytopenia – TUBB1-related thrombocytopenia – CYCS-related thrombocytopenia – Thrombocytopenia associated with sitosterolemia* Red=syndromic vs. Blue=non-syndromic *Autosomal recessive (all others dominant)

6. MYH9-RELATED DISEASE

7. MHY9-Related Disease MHY9 gene at chromosome 22q12-13 Autosomal dominant Non-syndromic  syndromic over time – Cataracts, sensorineural deafness, altered liver enzymes, and/or progressive glomerulonephropathy (ESRD) Associated features – Giant platelets No spontaneous bleeding

8. MYH9 Gene Encodes heavy chain of non-muscle myosin IIA – aka. Myosin 9 Non-muscle myosin IIA – Motor protein involved in cellular force contraction N-terminal motor domain – Mechanical translocation along actin filaments – Mutations here  severe thrombocytopenia and full syndrome Alpha-helical rod domain – Intermediate phenotype C-terminal non-helical domain – Mildest phenotype with only mild thrombocytopenia

9. MHY9-Related Disease: Pathophysiology Disease phenotype – Caused by dominant-negative effect of the mutated myosin-9 – NOT caused by the loss of myosin-9 function

10. MHY9-Related Disease: Pathophysiology Non-hematologic manifestions  uncertain Thrombocytopenia – Reduced number and branching of proplatelets Leads to fewer number of release platelets from megakaryocytes – Impaired migration of megakaryocytes from the osteoblastic niche to the vascular niche – Premature release of platelets away from the vascular sinusoids

11. MYH9-Related Disease Journal of Thrombosis and Haemostasis Volume 11, Issue 6, pages 1006-1019, 3 JUL 2013 DOI: 10.1111/jth.12196 http://onlinelibrary.wiley.com/doi/10.1111/jth.12196/full#jth12196-fig-0003 Volume 11, Issue 6, http://onlinelibrary.wiley.com/doi/10.1111/jth.12196/full#jth12196-fig-0003 Mutations in which domain(s) is/are most likely associated with this phenotypic morphology?

12. MHY9-Related Disease: Diagnosis Anti-myosin-9 antibodies – Used to identify the inclusion bodies Present in all neutrophils of all affected patients

13. MONOALLELIC BERNARD-SOULIER SYNDROME

14. Benard-Soulier Syndrome Genes Associated: – GP1BA gene at chromosome 17p13 – GB1BB gene at chromosome 22q11 – GP9 at chromosome 3q21

15. Glycoprotein Ib-IXA-V Receptor

16. SFKs in GPIb-IX-V proximal signaling. Yotis A. Senis et al. Blood 2014;124:2013-2024 ©2014 by American Society of Hematology

17. Biallelic Benard-Soulier Syndrome Autosomal recessive (>50 mutations described) Genes mutations in BOTH alleles of either – GP1BA gene at chromosome 17p13 – GB1BB gene at chromosome 22q11 – GP9 at chromosome 3q21 Non-syndromic Associated features – Giant platelets Spontaneous bleeding

18. Biallelic Benard-Soulier Syndrome BOTH alleles must be mutated – Heterozygotes Slightly enlarged platelets OR No phenotypic abnormality

19. Biallelic Benard-Soulier Syndrome: Pathophysiology Mutations prevent receptor assembly in the Golgi apparatus and migration to surface – Absence of GP 1B-IXA-V receptor – Decreased vWF binding – Decreased platelets adhesion at site of vascular injury Thrombocytopenia with giant platelets BUT – Bleeding is worse than the platelet numbers would suggest

20. Biallelic Benard-Soulier Syndrome: Pathophysiology Mutations prevent receptor assembly in the Golgi apparatus and migration to surface – Absence of GP 1B-IXA-V receptor – Decreased vWF binding – Decreased platelets adhesion at site of vascular injury Thrombocytopenia with giant platelets BUT – Bleeding is worse than the platelet numbers would suggest

21. Biallelic Benard-Soulier Syndrome: Diagnosis PFA-100 – Both collagen and ADP prolonged Platelet aggregation assays – Decreased aggregation with Ristocetin Flow cytometry – Absence of GP IB-IXA-V

22. Monoalleleic Bernard-Soulier Syndrome Autosomal dominant Genes Associated: – GP1BA gene at chromosome 17p13 – GB1BB gene at chromosome 22q11 – GP9 at chromosome 3q21 Non-syndromic Associated features – Giant platelets with GP1BA mutations – Large platelets with GP1BB and GP9 mutations No spontaneous bleeding

23. Monoalleleic Bernard-Soulier Syndrome Autosomal dominant – Far fewer mutations described Genes mutations in ONE allele of either – GP1BA gene at chromosome 17p13 – GB1BB gene at chromosome 22q11 Non-syndromic Associated features – Large to Giant platelets No spontaneous bleeding

24. Monoallelic Benard-Soulier Syndrome: Clinical Features Thrombocytopenia with giant platelets – Mean platelet count 81 x 10 9 /L No or trivial bleeding tendency usually – Less commonly more severe thrombocytopenia and bleeding seen 5% of cases in one series had been treated for ITP

25. Monoallelic Benard-Soulier Syndrome: Pathophysiology WHY monoallelic forms  uncertain – DiGeorge’s syndrome (22q11 microdeletion) has 50% expression but no giant plaletets or thrombocytopenia – Hypothesis Mutations must exert some dominant effect ?

26. Bernard-Soulier Syndrome: Why Macrothrombocytopenia? GP IB-IXA-V needed for platelet production – Inhibition blocks proplatelet formation Proplatelet tips are larger  -tubulin distribution altered – NO effect on differentiation or maturation

27. ANKRD26-RELATED THROMBOCYTOPENIA

28. ANKRD26-Related Thrombocytopenia Gene associated – ANKRD26 gene at chromosome 10p2 Autosomal dominant Non-syndromic Associated features – Normal size platelets – Increase risk of acute leukemia (?) No spontaneous bleeding

29. ANKRD26-related thrombocytopenia: Pathophysiology Mutations in the 5’-UTR of ANKRD26 on 10p2 How leads to disease physiology – uncertain – Possibly enhance expression of ANKRD26 Deletions and partial inactivations do not lead to thrombocytopenia – Bone marrow examinations Suggest possible dysmegakaryopoiesis Many smaller or dystrophic megakaryocytes – Hypolobulated nuclei

30. ANKRD26-related thrombocytopenia: Clinical Features Thrombocytopenia with normal size platelets – Mean platelet count 47 x 10 9 /L No or mild bleeding tendency usually Less commonly severe thrombocytopenia (<10 x 10 9 /L)

31. ANKRD26-Related Thrombocytopenia Journal of Thrombosis and Haemostasis Volume 11, Issue 6, pages 1006-1019, 3 JUL 2013 DOI: 10.1111/jth.12196 http://onlinelibrary.wiley.com/doi/10.1111/jth.12196/full#jth12196-fig-0004 Volume 11, Issue 6, http://onlinelibrary.wiley.com/doi/10.1111/jth.12196/full#jth12196-fig-0004

32. ANKRD26-related thrombocytopenia: Clinical Features 30-fold increase in acute leukemia (?) – Compared with the normal population

33. FAMILIAL PLATELET DISORDER AND PREDISPOSITION TO ACUTE MYELOGENOUS LEUKEMIA

34. Familial Platelet Disorder and Predisposition to AML RUNX1 gene at chromosome 21q22 Autosomal dominant Non-syndromic Associated features – Normal-size platelets – HIGH risk of AML and MDS No spontaneous bleeding

35. Familial Platelet Disorder and Predisposition to AML: Pathophysiology RUNX1 gene at chromosome 21q22 – Encodes for RUNX1 (aka CBF  2) Core Binding Factor – Heterodimeric transcription complex CBF  2 important for dimerization and DNA binding CBF  helps stabilize CBF  2 – Protecting it from degradation – Enhancing its DNA affinity Mutations usually lead to haploinsufficiency – Some dominant negative mutations also reported Even higher risk of hematologic malignancy

36. Familial Platelet Disorder and Predisposition to AML: Pathophysiology RUNX1 gene at chromosome 21q22 – Encodes for RUNX1 (aka CBF  2) Core Binding Factor – Heterodimeric transcription complex CBF  2 important for dimerization and DNA binding CBF  helps stabilize CBF  2 – Protecting it from degradation – Enhancing its DNA affinity – Regulates expression of many hematopoietic-specific genes Inversely regulates MYL9 and MYH10 Directly regulates MYH9 Thus suggests megakaryopoiesis may be effected in part by deregulation of myosin IIA and IIb  defective proplatelet formation Mutations usually lead to haploinsufficiency – Some dominant negative mutations also reported Even higher risk of hematologic malignancy

37. Familial Platelet Disorder and Predisposition to AML: Clinical Features Thrombocytopenia with normal size platelets – Platelets may look a little paler – Mild thrombocytopenia Reduced platelet aggregation with collagen and epi – Platelet counts NOT ALWAYS reduced Uncertain if also have qualitative platelet defects No or mild bleeding tendency usually Easy bruising since childhood in some but not all – Hemoglobin and WBC may be elevated

38. Familial Platelet Disorder and Predisposition to AML: Clinical Features Increased risk of hematologic malignancies – ESPECIALLY MDS and AML 40% of affected individual Mean age of onset 33 years

39. Inherited thrombocytopenias frequently diagnosed in adults Journal of Thrombosis and Haemostasis Volume 11, Issue 6, pages 1006-1019, 3 JUL 2013 DOI: 10.1111/jth.12196 http://onlinelibrary.wiley.com/doi/10.1111/jth.12196/full#jth12196-fig-0005 Volume 11, Issue 6, http://onlinelibrary.wiley.com/doi/10.1111/jth.12196/full#jth12196-fig-0005