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Satchwell T.J.,University of Bristol | Pellegrin S.,University of Bristol | Bianchi P.,Hematology and Transplantation Unit | Hawley B.R.,University of Bristol | And 9 more authors.
Haematologica | Year: 2013

Congenital dyserythropoietic anemia type II is an autosomally recessive form of hereditary anemia caused by SEC23B gene mutations. Patients exhibit characteristic phenotypes including multinucleate erythroblasts, erythrocytes with hypoglycosylated membrane proteins and an apparent double plasma membrane. Despite ubiquitous expression of SEC23B, the effects of mutations in this gene are confined to the erythroid lineage and the basis of this erythroid specificity remains to be defined. In addition, little is known regarding the stage at which the disparate phenotypes of this disease manifest during erythropoiesis. We employ an in vitro culture system to monitor the appearance of the defining phenotypes associated with congenital dyserythropoietic anemia type II during terminal differentiation of erythroblasts derived from small volumes of patient peripheral blood. Membrane protein hypoglycosylation was detected by the basophilic stage, preceding the onset of multinuclearity in orthochromatic erythroblasts that occurs coincident with the loss of secretory pathway proteins including SEC23A during erythropoiesis. Endoplasmic reticulum remnants were observed in nascent reticulocytes of both diseased and healthy donor cultures but were lost upon further maturation of normal reticulocytes, implicating a defect of ER clearance during reticulocyte maturation in congenital dyserythropoietic anemia type II. We also demonstrate distinct isoform and species-specific expression profiles of SEC23 during terminal erythroid differentiation and identify a prolonged expression of SEC23A in murine erythropoiesis compared to humans. We propose that SEC23A is able to compensate for the absence of SEC23B in mouse erythroblasts, providing a basis for the absence of phenotype within the erythroid lineage of a recently described SEC23B knockout mouse. © 2013 Ferrata Storti Foundation. Source

Boiocchi L.,New York Medical College | Boiocchi L.,University of Milan | Boiocchi L.,University of Brescia | Mathew S.,New York Medical College | And 6 more authors.
Modern Pathology | Year: 2013

Polycythemia vera and primary myelofibrosis share a propensity to progress toward a myelofibrotic late stage with overlapping clinical characteristics. Bone marrow features potentially useful for distinguishing the two entities have not been thoroughly investigated and, currently, clinical history is used for purposes of disease classification. This study describes in detail the morphologic features of 23 cases of post-polycythemic myelofibrosis and 15 cases of primary myelofibrosis with a similar degree of fibrosis, from two large medical centers. Cytogenetic results were available in 19 post-polycythemic myelofibrosis and in 13 primary myelofibrosis cases. JAK2 status and follow-up information was available in all cases. Cellularity was increased in both groups, but more so in post-polycythemic myelofibrosis than in primary myelofibrosis. In post-polycythemic myelofibrosis, most megakaryocytes retained polycythemia vera-like features including normally folded and/or hyperlobulated nuclei devoid of severe maturation defects; only in a few cases were rare tight clusters present. In primary myelofibrosis cases, megakaryocytes showed pronounced anomalies, including increased nuclear:cytoplasmic ratio, abnormal clumping of chromatin and frequent tight clustering. No differences in blast number (<1%) or in the myeloid:erythroid ratio were observed. Post-polycythemic myelofibrosis showed a higher degree of karyotypic alterations and higher percentage of cases with complex karyotype and/or two or more clones. Chromosome 1 defects were common in post-polycythemic myelofibrosis, whereas isolated del(20q) was the most common alteration in primary myelofibrosis. No survival differences were noted between the two groups. Post-polycythemic myelofibrosis cases retain a distinct megakaryocytic morphology that represents a useful clue for differential diagnosis. In addition, they more often display a complex karyotype than do primary myelofibrosis cases. These results suggest that myelofibrosis in polycythemia vera represents a form of progression characterized by profound genetic damage whereas in primary myelofibrosis it is an intrinsic part of the phenotypic manifestation of the disease, not necessarily associated with adverse cytogenetics. © 2013 USCAP, Inc. Source

Augello C.,University of Milan | Gianelli U.,University of Milan | Falcone R.,IRCCS Fondazione Ca Granda Ospedale Maggiore Policlinico Milan | Tabano S.,University of Milan | And 18 more authors.
Leukemia Research | Year: 2015

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterised by the clonal proliferation of the haematopoietic precursors together with the progressive development of bone marrow fibrosis. This stromal alteration is an important clinical issue and specific prognostic markers are not currently available. In bone marrow biopsies from 58 PMF patients, we explored the methylation pattern of genes encoding cytokines involved in the stromal reaction, namely platelet-derived growth factor-beta (PDGFB), transforming growth factor-beta (TGFB) and basic fibroblast growth factor (FGF2). We also evaluated the methylation profile of the Long Interspersed Nucleotide Element 1 (LINE-1). PDGFB, FGF2 and LINE-1, but not TGFB, were significantly differently methylated in PMF compared to controls. Significantly, PDGFB hypomethylation (<16%) was correlated with a favourable PMF prognosis (grade of marrow fibrosis, p= 0.03; International Prognostic Scoring Systems p= 0.01 and Dynamic International Prognostic Scoring Systems, p= 0.02). Although the basis of the association of PDGFB hypomethylation with favourable prognosis remains to be clarified, we speculate that hypomethylation in PMF could represent the effect of acquired somatic mutations in genes involved in epigenetic regulation of the genome. © 2014 Elsevier Ltd. Source

Vercellati C.,Hematology and Transplantation Unit | Marcello A.P.,Hematology and Transplantation Unit | Fermo E.,Hematology and Transplantation Unit | Barcellini W.,Hematology and Transplantation Unit | And 2 more authors.
Clinical Laboratory | Year: 2013

Background: Hereditary spherocytosis (HS) and pyruvate kinase (PK) deficiency are the most common causes of congenital hemolytic anemia. We describe a case of HS with defective PK activity initially misdiagnosed as PK deficiency. Methods: Hematologic investigation, SDS-PAGE analysis of red cell membrane proteins and sequencing of the PKLR gene were performed. Results: The molecular characterization of the PKLR gene showed a heterozygous mutation 994G > A (Gly332Ser) associated with the promoter substitution -148C > T, whose role in the pathophysiology of PK deficiency is debated. Further investigations revealed spectrin deficiency; the family study demonstrated that the hemolysis was exclusively attributable to HS. Conclusions: The present case pinpoints to the need for extensive family investigations to correctly diagnose chronic hemolytic anemia, in particular when molecular characterization does not fully explain the clinical phenotype. Source

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