Some myelodysplastic syndrome (MDS) patients present with macrothrombocytopenia due to impaired megakaryocyte (MK) differentiation. Transgenic mice that express the NUP98-HOXD13 (NHD13) fusion gene is a model for MDS and recapitulates the key features of MDS. The study investigated the hypothesis that expression of NHD13 disrupts actin architecture during MK differentiation leading to macrothrombocytopenia. To test the hypothesis, sternums were stained with hematoxylin and eosin, and evaluated by light microscopy to analyze MK morphology in vivo. NHD13 bone marrow (BM) contained many dysplastic MK. BM from wild type (WT) and NHD13 mice were also flushed, cultured in media supplemented with thrombopoietin only or with estrogen to induce proplatelet formation, and MK harvested after 5 days. Harvested MK and BM cores were processed and analyzed by transmission electron microscopy (TEM) to detail the ultrastructural features. TEM of MK revealed that NHD13 leads to formation of an irregular demarcation membrane system and fewer proplatelets. Cultured WT and NHD13 MK were also cytospun onto glass slides, labeled with fluorescent-tagged F-actin, α/β-tubulin and myosin IIa, and their cytoskeleton compared. Interestingly WT MK had actin either distributed evenly or predominantly in the periphery of the cytoplasm, NHD13 MK displayed only the former phenotype. Additionally, proplatelets lacked actin cytoplasmic extensions. The results from the present thesis demonstrate actin expression and architecture are impaired in dysplastic MK expressing the NHD13 leukemic fusion gene and leads to macrothromcytopenia. Understanding the molecular mechanisms of abnormal MK differentiation in MDS is important as many MDS patients die of hemorrhagic complications. / Master of Science

Background: Bernard Soulier Syndrome (BSS) is a rare, autosomal recessive inheritance disorder of platelet function. Estimated to affect one per one million, there are currently only 200 cases reported worldwide presenting more commonly in families with parental consanguinity. This syndrome occurs when there is a genetic defect in the subunits (GPIb-alpha, GPIB-beta, and GP9) that form the GPIb-IX-V complex. The result is inadequate binding to von Willebrand factor. The clotting cascade is, therefore, unable to begin, causing symptoms of excessive and prolonged bleeding. Objectives: We report a case with multiple episodes of exaggerated bleeding and easy bruising. Methods: We analyzed complete blood count, coagulation studies, platelet aggregation assays, platelet glycoprotein expression by flow cytometry, as well as screened both patient and parents for relevant genes responsible for BSS. Results: 14-month-old Caucasian male born at 38w3d gestational age, non-consanguineous parents with multiple episodes of exaggerated bleeding and easy bruising from minor injuries. His symptoms started early in life with excessive bleeding after circumcision. No history of intramuscular, joint, or intracranial bleeding. Complete blood counts showed macrothrombocytopenia (98 X109 /L MPV 12.3 fl) no leukocyte inclusion bodies on peripheral smear. Coagulation tests (prothrombin time, activated partial thromboplastin time, vWF antigen, and vW-Ristocetin cofactor activity, platelet function assay) were normal. Platelet glycoprotein expression by flow cytometry revealed significantly reduced binding of monoclonal antibodies to platelet GPIb and normal GPIIb/IIIa. Comprehensive platelet disorder panel revealed two clinically significant variants missense mutations in the GP9 gene (P.Cys135 Tyr and P.Asn61Ser) These variants were on opposite alleles and results were consistent with the diagnosis of Bernard Soulier syndrome (BSS). The mother reported heavy menstrual cycles, the father had no significant bleeding symptoms, and both parents had normal platelet counts. Target genetic testing identified these two distinct missense mutations from both Mother and Father of the child. Conclusion: The two rare variants occurring on the gene for GPIX (GP9) increase the number of known genetic defects associated with the manifestation of Bernard Soulier Syndrome.

Inherited thrombocytopenias, including inherited giant platelet disorders (IGPD) or macro thrombocytopenias are relatively rare, but their prevalence is likely underestimated from complexities of diagnosis and a spectrum of subclinical phenotypes. Harris platelet syndrome (HPS) is the most common IGPD reported from the Indian subcontinent. Of note there are an increased number of hemoglobinopathies reported from the geographic location. We analysed red blood cell and platelet indices of blood donors with HPS from the north eastern part of India and compared them with blood indices of blood donors of south India. We found a statistically significant lower platelet count in blood donors with HPS (median, range) 132 (71-267) vs. 252 (160-478) as compared to donors from south India (P < 0.001). Mean platelet volume (MPV) was higher in donors with HPS 13.1, (range 12-21.9 fl) as compared to donors from south India 7.35 (range 6-9.2 fl) (P < 0.001). This study showed that blood donors with HPS had a low median platelet bio-mass 0.17 (0.10-0.38%) vs. 0.19 (0.13-0.28%) in donors from south India. The platelet distribution width (PDW) was 17.4 (14.9-19.6) in donors with HPS vs. 16.38 (15.2-18.5) in south Indian blood donors (P < 0.001). Thirty-three donors with HPS had a normal platelet count with MPV more than 12 fL. Only donors with HPS had giant platelets and thrombocytopenia on peripheral blood smear examination. None of these donors had Dohle body inclusion in their leukocytes. Compared to donors from south India, donors with HPS had a significantly lower hemoglobin 13.8 (12-16.3 gm/dL) vs. 14.8 (12-18) respectively (P < 0.001) while red distribution width (RDW) was higher in HPS 13.6 (11.5-16.7) vs. 12.8 (11.4-15.1). However we did not find any statistically significant difference in MCV, MCH, MCHC between the two groups. Peripheral blood smear did not show any obvious abnormal red blood cell morphology. In the blood donors with HPS we found a statistically higher MPV, RDW and a lower platelet count and platelet biomass. A population-based study will be helpful in determining the existence of any hemoglobinopathies among subjects with HPS.

Sitosterolemia (STSL) is a sterol storage disorder characterized by very high plasma plant sterol (PS) and 5α-stanol levels, and leads to premature atherosclerosis, xanthomas, macrothrombocytopenia and endocrine disruption. Ezetimibe (EZE), a sterol absorption inhibitor, reduces plasma PS levels in STSL but its effect on tissue pool of sterols has not been investigated yet. The research objectives were to assess if EZE reduces whole body sitosterol and cholesterol pool sizes, improves cholesterol homeostasis, enhance hematologic profile and reduce endocrine disruption in STSL. EZE effects on circulating levels of cholestanol and its precursors (cholesterol and bile acid derivative 7α-hydroxy-4-cholesten-3-one, 7α-H-C4) relative to exogenous stanols (sitostanol) were also studied. Eight STSL patients were taken off EZE for 14 wks. After 4 wks off EZE they received intravenous doses of D7-sitosterol and 18O-cholesterol for sterol pool sizes assessments, and oral doses of 13C-cholesterol and deuterium oxide to measure fractional cholesterol absorption and synthesis rates. EZE (10 mg/d) was resumed and stable isotopes testing repeated. Measurement parameters included isotopic sterol enrichments, blood cell count, plasma and red blood cell (RBC) PS, cholesterol and its precursor (lathosterol), 5α-stanols and plasma 7α-H-C4, and thyroid hormones levels. EZE reduced plasma levels of sitosterol and total cholesterol, whole body sitosterol and cholesterol pool sizes and fractional cholesterol absorption rate while increasing cholesterol synthesis, production and clearance rates. EZE increased platelet count and decreased platelet size without affecting RBC indices of size or mass. A substantial decrease in circulating sitostanol but moderate decrease of cholestanol was noted with EZE. EZE increased lathosterol but not 7α-H-C4, suggesting increases in cholesterol biosynthesis and thus precursor availability for synthesis of cholestanol. In summary, EZE reduces body stores of PS and cholesterol, and increases cholesterol turnover by reducing cholesterol absorption and enhancing its synthesis and clearance. EZE reduces circulating PS and 5α-stanol levels, and improves macrothrombocytopenia and thyroid disruption. Endogenous cholestanol in STSL is mainly derived from cholesterol but not bile acid synthesis pathway. These data suggest that EZE may reduce the risks of developing premature atherosclerosis, bleeding and hormone disruption, thereby reinforcing the rationale for the use of EZE in treatment of STSL. / February 2015