Olcay L, et al: Hematopoiesis in Families with Congenital NeutropeniaTurk J Hematol 2018;35:229-259Figure 3. Cell cycle patterns of two sibling patients, their mother, and their father: A) normal cell cycle (patient AG, lymphocyte; siblingof ZG); B) G1 arrest and pre-G1 peak showing apoptosis (patient ZG; sibling of AG); C) G1 arrest and apoptosis (mother DG, lymphocyte);D) normal cell cycle (mother DG, granulocyte); E) G1 arrest and apoptosis (father SG, lymphocyte); F) normal cell cycle (father SG,granulocyte) (the patients’ granulocytes could not be evaluated due to granulocytopenia). G-H-I: The leukocytes of two sibling patientsand their mother stained by SA-β-gal, as blue granules, from peripheral blood culture (400 x ). The leukocytes of patient AG (G), patientZG (H), and their mother DG (I). These patients were members of a family evaluated for cell cycles.240
Turk J Hematol 2018;35:229-259Olcay L, et al: Hematopoiesis in Families with Congenital Neutropeniaerythroblasts, or apoptotic cells. Necrosis of cells that hadphagocytosed other cells was also evident (Figures 6A, 6B, 6C).MegakaryopoiesisMegakaryocytes with asynchrony in nucleo-cytoplasmicmaturation or those undergoing emperipolesis or transformed/transforming to naked megakaryocyte nuclei were striking.Additionally, naked megakaryocyte cytoplasm just aftercompleting thrombocyte release, many megakaryoblasts, andnecrotic, apoptotic, or dysplastic megakaryocytes were also seenin the bone marrow examinations of the patients (Figures 6A,6B, 6C).Thrombocytes and Thrombocyte FunctionsThrombocytes with heterogeneous size, abnormal shape,and/or giant forms were observed on the peripheral bloodsmears of both the parents and patients. Giant and dysplasticthrombocytes were also evident in many patients’ bone marrowunder light microscope (Figures 6A, 6B, 6C).The mean dense granule number per thrombocyte was 2.01±1.19(0.37-3.55) in the patients (n=12), 2.27±1.33 (0.22-4.47) inthe parents (n=20), and 3.32±0.40 (2.78-3.82) in the healthycontrols (n=5), and these were comparable with each other(p=0.147). However, the percentage of patients, parents, andcontrols who had fewer than 2 dense granules per thrombocytewas 50%, 35%, and 0% respectively (Supplemental Figure 1).Ultrastructural examination showed that the thrombocytes hada reduced number of dense granules that were heterogeneousin size, shape, and composition. The open canalicular system(OCS) was enlarged and contained unevacuated components inpatients (Figures 4H, 4I, 4J, 4K, and 7A - Case 2) and parents(Figures 5G and 5I).In vitro bleeding time was prolonged in patients and parentsby 37.5% and 18.8% with collagen-epinephrine cartridges andby 33.3% and 12.5% with collagen-ADP cartridges, respectively,vs. 0% in the control group with both cartridges. While invitro bleeding times in patients and parents were comparable(p=0.293 and 0.233, respectively), only the in vitro bleedingtime with collagen-ADP in patients was longer than in thecontrol (p=0.031) (Supplemental Table 4).Up to 63.6% and 44.4% of the aggregation results performedwith various reactive substances in patients and their familymembers displayed abnormalities (Table 3; Supplemental Tables5 and 6).Thrombocyte aggregation at the 2 nd , 8 th , and 14 th minutes underTEM (Figure 7) revealed a lack of adhesion and a lack of orinadequate secretion as also seen in Figures 4H, 4I, 4J, 4K, 5G,and 5I, with delayed or defective centralization, developmentof pseudopods, and/or secretion, abnormal degranulation,dissociation phenomenon [23,24], and abnormal amoeboidcytoplasmic protrusions (Supplemental Results).There was inconsistency between the presence of hemorrhagicdiathesis and abnormality of laboratory tests (aggregationtests, dense granule number in thrombocytes, in vitro bleedingtime, thrombocyte ultrastructure) and vice versa. Not all theseabnormalities coexisted all together (Table 3; SupplementalTable 6).Genetic MutationsFourteen of 15 patients and 9 of 21 parents were evaluated forgenetic mutations. Patients had homozygous [c.130_131insA(p.W44*)] mutation in the second exon of the HAX1 gene (n=6),heterozygous ELANE mutations [c.597+5G>A and c.416C>T(p.P139L) (n=2)], and homozygous G6PC3 mutation [c.194A>C(p.E65A), n=2], which is a novel mutation in the literature andis predicted to be disease-causing by SIFT and MutationTasterin silico analysis software [in submission]. Five had unidentifiedmutations. No tested patient had CSF3R mutation. ELANEc.597+5G>A splicing mutation was predicted to be diseasecausingby NNSPLICE, GeneSplicer, and Human Splicing Finderin silico prediction tools.Patients with HAX1 displayed coexistent homozygousc.159T>C polymorphism in the second exon of the HAX1gene. Three patients with other mutations were heterozygousfor this polymorphism (Tables 1 and 3). Both the mother andfather of AY, MNY, and HY and the mother of EÇ were foundheterozygous for HAX1 [c.130_131insA (p.W44*)]. The parentsof the two patients with heterozygous ELANE mutation revealedno mutation in the ELANE gene and their buccal mucosa cellsdid not reveal mosaicism.DiscussionIn this study, we showed that non-granulocytic blood cells werealso affected and that morphologic and functional changesoccurred in patients with SCN and in their non-neutropenicfamily members, and cell death mechanisms other thanapoptosis also operated.Apoptosis and Secondary Necrosis in Granulocytic and Non-Granulocytic CellsIt has been reported that in SCN and other neutropenic states,accelerated apoptosis of bone marrow granulocytic progenitorcells [1,2,6,25,26,27,28,29,30,31] and lymphocyte apoptosis[7,8] took place through different mechanisms. In our study,apoptosis was demonstrated not only in granulocytes andlymphocytes but also in monocytes by elevated annexin V,ultrastructural appearance, and a pre-G1 peak in cell cycleanalysis. The absence of a pre-G1 peak is not enough to exclude241
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AUTHOR INDEX 2018Olga Meltem Akay..
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SUBJECT INDEX 2018Thalassemia / Tal