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de Siqueira, Lucia Helena, Miriam P. Beltrame, Fernanda P. G. Cunha, Marina P. Colella, Joyce M. Annichino-Bizzacchi, Erich V. de Paula, and Margareth C. Ozelo. "Six Novel Mutations Identified in the Glycoproteins Ib Alpha, Ib Beta and IX Genes Among Twenty-Two Unrelated Patients with Bernard-Soulier Syndrome in Brazil." Blood 118, no. 21 (November 18, 2011): 1156. http://dx.doi.org/10.1182/blood.v118.21.1156.1156.
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Abstract Abstract 1156 Introduction: Bernard-Soulier syndrome (BSS) is a rare hereditary bleeding disorder, caused by mutations within the glycoprotein (GP) Ib alpha, GP Ib beta and GP IX genes that encode three of four subunits of the platelet GP Ib-V-IX adhesion receptor. In the present study we evaluated the mutations involved in the diagnosis of BSS from twenty-two unrelated patients. Patients and Methods: All patients were followed in two large bleeding disorder reference centers in Brazil. The diagnosis of BSS was established based on the presence of mucocutaneous bleeding and macrotrombocytopenia, and was confirmed by platelet ristocetin aggregation and flow cytometry for the platelet GP Ib alpha (CD 42b), GP Ib beta (CD 42c), and GP IX (CD 42a). Available first- and second-degree relatives were also contacted for clinical, laboratory and molecular evaluation. Genomic DNA from all index cases was used for sequence analysis of the three genes, GP1BA, GP1BB and GP9, and the results were confirmed in relatives when available. Results: Twenty-two unrelated patients with the confirmed diagnosis of BSS were enrolled in this study. Among twenty-two index cases, twenty-one had one or two mutations identified, including six novel mutations. We also identified two mutations that have been previously reported, GP1BA C209S (Simsek, et al., 1994), and GP9 A140T (Wang, et al., 2004). The six novel mutations correspond to conserved regions, and they consist of two mutations in the GP1BA (L51R, and L99P), three in GP1BB (M-25A, L72R, and L112P), and one in GP9 (P52Q). One of these novel mutations, the GP1BB L112P was observed in twelve of twenty-two unrelated cases. PCR-restriction fragment length analysis of genomic DNA from a hundred normal unrelated controls were performed to evaluate the presence of GP1BA L99P and GP1BB L112P mutations by using the AlwN I and Alu I restriction enzymes, respectively. All controls were negative for both mutations. Interestingly, when we analyzed the relatives of the index cases indentified with the mutations GP1BA L99P and GP1BB M-25A, we found evidence of mild macrotrombocytopenia in the heterozygote carriers of these mutations. The relatives heterozygous for GP1BB L112P showed normal platelet count and morphology. However, in three index cases with mild macrotrombocytopenia, GP1BB L112P in heterozygosity was the only mutation identified. Furthermore, site-directed mutagenesis was carried out to evaluate the expression of the novel mutations GP1BA L51R, and GP1BA L99P. Compared to the GP1BA wild-type, both mutations were only minimally expressed in CHO bIX cells, which stably express GP Ib beta and GP IX. Conclusions: We identified in this study eight distinct mutations among twenty-two unrelated SBS patients, including six novel mutations. The GP1BB L112P mutation was found in twelve of the twenty-two index cases, suggesting that this could be due to a founder effect. Identifying such a frequent mutation in this population of BSS patients will be helpful for genetic diagnosis of this condition in Brazil. Furthermore these mutations significantly add to the mutation database of BSS and will inevitably provide insights into the function of GP Ib-V-IX. Disclosures: No relevant conflicts of interest to declare.
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Barozzi, Serena, Valeria Bozzi, Daniela De Rocco, Tania Giangregorio, Patrizia Noris, Anna Savoia, and Alessandro Pecci. "A Novel Mutation in GP1BB Reveals the Role of the Cytoplasmic Domain of GPIbβ in the Pathophysiology of Bernard-Soulier Syndrome and GPIb-IX Complex Assembly." International Journal of Molecular Sciences 22, no. 19 (September 22, 2021): 10190. http://dx.doi.org/10.3390/ijms221910190.
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Bernard-Soulier syndrome (BSS) is an autosomal-recessive bleeding disorder caused by biallelic variants in the GP1BA, GP1BB, and GP9 genes encoding the subunits GPIbα, GPIbβ, and GPIX of the GPIb-IX complex. Pathogenic variants usually affect the extracellular or transmembrane domains of the receptor subunits. We investigated a family with BSS caused by the homozygous c.528_550del (p.Arg177Serfs*124) variant in GP1BB, which is the first mutation ever identified that affects the cytoplasmic domain of GPIbβ. The loss of the intracytoplasmic tail of GPIbβ results in a mild form of BSS, characterized by only a moderate reduction of the GPIb-IX complex expression and mild or absent bleeding tendency. The variant induces a decrease of the total platelet expression of GPIbβ; however, all of the mutant subunit expressed in platelets is correctly assembled into the GPIb-IX complex in the plasma membrane, indicating that the cytoplasmic domain of GPIbβ is not involved in assembly and trafficking of the GPIb-IX receptor. Finally, the c.528_550del mutation exerts a dominant effect and causes mild macrothrombocytopenia in heterozygous individuals, as also demonstrated by the investigation of a second unrelated pedigree. The study of this novel GP1BB variant provides new information on pathophysiology of BSS and the assembly mechanisms of the GPIb-IX receptor.
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Tang, Jingrong, Sara Stern-Nezer, Po-Ching Liu, Ludmila Matyakhina, Michael Riordan, Naomi Luban, Peter Steinbach, and Stephen Kaler. "Mutation in the leucine-rich repeat C-flanking region of platelet glycoprotein Ibβ impairs assembly of von Willebrand factor receptor." Thrombosis and Haemostasis 92, no. 07 (2004): 75–88. http://dx.doi.org/10.1160/th04-02-0071.
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SummaryWe describe a syndrome of thrombocytopenia, bleeding episodes, congenital heart disease and facial dysmorphism in a newborn infant, and trace the cause to mutations on chromosome 22 that involve the gene for platelet glycoprotein Ibβ (GPIbβ, Human Genome Organisation gene symbol GPIBB), a critical component of the von Willebrand factor (vWF) receptor. Fluorescence in situ hybridization in transformed lymphoblasts revealed hemizygous microdeletion of 22q11.2 containing the GP1BB locus. DNA sequencing revealed a C to T transition in the patient’s remaining GP1BB allele, predicting a novel proline to serine substitution (Pro96Ser) in the carboxyterminal flanking domain of a leucine-rich repeat. We characterized the mutant GP1BB allele by expression in a cell line (CHOαIX) that stably expresses two other components of the vWF receptor, GPIbα and GPIX. Flow cytometry and confocal imaging of transfected CHOαIX cells demonstrated that P96S GPIbβ abrogates surface assembly of the complex, consistent with platelet flow cytometry studies in the patient. Based on sequence homology to the known crystal structures of two other leucine-rich repeat proteins, the human Nogo receptor and GPIbα, we propose a new structural model of GPIbβ. The model refutes earlier assumptions about cysteine-cysteine interactions in the amino-terminal region of GPIbβ, and predicts a hydrophobic patch the burial of which may contribute to proper conformation of the fully assembled vWF receptor complex.
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Mekchay, Ponthip, Praewphan Ingrungruanglert, Kanya Suphapeetiporn, Darintr Sosothikul, Wilawan Ji-au, Supang Maneesri Le Grand, Nipan Israsena, and Ponlapat Rojnuckarin. "Study of Bernard–Soulier Syndrome Megakaryocytes and Platelets Using Patient-Derived Induced Pluripotent Stem Cells." Thrombosis and Haemostasis 119, no. 09 (July 28, 2019): 1461–70. http://dx.doi.org/10.1055/s-0039-1693409.
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AbstractBernard–Soulier syndrome (BSS) is a hereditary macrothrombocytopenia caused by defects in the glycoprotein (GP) Ib-IX-V complex. The mechanism of giant platelet formation remains undefined. Currently, megakaryocytes (MKs) can be generated from induced pluripotent stem cells (iPSCs) to study platelet production under pharmacological or genetic manipulations. Here, we generated iPSC lines from two BSS patients with mutations in different genes (GP1BA and GP1BB: termed BSS-A and BSS-B, respectively). The iPSC-derived MKs and platelets were examined under electron microscopy and stained by immunofluorescence to observe proplatelet formation and measure platelet diameters which were defined by circumferential tubulin. BSS-iPSCs produced abnormal proplatelets with thick shafts and tips. In addition, compared with the normal iPSCs, the diameters were larger in platelets derived from BSS-A and BSS-B with the means ± standard deviations of 4.34 ± 0.043 and 3.88 ± 0.045 µm, respectively (wild-type iPSCs 2.61 ± 0.025 µm, p < 0.001). Electron microscopy revealed giant platelets with the abnormal demarcation membrane system. Correction of BSS-A and BSS-B-iPSCs using lentiviral vectors containing respective GP1BA and GP1BB genes improved proplatelet structures and platelet ultrastructures as well as reduced platelets sizes. In conclusion, the iPSC model can be used to explore molecular mechanisms and potential therapy for BSS.
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Savoia, Anna, Shinji Kunishima, Patrizia Noris, Nuria Pujol-Moix, Dermot Kenny, Nurit Rosenberg, Margaret L. Rand, et al. "International Consortium for the Study of Clinical and Molecular Aspects of Bernard-Soulier Syndrome." Blood 118, no. 21 (November 18, 2011): 707. http://dx.doi.org/10.1182/blood.v118.21.707.707.
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Abstract Abstract 707FN2 Bernard-Soulier syndrome (BSS) is an extremely rare inherited bleeding disorder characterized by a defect of the GPIb/IX/V complex, which is essential for hemostasis, as the GPIbα subunit binds to subendothelial von Willebrand factor. Since the identification of the first mutation in 1990, almost one hundred cases carrying mutations in the GP1BA, GP1BB, and GP9 genes have been described. Most of the mutations prevent the coordinated association of the complex or binding to the von Willebrand factor. BSS is usually transmitted as a recessive trait with giant platelets and severe bleeding tendency. However, there are families with a dominant mild form, in which the affected individuals have only moderate macrothrombocytopenia and bleeding tendency. A correct definition of the clinical and laboratory features, together with accurate genotype/ phenotype correlation studies, remains essential for understanding the molecular basis of the disease and managing patients appropriately. Moreover, it is important to understand the variability of clinical manifestations. Since BSS is rare with an estimated prevalence of 1:1,000,000, an International Consortium has recently been established to collect a large series of cases and families worldwide. At present, the Consortium has been compiling data from 165 unrelated families, of which 50% have not been previously described. In this cohort, the molecular genetic testing reveals more than 30 novel mutations, confirming the wide spectrum of alterations responsible for the disease. Data from 65 unrelated families (69 patients) mainly from France, Italy and Japan show that 23 have mutations in GP9 and 29 in GP1BB. In the remaining 13 families, the defective gene is GP1BA. In agreement with the view that BSS is an extremely rare disease, 53 probands carried homozygous mutations, 10 are compound heterozygous, and 2 hemizygous because of a 22q11 deletion of the DiGeorge syndrome. The mean age of patients at diagnosis was 18 years (range 0–75 years) of which 27 were males and 38 females. Misdiagnosis of autoimmune thrombocytopenia was frequent and 26 patients were previously treated with steroids, intravenous immunoglobulins and/or splenectomy. Except two Japanese cases without any bleeding manifestations, patients presented with a variable bleeding diathesis measured by the World health Organization bleeding scale: grades 1, 2, 3 and 4 in 9, 18, 19 and 10 patients, respectively. The mean platelet count was 64×109/L (range 24–130) as determined by microscopy. In contrast, using a cell counter, thrombocytopenia was more severe (45×109/L; range 5–125). The mean platelet mean diameter was larger than in controls and varied from 2.9 to 7.5 mm. Ristocetin-induced platelet agglutination was absent or lower than 22% of normal response in all patients. Flow cytometry revealed a defective expression of the GPIb/IX/V expression in all patients. Correlating between expression data and gene affected, we found that the expression of GP1ba was often undetectable in patients with GP1BA mutations whereas it was higher, 8% and 17%, in patients with mutations of GP1BB and GP9, respectively. Instead, the GPIX mean level was 14%, 8% and 25% in patients with GP9, GP1BB and GP1BA mutations. The expression of GPV was higher than that of the other subunits, being more than 30% regardless of which gene was mutated. This is the largest cohort of BSS patients characterized to date. These patients together with the other 100 cases not yet included in the BSS database will enable correlations of the molecular genetic defects, receptor expression and clinical manifestations observed in BSS patients. Disclosures: Zieger: CSL Behring Hattersheim: Research Funding.
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Blanco-Luquin, Idoia, Blanca Acha, Amaya Urdánoz-Casado, Eva Gómez-Orte, Miren Roldan, Diego R. Pérez-Rodríguez, Juan Cabello, and Maite Mendioroz. "NXN Gene Epigenetic Changes in an Adult Neurogenesis Model of Alzheimer’s Disease." Cells 11, no. 7 (March 22, 2022): 1069. http://dx.doi.org/10.3390/cells11071069.
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In view of the proven link between adult hippocampal neurogenesis (AHN) and learning and memory impairment, we generated a straightforward adult neurogenesis in vitro model to recapitulate DNA methylation marks in the context of Alzheimer’s disease (AD). Neural progenitor cells (NPCs) were differentiated for 29 days and Aβ peptide 1–42 was added. mRNA expression of Neuronal Differentiation 1 (NEUROD1), Neural Cell Adhesion Molecule 1 (NCAM1), Tubulin Beta 3 Class III (TUBB3), RNA Binding Fox-1 Homolog 3 (RBFOX3), Calbindin 1 (CALB1), and Glial Fibrillary Acidic Protein (GFAP) was determined by RT-qPCR to characterize the culture and framed within the multistep process of AHN. Hippocampal DNA methylation marks previously identified in Contactin-Associated Protein 1 (CNTNAP1), SEPT5-GP1BB Readthrough (SEPT5-GP1BB), T-Box Transcription Factor 5 (TBX5), and Nucleoredoxin (NXN) genes were profiled by bisulfite pyrosequencing or bisulfite cloning sequencing; mRNA expression was also measured. NXN outlined a peak of DNA methylation overlapping type 3 neuroblasts. Aβ-treated NPCs showed transient decreases of mRNA expression for SEPT5-GP1BB and NXN on day 9 or 19 and an increase in DNA methylation on day 29 for NXN. NXN and SEPT5-GP1BB may reflect alterations detected in the brain of AD human patients, broadening our understanding of this disease.
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Sivapalaratnam, Suthesh, Sarah K. Westbury, Jonathan C. Stephens, Daniel Greene, Kate Downes, Anne M. Kelly, Claire Lentaigne, et al. "Rare variants in GP1BB are responsible for autosomal dominant macrothrombocytopenia." Blood 129, no. 4 (January 26, 2017): 520–24. http://dx.doi.org/10.1182/blood-2016-08-732248.
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Sivapalaratnam, Suthesh, Willem Ouwehand, Bridge Consortium, and ThromboGenomics Consortium. "Rare Variants in GP1BB underlie Autosomal Dominant Macrothrombocytopenia; Findings of Large Unique Bleeding and Platelet Disorders Cohort." Blood 128, no. 22 (December 2, 2016): 1359. http://dx.doi.org/10.1182/blood.v128.22.1359.1359.
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Abstract The incidence of inherited rare bleeding, thrombotic and platelet disorders (BPD) is estimated to be 200-250 per million individuals. For at least 15% of these cases the molecular basis is unresolved (Lentaigne et al, Blood, 2016). We aim to discover the genetic basis of these unresolved BPDs, to improve diagnosis and treatment. In addition this will increase our knowledge of the molecular pathways of megakaryopoiesis, haemostasis and thrombus formation. For this purpose we have established a prospective BPD cohort, which at time of writing consist of 1,378 probands, 123 affected relatives and 41 unaffected relatives. After consenting, all these individuals have been precisely phenotyped using human phenotype ontology (HPO) terms (Westbury et al, Genome Medicine, 2015). This includes clinical parameters, laboratory results and pedigree history. Ten thousand DNA samples from the BPD cases, patients with other rare diseases and their close relatives, who were all enrolled in the NIHR BioResource were analysed by whole genome sequencing (Turro et al, Science Trans. Med, 2016). We applied phenotype similarity regression to identify statistical associations between presence of a coding variants with consequences in a gene and similarity to a latent HPO-coded phenotype (Greene et al, AJHG, 2016). We identified a strong statistical association between presence of 8 unique rare coding DNA variants with consequences in GP1BB and 8 probands with macrothrombocytopenia (SimReg posterior probability = 0.93 with inferred characteristic phenotype preferentially included the term "Increased mean platelet volume", Fisher's p = 2.10 x 10-6). We sought to validate these discovery findings through identification of further cases in the cohorts of 75 and 301 macrothrombocytopenia cases from the ThromboGenomics consortium (Simeoni et al, Blood, 2016) and the Nagoya Medical Center in Japan, respectively. Three additional variants in GP1BB were identified in 9 individuals from 8 pedigrees. Systematic review of the sequencing results of 27 BPD genes (including GP1BA, GP9) implicated in thrombocytopenia in 10 probands did not reveal any alternative variants that could plausibly explaining the phenotype. In aggregate 59 affected macrothrombocytopenia cases were observed in 16 pedigrees with 9 unique GP1BB variants, with the Y113C variant, which was observed in 6 pedigrees thought to be a Japanese founder variant. The means of the count and volume of platelets of the probands was 104.6 x109/l (range 47-172 x109/l) and 12.6 fL, respectively. Inspection of blood smears revealed anisocytosis with a small number of giant platelets and electron micrograph images were reminiscent of those from platelets of a patient with Bernard Soulier syndrome (BSS). In 11 pedigrees measurement by cytometry showed reduced levels of the GpIb/IX/V complex on the platelets of 8 genetically independent individuals and bleeding diathesis was reported in 7 of 16 pedigrees. Altogether, we identified 9 unique variants in the GP1BB gene, which encodes the 202 amino acid long Type 1 transmembrane protein GpIb▢, which together with GpIba, GpV and GpIX form the receptor complex for von Willebrand Factor on megakaryocytes and platelets. They result in a disruption of the canonical methionine start codon, another resulting in a premature stop at residue 46, 5 missense variants at L16P, G43W, T68M, Y113C and L132Q, a deletion removing PAL at residues 79-81, and finally a frameshift in the codon for residue A150 leading to an alternative open reading frame predicted to result in a protein of 193 instead of 202 amino acids long. All 9 variants but the G43W one, which was observed in one of the 61,000 ExAC subjects were unobserved in the ExAC database. In summary before our study there was only one isolated report of a Gp1ba-R42C variant assumed to be causal of macrothrombocytopenia, but no segregation study was performed to corroborate this observation. Our findings in 16 pedigrees with 59 subjects with macrothrombocytopenia provide robust statistical and convincing co-segregation evidence that some variants in GP1BB if present as a single alleleexert a dominant effect on the count and volume of platelets, resulting in some pedigrees in a bleeding diathesis rejecting the dogma that BSS is mainly an autosomal recessive disorder. Disclosures No relevant conflicts of interest to declare.
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Bartsch, Ingrid, Kirstin Sandrock, Francois Lanza, Paquita Nurden, Ina Hainmann, Anna Pavlova, Andreas Greinacher, et al. "Deletion of human GP1BB and SEPT5 is associated with Bernard-Soulier syndrome, platelet secretion defect, polymicrogyria, and developmental delay." Thrombosis and Haemostasis 106, no. 09 (2011): 475–83. http://dx.doi.org/10.1160/th11-05-0305.
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SummaryThe bleeding disorder Bernard-Soulier syndrome (BSS) is caused by mutations in the genes coding for the platelet glycoprotein GPIb/IX receptor. The septin SEPT5 is important for active membrane movement such as vesicle trafficking and exocytosis in non-dividing cells (i.e. platelets, neurons). We report on a four-year-old boy with a homozygous deletion comprising not only glycoprotein Ibβ (GP1BB) but also the SEPT5 gene, located 5’ to GP1BB. He presented with BSS, cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect. The homozygous deletion of GP1BB and SEPT5, which had been identified by PCR analyses, was confirmed by Southern analyses and denaturing HPLC (DHPLC). The parents were heterozygous for this deletion. Absence of GPIbβ and SEPT5 proteins in the patient’s platelets was illustrated using transmission electron microscopy. Besides decreased GPIb/IX expression, flow cytometry analyses revealedimpaired platelet granule secretion. Because the bleeding disorder was extremely severe, the boy received bone marrow transplantation (BMT) from a HLA-identical unrelated donor. After successful engraftment of BMT, he had no more bleedingepisodes. Interestingly, also his mental development improved strikingly after BMT. This report describes for the first time a patient with SEPT5 deficiency presenting with cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect.
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Louzil, Jan, Jana Stikarova, Dana Provaznikova, Ingrid Hrachovinova, Tereza Fenclova, Jan Musil, Martin Radek, et al. "Diagnosing Czech Patients with Inherited Platelet Disorders." International Journal of Molecular Sciences 23, no. 22 (November 19, 2022): 14386. http://dx.doi.org/10.3390/ijms232214386.
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A single-center study was conducted on 120 patients with inherited disorders of primary hemostasis followed at our hematological center. These patients presented a variety of bleeding symptoms; however, they had no definitive diagnosis. Establishing a diagnosis has consequences for the investigation of probands in families and for treatment management; therefore, we aimed to improve the diagnosis rate in these patients by implementing advanced diagnostic methods. According to the accepted international guidelines at the time of study, we investigated platelet morphology, platelet function assay, light-transmission aggregometry, and flow cytometry. Using only these methods, we were unable to make a definitive diagnosis for most of our patients. However, next-generation sequencing (NGS), which was applied in 31 patients, allowed us to establish definitive diagnoses in six cases (variants in ANKRD26, ITGA2B, and F8) and helped us to identify suspected variants (NBEAL2, F2, BLOC1S6, AP3D1, GP1BB, ANO6, CD36, and ITGB3) and new suspected variants (GFI1B, FGA, GP1BA, and ITGA2B) in 11 patients. The role of NGS in patients with suspicious bleeding symptoms is growing and it changes the diagnostic algorithm. The greatest disadvantage of NGS, aside from the cost, is the occurrence of gene variants of uncertain significance.
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Wunderlich, Frank, Denis Delic, Daniela Gerovska, and Marcos J. Araúzo-Bravo. "Vaccination Accelerates Liver-Intrinsic Expression of Megakaryocyte-Related Genes in Response to Blood-Stage Malaria." Vaccines 10, no. 2 (February 14, 2022): 287. http://dx.doi.org/10.3390/vaccines10020287.
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Erythropoiesis and megakaryo-/thrombopoiesis occur in the bone marrow proceeding from common, even bipotent, progenitor cells. Recently, we have shown that protective vaccination accelerates extramedullary hepatic erythroblastosis in response to blood-stage malaria of Plasmodium chabaudi. Here, we investigated whether protective vaccination also accelerates extramedullary hepatic megakaryo-/thrombopoiesis. Female Balb/c mice were twice vaccinated with a non-infectious vaccine before infecting with 106 P. chabaudi-parasitized erythrocytes. Using gene expression microarrays and quantitative real-time PCR, transcripts of genes known to be expressed in the bone marrow by cells of the megakaryo-/thrombocytic lineage were compared in livers of vaccination-protected and unprotected mice on days 0, 1, 4, 8, and 11 p.i. Livers of vaccination-protected mice responded with expression of megakaryo-/thrombocytic genes faster to P. chabaudi than those of unvaccinated mice, evidenced at early patency on day 4 p.i., when livers exhibited significantly higher levels of malaria-induced transcripts of the genes Selp and Pdgfb (p-values < 0.0001), Gp5 (p-value < 0.001), and Fli1, Runx1, Myb, Mpl, Gp1ba, Gp1bb, Gp6, Gp9, Pf4, and Clec1b (p-values < 0.01). Together with additionally analyzed genes known to be related to megakaryopoiesis, our data suggest that protective vaccination accelerates liver-intrinsic megakaryo-/thrombopoiesis in response to blood-stage malaria that presumably contributes to vaccination-induced survival of otherwise lethal blood-stage malaria.
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Cutler, Jacky, Mike Mitchell, Hady Goubran, and Geoffrey F. Savidge. "Familial Bernard-Soulier Syndrome Due to a Novel Ins/Del Mutation in Glycoprotein IX." Blood 106, no. 11 (November 16, 2005): 2178. http://dx.doi.org/10.1182/blood.v106.11.2178.2178.
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Abstract Bernard-Soulier syndrome (BSS) is a rare autosomal recessive bleeding problem, characterised by thrombocytopaenia, large dysfunctional platelets, and defective von-Willebrand factor dependent platelet aggregation. It is caused by a qualitative or quantitative abnormality of the platelet glycoprotein (GP) 1b/IX/V complex. Of the components of this complex, defects have been identified in all but GPV; in order of frequency of mutation GP1bα >GPIX >GP1bβ. Classically, mutations in any of these chains will lead to a total absence of the complex from the platelet surface, but variant forms of BSS have been described in which there is an inbalance in the expression of GPIX in relation to GP1b and GPV chains. We present a case study of a familial BSS. The proband was originally referred to this centre with a diagnosis of probable type 3 von Willebrand disease. He presented with lifelong epistaxis and oral bleeding, which have been variously treated with whole blood, F8 concentrate and platelet transfusion. Two unrelated surgical procedures were uneventful. Laboratory investigations showed a platelet count of 22 x109 /L, normal vWF antigen, activity and collagen binding, and abnormal platelet function screens in the presence of both ADP and epinephrine. Platelet glycoprotein analysis by FACs failed to detect any expression of GP1bα, GP1bβ or GPIX. Following these laboratory investigations the diagnosis was amended to BSS. The patient is the eldest of five children of consanguinous (first cousin) parents of Egyptian origin. One sister has a bleeding diathesis of approximately equal severity to the proband. The mother and another sister presented with much less pronounced bleeding. The remaining family members are asymptomatic; however, one sister is pre-pubescent, and may present with menorraghia in the future. Genetic analysis of the GP1bα, GP1bβ and GPIX genes was undertaken in the proband, and a novel ins/del mutation identified in the only coding exon of GPIX. Nucleotides 1717–1721 (TGTAC) are replaced with alternate bases (GTGGG) of unknown origin. This mutation results in the replacement of cysteine-8 and threonine-9 with valine -8 and glycine-9. The loss of the cysteine residue at codon 8 disrupts a putative disulphide bond between cysteine-8 and cysteine -12, thereby impairing correct assembly and anchoring of GPIX on the platelet surface. Mutations affecting the conserved cysteine residues of each subunit of the GP1b/IX complex have been reported, and have a significant effect on the biosynthesis and function of the complex. Analysis of family members determined that both parents, and two siblings are heterozygous for this mutation, while the equally symptomatic sister and the youngest child are homozygous. Heterozygosity for this novel ins/del mutation is unlikely to explain the mild bleeding diathesis seen in some female members of this family. Phenotypic investigations are ongoing, but thus far have proved inconclusive in demonstrating a coexisting haemostatic disorder, such as type 1vWD or mild FVII deficiency.
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Ferrari, Silvia, Anna M. Lombardi, Irene Cortella, Maria A. Businaro, Antonella Bertomoro, Irene Di Pasquale, and Fabrizio Fabris. "New heterozygous variant in GP1BB gene is responsible for an inherited form of macrothrombocytopenia." British Journal of Haematology 184, no. 5 (March 12, 2018): 855–58. http://dx.doi.org/10.1111/bjh.15176.
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Oved, Joseph H., Michele P. Lambert, M. Anna Kowalska, Mortimer Poncz, and Konrad Karczewski. "Analysis of the Frequency of Spontaneous, Functionally-Significant Mutations in Genes Associated with Platelet Disorders in >120,000 Healthy Individuals." Blood 132, Supplement 1 (November 29, 2018): 2438. http://dx.doi.org/10.1182/blood-2018-99-115567.
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Abstract Inherited platelet disorders (IPD) are increasingly recognized as the cause of clinical bleeding. Advances in genomic technologies have identified a growing number of platelet-associated genes that are currently vetted with phenotypic correlates. These platelet-associated genes are a disparate group, including transcription or related nuclear factors, cytoskeletal proteins, surface receptors, intracellular proteins and granule forming proteins. At present the prevalence of each inherited platelet-associated disorder and the disorders in aggregate are not well defined. We leveraged the recent curating of 123,136 high quality exomes from a cross section of the general population in the form of the genome aggregation database (gnomAD) for analysis. We used the loss-of-function transcript effect estimator (LoFTEE) in conjunction with the gnomAD dataset to study loss of function (LoF) variants in genes of interest. With this set of predicted LoF variants, we generated a LoF frequency for each gene of interest taking into account whether the heterozygote or homozygote state is sufficient and/or necessary for clinical phenotype. These data are analyzed to determine whether each platelet-associated gene is relatively tolerant or intolerant to LoF mutations in the context of their clinical phenotypes. By this analysis, we found approximately 800 novel LoF variants in platelet-related genes in this population of >120,000 individuals. Affected genes known to cause disease phenotype in the heterozygous state (n=33) accounted for 27% of the mutations analyzed. With these data, we calculated the frequency of IPD in the general population secondary to LoF mutations and estimated the relative impact of dominant versus recessive cases of IPD. We demonstrate that the majority of manifest cases of IPD will be due to the dominantly inherited, haploinsufficient IPDs. The transcription factor gene subset (9 of the IPD associated genes) was the most intolerant to LoF variants based on ratio of observed vs. expected number of variants (pLI measurement). Interestingly, the severity of the platelet dysfunction and resultant bleeding from LoF mutations in this subset of genes is not directly related to their intolerance of these mutations. For instance, heterozygous LoF of RUNX1 result in a mild-moderate bleeding disorder; however, a pLI of 0.819 indicates this gene is moderately to very intolerant of LoF variants. These same LoF variants in RUNX1 predispose to myelodysplastic syndromes with a high risk of myeloid leukemia in the form of familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML), and likely this is driving LoF intolerance. Cytoskeletal protein encoding genes represent another subset of mostly LoF intolerant platelet-related genes. Intracellular protein encoding genes and granule protein genes have varied tolerance and platelet-associated receptor protein genes as a subgroup were most tolerant of haploinsufficiency. There were some genes with similar clinical bleeding phenotypes that had divergent tolerance to LoF. For instance, GP9 and GP1BB both cause Montreal Platelet Syndrome in the haploinsufficient state and had moderate intolerance to LoF mutations (pLI GP9 = 0.804; pLI GP1BB = 0.575). In contrast, while LoF mutations in GP1BA cause the same bleeding phenotype, this gene is much more tolerant to haploinsufficiency (pLI = 0.0002). These data indicate that perhaps there is another unidentified adverse condition associated with GP9 and GP1BB that is driving increased haploinsufficiency intolerance. In summary, we present a comprehensive analysis of known platelet-associated genes, the frequency of LoF mutations in these genes and their relative tolerance of the haploinsufficient state. These data generate an incidence of IPDs of ~0.18% in the general population. Importantly, these data also inform the driving mechanisms of LoF intolerance as there are defective genes resulting in similar bleeding phenotypes, but divergent tolerance to haploinsufficiency, indicating that further investigation is warranted for additional biology. Disclosures Lambert: CSL: Consultancy; Rigel: Consultancy; Sysmex: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Summus: Consultancy; Bayer: Membership on an entity's Board of Directors or advisory committees; Shionogi: Consultancy; Educational Concepts in Medicine: Consultancy. Poncz:Incyte Corporation: Consultancy, Research Funding.
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London, Fredda S. "The PAR-1-Stimulated Platelet Subpopulation That Binds Factor Xa Also Expresses Matrixmetalloproteinase and Calpain Activities Resulting in Population-Specific GP1bα Shedding and Platelet Vesiculation." Blood 106, no. 11 (November 16, 2005): 3563. http://dx.doi.org/10.1182/blood.v106.11.3563.3563.
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Abstract We previously showed that only a subpopulation of platelets responds to stimulation by strong agonists with exposure of coagulation factor binding sites. The subpopulation of SFLLRN-amide or collagen-stimulated platelets that bound coagulation factor Xa showed 50% decreased surface glycoprotein 1bα (GP1bα) and 60% reduced forward scatter relative to unstimulated platelets. While collagen-stimulated Xa-negative platelets lost no GP1b/IX/V surface density, the PAR-1-stimulated Xa-negative population lost 25% of both GP1bα and GPIX surface density signifying decreased GP1b/IX/V complex following PAR-1 stimulation. The PAR-1-stimulated Xa-positive platelets showed a discrepancy between the surface densities of GPIX and GP1bα that suggested a procoagulant subpopulation-specific shedding of GP1bα. While Xa-positive PAR-1-stimulated platelets pretreated with 10μM GM6001, a nonspecific inhibitor of matrixmetalloproteinases, recovered 30% of the lost surface GP1bα, bringing the GP1bα /GPIX ratio to ~1, collagen-stimulated loss of GP1bα on Xa-positive platelets was little affected by GM6001 pretreatment, and GP1bα on Xa-negative platelets was unaffected by GM6001 pretreatment. While inhibition of calpains by pretreatment of platelets with 100μM calpeptin had little effect on agonist-stimulated GP1bα shedding, calpeptin pretreatment resulted in 50% decreased loss of GP1bα/GPIX from PAR-1-stimulated Xa-negative platelets, in a 35–45% loss of the Xa-positive subpopulation resulting from PAR-1 stimulation, in 25% increased forward scatter from all agonist-induced Xa-positive populations, and in a 40–50% decrease in positive events occurring outside the defined platelet region. On the contrary, GM6001 inhibition of GP1bα shedding had no effect on either the size of the Xa-positive subpopulation, on the density of binding sites or on forward scatter. Platelet vesiculation would produce smaller platelets with decreased forward scatter, and microparticles. Calpain, previously implicated in platelet vesiculation, appears to mediate specific vesiculation of Xa-positive platelets. In summary, PAR-1 agonists and collagen recruit a platelet subpopulation to undergo specific membrane changes exposing binding sites for coagulation enzymes factors IXa and Xa. This procoagulant subpopulation recruited by PAR-1 signaling is also notable for specific matrixmetalloprotease activities leading to procoagulant population-specific GP1bα shedding. Calpain activity mediates membrane vesiculation of procoagulant platelets resulting from both collagen- and PAR-1-stimulation leading to smaller procoagulant platelets and to platelet microparticles.
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Al-Numair, Nouf, Khushnooda Ramzan, Laila Alquait, Meshal Alshehri, Faiqa Imtiaz, and Tarek Owaidah. "A homozygous loss-of-function mutation in GP1BB causing variable clinical phenotypes in a family with Bernard–Soulier syndrome." Blood Coagulation & Fibrinolysis 32, no. 5 (March 1, 2021): 352–55. http://dx.doi.org/10.1097/mbc.0000000000001027.
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Vaisvilas, M., V. Dirse, B. Aleksiuniene, I. Tamuliene, L. Cimbalistiene, A. Utkus, and J. Rascon. "Acute pre-B lymphoblastic leukemia and congenital anomalies in a child with a de novo 22q11.1q11.22 duplication." Balkan Journal of Medical Genetics 21, no. 1 (October 29, 2018): 87–91. http://dx.doi.org/10.2478/bjmg-2018-0002.
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Abstract Microdeletions and microduplications are recurrent in the q11.2 region of chromosome 22. The 22q11.2 duplication syndrome is an extremely variable disorder with a phenotype ranging from severe intellectual disability, facial dysmorphism, heart defects, and urogenital abnormalities to very mild symptoms. Both benign and malignant hematological entities are rare. A male patient was diagnosed with mild facial dysmorphia, congenital heart anomalies shortly after birth and acute bowel obstruction due to malrotation of the intestine at the age of 3 years. A whole-genome single nucleotide polymorphism (SNP) array revealed a de novo 6.6 Mb duplication in the 22q11.1q11.22 chromosomal region. A year later, the patient was diagnosed with acute pre-B lymphoblastic leukemia (pre-B ALL). Five genes, CDC45, CLTCL1, DGCR2, GP1BB and SEPT5, in the 22q11.1q11.22 region are potentially responsible for cell cycle division. We hypothesized that dosage imbalance of genes implicated in the rearrangement could have disrupted the balance between cell growth and differentiation and played a role in the initiation of malignancy with a hyperdiploid leukemic clone, whereas over-expression of the TBX1 gene might have been responsible for congenital heart defects and mild facial dysmorphia.
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Szelenberger, Rafał, Michał Seweryn Karbownik, Michał Kacprzak, Ewelina Synowiec, Sylwia Michlewska, Michał Bijak, Marzenna Zielińska, Alina Olender, and Joanna Saluk-Bijak. "Dysregulation in the Expression of Platelet Surface Receptors in Acute Coronary Syndrome Patients—Emphasis on P2Y12." Biology 11, no. 5 (April 22, 2022): 644. http://dx.doi.org/10.3390/biology11050644.
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The pathological conditions caused by blood platelet activation constitute a fundamental core in the pathogenesis of Acute Coronary Syndrome (ACS). The hyperactivity of platelets in ACS is well-documented, but there is still little research into the molecular basis of phenotypic changes in platelet functionality. To expand the knowledge of this phenomenon, we analyzed the disturbances in the expression of several key platelet receptors and the aspect of regulating potential abnormalities. Platelet surface receptors are responsible for maintaining the hemostatic balance, platelet interaction with immune cells, and support of the coagulation cascade leading to occlusion of the vessel lumen. Due to their prominent role, platelet receptors constitute a major target in pharmacological treatment. Our work aimed to identify the molecular alteration of platelet surface receptors, which showed augmented mRNA expression of P2Y12, GP1BB, ITGA2B, and ITGB3 and increased protein concentrations of P2Y12 and GP IIb/IIIa in ACS. The upregulation of the P2Y12 level was also confirmed by confocal and cytometric visualization. Furthermore, we evaluated the expression of two microRNAs: miR-223-3p and miR-126-3p, which were suggested to regulate platelet P2Y12 expression. Results of our study present new insight into the molecular background of ACS.
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Peck, Rachel C., Sarah Westbury, Lucy Fitzgibbon, Neil V. Morgan, Jose Rivera, Walter H. Kahr, Nihr BioResource, et al. "Comprehensive Description of Monoallelic GP1BA Variants Associated with Thrombocytopenia." Blood 136, Supplement 1 (November 5, 2020): 34–35. http://dx.doi.org/10.1182/blood-2020-133987.
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GP1BA encodes the glycoprotein (GP) 1bα subunit of the GP1b/IX/V complex, the platelet receptor for von Willebrand factor (VWF) and a mediator of outside-in signalling that contributes to platelet activation. Biallelic variants in GPIBA that prevent surface expression of GPIB/IX/V cause the recessive disorder Bernard Soulier Syndrome (BSS; OMIM #23100) in which there is often severe bleeding caused by thrombocytopenia and platelet dysfunction. Thrombocytopenia has also been associated with monoallelic missense variants in the GP1bα R loop (residues 227-242) which enhance VWF binding (platelet-type von Willebrand disease (pVWD); OMIM #177820). Other monoallelic GPIBA missense variants such as the Bolzano variant (p.Ala172Val; OMIM #153670) have been associated with dominant thrombocytopenia without altered VWF levels. In order to better describe the relationship between monoallelic GPIBA variants and thrombocytopenia we evaluated data from the NIHR BioResource for Rare Diseases, (NBR-RD) and the Inherited Platelet Disorders programmes at the Universities of Birmingham (UK), Murcia (Spain) and Toronto (Canada) in which patients with unexplained platelet disorders underwent diagnostic next generation sequencing. Using the BeviMed method for genetic association we compared the genotypes at rare nonsynoymous variants of 105 unrelated thrombocytopenia cases (platelet count (PLT) <130x109/L or the human phenotype ontology term 'HP 001873 thrombocytopenia') in the NBR-BR with those of 10,152 unrelated participants without thrombocytopenia. There was a strong statistical association (posterior probability 0.97) between dominantly inherited GPIBA variants and thrombocytopenia. Considering the five variants within the NBR-BR that drove this association alongside genotype data from the other case collections, we identified a total of 21 different monoallelic GPIBA coding variants that were rare (gnomAD allele frequency <1/1000) and had a CADD pathogenicity score >15. These were present in 29 index cases (18 females, median age 37 years) with unexplained thrombocytopenia and included the variant predicting the p.Asn150Ser substitution which was identified in seven independent index cases. The variants comprised 18 missense, two inframe insertions (one with an additional missense change) and one insertion resulting in a frame shift. Seven had been previously associated with thrombocytopenia. Sixteen of the variants predicted amino acid substitutions or single residue insertions within the GP1bα leucine rich repeat region (LRR; residues 48-200) or in adjacent regions of the LRR N- and C-terminal caps. Cases with variants in the LRR region typically displayed mild mucocutaneous bleeding, abnormal bleeding after minor trauma or surgery and heavy menstrual bleeding. The platelet count was typically 60-100 x109/L and mean platelet volume greater than 11 fL. There were two missense variants in the GP1bα R loop that have been previously associated with pVWD and two further previously unreported missense variants in the cytoplasmic domain. The final variant was a monoallelic single nucleotide insertion predicted to cause a frame shift and absent expression of GP1bα and has been previously reported as a biallelic variant associated with BSS. These data indicate that in a multicentre cross-sectional cohort of cases with otherwise unexplained thrombocytopenia, monoallelic missense GPIBA variants affecting the LRR or adjacent regions were more prevalent than R loop variants associated with pVWD or variants causing absent GP1bα expression associated with biallelic BSS. The LRR region contains the GP1bα-VWF binding site which is predicted to be disrupted by the missense variants observed in this study. The LRR region is also the location of the GP1bα Bolzano variant and six other monoallelic variants previously associated with thrombocytopenia in previous single case reports. These observations extend the repertoire of GPIBA variants associated with thrombocytopenia and although requiring experimental confirmation of pathogenicity, suggest a common molecular pathogenesis for dominant GPIBA-associated thrombocytopenia in which reduced circulating platelet numbers arises from defective GP1bα-VWF interactions. Figure Disclosures Turro: Tachyon Ventures: Other: Partner & Scientific Director.
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Gissi, Davide, Viscardo Fabbri, Andrea Gabusi, Jacopo Lenzi, Luca Morandi, Sofia Melotti, Sofia Asioli, et al. "Pre-Operative Evaluation of DNA Methylation Profile in Oral Squamous Cell Carcinoma Can Predict Tumor Aggressive Potential." International Journal of Molecular Sciences 21, no. 18 (September 14, 2020): 6691. http://dx.doi.org/10.3390/ijms21186691.
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Background: Prognosis of oral squamous cell carcinoma (OSCC) is difficult to exactly assess on pre-operative biopsies. Since OSCC DNA methylation profile has proved to be a useful pre-operative diagnostic tool, the aim of the present study was to evaluate the prognostic impact of DNA methylation profile to discriminate OSCC with high and low aggressive potential. Methods: 36 OSCC cases underwent neoplastic cells collection by gentle brushing of the lesion, before performing a pre-operative biopsy. The CpG islands methylation status of 13 gene (ZAP70, ITGA4, KIF1A, PARP15, EPHX3, NTM, LRRTM1, FLI1, MiR193, LINC00599, MiR296, TERT, GP1BB) was studied by bisulfite Next Generation Sequencing (NGS). A Cox proportional hazards model via likelihood-based component-wise boosting was used to evaluate the prognostic power of the CpG sites. Results: The boosting estimation identified five CpGs with prognostic significance: EPHX3-24, EPHX3-26, ITGA4-3, ITGA4-4, and MiR193-3. The combination of significant CpGs provided promising results for adverse events prediction (Brier score = 0.080, C-index = 0.802 and AUC = 0.850). ITGA4 had a strong prognostic power in patients with early OSCC. Conclusions: These data confirm that the study of methylation profile provides new insights into the molecular mechanisms of OSCC and can allow a better OSCC prognostic stratification even before surgery.
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Mao, Xiaohong, Xin Zhang, Xiaowei Zheng, Yongwu Chen, Zixue Xuan, and Ping Huang. "Curcumin suppresses LGR5(+) colorectal cancer stem cells by inducing autophagy and via repressing TFAP2A-mediated ECM pathway." Journal of Natural Medicines 75, no. 3 (March 13, 2021): 590–601. http://dx.doi.org/10.1007/s11418-021-01505-1.
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Abstract Colorectal cancer stem cells (CSCs) have the potential for self-renewal, proliferation, and differentiation. And LGR5 is a stem cell marker gene of colorectal cancer. Curcumin can suppress oncogenicity of many cancer cells, yet the effect and mechanism of curcumin in LGR5(+) colorectal cancer stem cells (CSCs) have not been studied. In this study, we studied the effect of curcumin on LGR5(+) colorectal CSCs using the experiments of tumorsphere formation, cell viability and cell apoptosis. Then autophagy analysis, RNA-Seq, and real-time PCR were used to identify the mechanism responsible for the inhibition of LGR5(+) colorectal CSCs. Our results showed that curcumin inhibited tumorsphere formation, decreased cell viability in a dose-dependent manner, and also promoted apoptosis of LGR5(+) colorectal CSCs. Next, we found curcumin induced autophagy of LGR5(+) colorectal CSCs. When LGR5(+) colorectal CSCs were co-treated with curcumin and the autophagy inhibitor (hydroxychloroquine), curcumin-induced cell proliferation inhibition decreased. In addition, we also found that curcumin inhibited the extracellular matrix (ECM)-receptor interaction pathway via the downregulation of the following genes: GP1BB, COL9A3, COMP, AGRN, ITGB4, LAMA5, COL2A1, ITGB6, ITGA1, and TNC. Further, these genes were transcriptionally regulated by TFAP2A, and the high expression of TFAP2A was associated with poor prognosis in colorectal cancer. In conclusion, curcumin suppressed LGR5(+) colorectal CSCs, potentially by inducing autophagy and repressing the oncogenic TFAP2A-mediated ECM pathway. Graphic abstract
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Markham, Stephen J., Lisa Bevilaqua, Haley Zarrin, Donna McDonald-McGinn, Elaine Zackai, and Michele P. Lambert. "Detecting 22q11.2 Deletion Syndrome Using Flow Cytometry." Blood 124, no. 21 (December 6, 2014): 4207. http://dx.doi.org/10.1182/blood.v124.21.4207.4207.
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Abstract The 22q11.2 deletion syndrome (DS) is a multisystem chromosomal deletion disorder with pleotropic phenotypic presentations that may be subtle. We examine retrospectively whether we can support the diagnosis of 22q11.2DS by flow cytometric analysis of platelets for surface CD42 (GPIb/V/IX) as ~89% of these patients (with the classical deletion) should have a deficiency of GPIbbeta due to loss of one copy of the GPIBB gene. Loss of one copy of GPIBB may result in macrothrombocytopenia and a mild bleeding diathesis. We characterized the bleeding manifestations and the level of expression of the CD42 complex in patients with known 22q11.2 deletion to determine if CD42 levels could be used to assess bleeding risk. We evaluated 56 patients with 22q11.2 deletion and compared them to age-matched controls with normal platelet counts and no bleeding as well as with controls evaluated for concern for platelet disorder (platelet disorder subjects – PDS), mostly other thrombocytopenias. Comparisons were also made for other platelet parameters. Expression of the CD42b (GP1bb) varied in individuals with the 22q11.2 deletion. Although only 30/56 (54%) subjects had low CD42 expression (<70% control), CD42 expression overall in the 22q11.2 deleted patients was significantly lower than in PDS (69±5% vs 93±5% of PDS, p<0.002) or in normal controls (69±5% vs 100±5% of WT, p<0.001). For the total population (22q11.2 DS and PDS subjects), MPV and platelet count were inversely related (R2 0.18, p<0.001), but there was no correlation with either CD41 or CD42 expression. There was no significant difference in MPV or platelet count between PDS and 22q11.2DS subjects. When compared to normal control subjects, MPV was significantly higher in 22q11.2DS subjects compared to normal controls (9.5±0.3 vs 7.8±0.3, p< 0.001) and the platelet count was significantly lower (187±12 vs 295±16, p<0.001). We also examined the expression of CD41 (GPIIb) in subjects with 22q11.2DS as it would be expected that in macrothrombocytes expression levels of CD41 might be increased compared to controls since the platelets are larger than control platelets. However, there was no overall increase in CD41 expression compared to PDS subjects. However, the ratio of normalized CD41 expression to CD42 expression in subjects with 22q11.2 deletion was inversely correlated with MPV (-0.31, p=.02) and was significantly lower than in other platelet disorders (5.2±0.6 vs 4.3±0.6, p<0.05). Bleeding manifestations were reported in 19/56 patients with 22qDS and of these 9/19 (47%) had low expression of CD42b and did not correlate with level of expression of CD42 (p NS). Taken together these data demonstrate that macro-thrombocytopenia (MPV >9, plt <150) with low expression of CD42 (<70% control) and high normalized CD41:CD42 ratio (>7), the diagnosis of 22q11.2 deletion syndrome results in a specificty of 85% to detect 22q11.2DS with a positive predictive value of 67% when at least 3 of the 4 criteria are present. We suggest that if subjects evaluated for thrombocytopenia manifest this constellation, appropriate genetic testing should be considered and sent, especially given that the manifestations of the disorder can be subtle, but the consequences for management are significant. Future studies will focus on prospectively evaluating all subjects with the combination of decreased CD42 expression, macrothrombocytopenia and low normalized CD42:CD41 ratio to see if this combination can predict the presence of 22q11.2 deletion. Disclosures No relevant conflicts of interest to declare.
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Rivera, Candido E., Prakash Vishnu, Gretchen Schaef Johns, Rajiv K. Pruthi, and Dong Chen. "Identification of a Novel Heterozygous Mutation (c.2213T>G;p.Leu738Arg) in Platelet Glycoprotein ITGB3 gene in a Patient with Glanzmann's Thrombasthenia." Blood 132, Supplement 1 (November 29, 2018): 1158. http://dx.doi.org/10.1182/blood-2018-99-117995.
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Abstract BACKGROUND: Glanzmann's thrombasthenia (GT) is an inherited platelet disorder (IPD) that leads to clinically significant bleeding. Platelets from patients with GT can show qualitative or quantitative defects of platelet membrane glycoprotein (GP) IIb/IIIa complex. Most GT are caused by autosomal recessive genetic defects in ITGA2B and ITGB3 (genes for GPIIb and GPIIIa, respectively) with rare cases showing an autosomal dominant pattern. Accurate diagnosis of GT requires a constellation of both phenotypic and genetic studies. Here we report a unique case of autosomal dominant GT resulted from thorough phenotypic and genetic studies. PATIENTS AND METHODS: A 19 year-old woman was recently evaluated for life-long history of easy bruising and severe menorrhagia that was only partially responsive to medroxyprogesterone. She has severe thrombocytopenia first recognized shortly after birth with a platelet count around 20x109/L. Platelet size was normal. She was presumed to have immune-mediated thrombocytopenia (ITP) since early infancy, but lacking responsiveness to the conventional treatments of ITP including immunomodulation, splenectomy and thrombopoietin receptor agonists. Family history was negative for a bleeding diathesis. Both von Willebrand factor antigen and activity were within normal range. Bone marrow aspirate and biopsy with associated chromosome studies were all normal. Platelet surface glycoprotein assessment by flow cytometry using antibodies to GP IIb, IIIa, Ia, Ib-a, VI, IX, and whole exome sequencing (WES) utilizing a predefined list of 53 clinically significant genes* related to genetically IPDs were performed. Due to thrombocytopenia platelet aggregation studies could not be performed. METHODS/RESULTS: Platelet surface expression of GPIIb (CD41) and GPIIIa (CD61) were markedly decreased suggestive of a variant of GT (Figure). WES performed with Illumina HiSeq 2500 sequencing system by using Agilent SureSelelct CRE kit V1 to capture and target the exonic regions showed presence of a heterozygous mutation in ITGB3 gene (c2213T>G; p.Leu738Arg). By in silico prediction modeling, this mutation is likely to be pathogenic and results in the substitution of hydrophobic leucine with hydrophilic arginine in the transmembrane domain of the β3 subunit of alpha IIb/beta 3 integrin (αIIbβ3), the platelet receptor that binds to fibrinogen. Interestingly, GPVI is also decreased which may be associated with GPIIb and GPIIIa deficiency or due to accelerated shedding since no GPVI mutation was identified. CONCLUSION: We describe a patient with GT associated with a novel heterozygous autosomal dominant mutation in ITGB3 gene with substitution of leucine with arginine (c2213T>G; p.Leu738Arg). This deletion caused a low expression of αIIbβ3 integrin on her platelets surface and severe thrombocytopenia. This case underscores the importance of an integrated phenotyping and genotyping approach to render a definitive diagnosis of an IPD. *Platelet exome gene list: ABCG5, ABCG8, ACBD5, ACTN1, ANKRD26, ANO6, AP3B1, BLOC1S3, BLOC1S6, CYCS, DTNBP1, ETV6, FLI1, FLNA, GATA1, GFI1B, GP1BA, GP1BB, GP6, GP9, HOXA11, HPS1, HPS3, HPS4, HPS5, HPS6, ITGA2B, ITGB3, LYST, MASTL, MPL, MYH9, NBEAL2, ORAI1, PSRX1, P2RY12, PLA2G4A, PRKACG, RASGRP2, RBM8A, RUNX1, STIM1, STX11, STXBP2, TBXA2R, TBXAS1, THPO, TUBB1, UNC13D, VIPAS39, VPS33B, VPS45, WAS Figure Figure. Disclosures No relevant conflicts of interest to declare.
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Arter, Zhaohui Liao, Caitlin Yatogo, Michael C. Chicka, and Jeffrey L. Berenberg. "The Mystery of "Magic Blood" - Familial Macrothrombocytopenia Associated with a Novel Variant in GP1BA Gene." Blood 134, Supplement_1 (November 13, 2019): 2380. http://dx.doi.org/10.1182/blood-2019-122384.
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Introduction: Inherited macrothrombocytopenias comprise a heterogeneous group of rare inherited disorders characterized by decreased platelet count with enlarged platelet size. Glycoprotein Ib (GP Ib) is a platelet surface membrane glycoprotein that is encoded by GP1BA gene, and functions as a receptor for von Willebrand factor (VWF). Mutations in the GP1BA gene are seen in Bernard-Soulier syndrome (BSS). Here we describe a family with an isolated giant platelet disorder and a novel variant in the GP1BA gene following an autosomal dominant mode of inheritance. In our kindred this variant was not associated with clinical history or specific laboratory evidence of BSS. Case Presentation: 34-year-old female reported first being diagnosed with macrothrombocytopenia at the age of 13 on routine blood work. The patient and 11 of her relatives spanning 5 generations have reported asymptomatic macrothrombocytopenia (Figure 1), described as "Magic Blood" by her family. Her platelet count fell below 20,000/microL during one of her pregnancies. She was treated as idiopathic thrombocytopenic purpura (ITP) and was given corticosteroids to increase platelet count without improvement. On presentation, patient's complete blood count was significant for low platelet at 55,000/microL and macrothrombocytes were observed on blood smear. Methods/Results: VWF assays, including Factor VIII activity, VWF Ag, and Ristocetin Cofactor, were within normal limits. VWF multimer analysis revealed a normal pattern and distribution of bands. Patient had a normal platelet aggregation in response to ADP, Collagen, Epinephrine, Ristocetin and Arachidonate. Flow Cytometry detected normal GP Ib and GP IIb/ IIIa expression. Whole exome sequencing and copy number analysis of 29 genes associated with thrombocytopenia revealed a c.97T>G substitution in the GP1BA gene predicted to result in the amino acid substitution p.Cys33Gly (Figure 2). To our knowledge, this variant has not been reported in the literature or public databases. To confirm this novel variant is the cause of the familiar macrothrombocytopenia, two relatives with macrothrombocytopenia, a maternal uncle and a first cousin, also underwent genetic testing and were found to have the same variant. Discussion: Variants in GP1BA are associated with both autosomal dominant and recessive forms of BSS and with autosomal dominant platelet-type VWD. Our kindred is surprisingly asymptomatic given the location and specific amino acid substitution generated by the variant. Amino acid residue p.Cys33 resides in an extracellular N-terminal domain that is critical for VWF binding and proper assembly of the GP1B-IX complex. A heterozygous substitution involving the same amino acid residue defined as p.Cys33Arg was observed in two patients with macrothrombocytopenia with no reported bleeding complications (MC, unpublished data). Substitution of a nearby cysteine residue defined as p.Cys20Gly has been reported in a case of monoallelic chronic macrothrombocytopenia without bleeding diathesis similar to our patient. Amino acid residues p.Cys20 and p.Cyc33 are highly conserved among divergent species and substitution of these amino acid residues appears to not be tolerated. Substitution of other cysteine amino acid residues in the extracellular domain of the GP1BA protein (p.Cys81 and p.Cys225) has also been reported in patients with biallelic BSS, suggesting that perturbation of cysteine amino acid residues is likely to affect protein structure and function. Conclusion:We think the p.Cys33Gly substitution found in our patient and her relatives is likely to be a primary cause of monoallelic GP1BA-associated macrothrombocytopenia. It is important to distinguish inherited macrothrombocytopenia from ITP in order to avoid unnecessary and potentially toxic treatment. Disclosures No relevant conflicts of interest to declare.
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Lian, Zheng, Milind Mahajan, Vincent Schulz, Erol Gulcicek, Diane Krause, and Sherman M. Weissman. "Intermediate Steps In Erythroid, Megakaryocytic and Myeloid Lineage Specification." Blood 116, no. 21 (November 19, 2010): 4778. http://dx.doi.org/10.1182/blood.v116.21.4778.4778.
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Abstract Abstract 4778 Normal human CD34 positive precursor cells can be expanded in vitro and converted with high efficiency into maturing erythroid cells or myeloid cells by treating the cells with erythropoietin (EPO) or with G-CSF, respectively. The maturation of megakaryocytic cells can also be induced from human CD34 positive precursor cells by treatment with thrombopoietin (TPO) with lower efficiency. The induction time varies from 9 to 12 days depending on the lineages. Total RNAs were extracted every other day during the induction period for the purpose of gene profiling at intermediate steps of the differentiation. The samples were analyzed with the “Whole Transcriptome Shotgun Sequencing (RNA-seq)” method. Comparison of the gene expression among these three lineages at different stages of differentiation shows complex changes of a substantial number of genes in categories including transcription factors, cytokines etc. Basically, many genes change similarly in both erythroid and megakaryocytic lineage differentiation, the number of this shared genes are much less when comparing myeloid cells with one of these lineages. Initially during erythropoietin treatment, a large number of megakaryocyte mRNAs are upregulated to levels comparable to those of the erythroid specific genes, or to those seen in precursor cells treated with thrombopoietin. After several days of erythropoietin treatment, most of the megakryocyte related genes are down regulated, while there is continued upregulation of erythroid genes. Unlike many other megakaryocytic genes, the level of mRNA for the thrombopoietin receptor MPL is rapidly down-regulated suggesting that erythroid lineage commitment occurs prior to silencing of many megakaryocytic genes. Comparison of the linage specific genes identified groups of cell surface proteins which may be applied as new markers for sorting various cell populations. Some of these include DARC, LEPR, TFRC, BMPR2, TFR2, AXL, EPHB4, IL15RA, TNFRSF19, FZD5, ULBP1, CELSR2, RYK, MRGPRE, CRY1 which are specific for erythroid lineage; FPR1, EVI2A, CD14, and others totaling 50 proteins for myeloid lineage and more than 80 highly expressed surface markers such as GP1BA, F2RL3, GP1BB, ADRA2A, GABRE unique for megakaryocytic lineage. In parallel, proteomic analysis was carried out with an Isotope coded affinity tag (ICAT)-based protein profiling and Multiplexed Isobaric Tagging Technology (iTRAQ™). Statistical analysis shows good correlation between the relative changes of RNA and protein expression levels during the erythropoiesis, although there were large discrepancies in the relative levels of mRNA and protein at any one time point. Only those genes with very low or very high RNA values show the poor correlations. CTCF and its associated factor Rad21 are involved in the establishment of enhancer boundary elements and chromosomal conformation. We have begun genome wide analysis of these factors during erythroid and myeloid cell growth and differentiation. In this project, we also aimed to investigate the modulation mechanisms of CTCF during the CD34 positive cells differentiation into different lineages. Taking the advantage of the ChIP-Sequencing (ChIP-Seq) technology, we found that progressive changes in the sites of binding of the DNA binding protein CTCF occurs, and are not limited to the regions embedding erythroid genes, suggesting that there is a lineage specific global reorganization of chromatin. In addition to transcriptional controls, there are prominent post-transcriptional effects regulating the expression of key transcription factors including CTCF. Through this study, the regulation mechanisms of the chromatin remodeling, signal pathways and other, molecular events during hematopoiesis will be described in detail. Disclosures: No relevant conflicts of interest to declare.
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Onundarson, Pall Torfi, Elisabet R. Birgisdottir, Gudrun Bragadottir, Bylgja Hilmarsdottir, Brynja Gudmundsdottir, Brynjar Vidarsson, and Magnus K. Magnusson. "Bernard-Soulier in Iceland. Bleeding Symptoms and Platelet Parameters in Patients, Carriers and Controls." Blood 108, no. 11 (November 16, 2006): 1095. http://dx.doi.org/10.1182/blood.v108.11.1095.1095.
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Abstract Bernard Soulier syndrome is a rare platelet disorder characterized by macrothrombocytopenia and absence of platelet aggregation in response to ristocetin due to inherited structural abnormalities in the glycoptein 1B complex. Due to the rarity of this disorder no systematic controlled evaluation has been reported to define the hematological abnormalities and clinical symptoms of these patients. In order to evaluate these findings associated with both Bernard Soulier syndrome and carrier status of glycoprotein 1B-alpha (GP1Bα) mutations we have conducted a nation-wide study in Iceland. All patients with Bernard Soulier syndrome diagnosed in Iceland are referred to the Landspitali University Hospital hemostasis center, a total of twelve patients. Of these, ten were available for the study along with 21 heterozygote carriers (first or second degree relatives) and 25 normal controls. All participating subjects answered a detailed questionnaire on bleeding history and had blood tests, including a complete blood count, coagulation tests, platelet aggregation, PFA-100 closure times, platelet flow cytometry, and DNA analysis to define the underlying GP1Bα mutational status. A diagnosis of Bernard Soulier syndrome was confirmed by platelet ristocetin aggregation and flow cytometry in all patients. Of these ten patients, seven were homozygous for a specific Icelandic mutation, T283 to C (Cys65 to Arg) in the conserved leucine-rich repeats within the ligand-binding region of platelet GP1Bα. Two patients had compound heterozygosity, Cys65 to Arg and a previously described Swedish mutation (Karlstad mutation), G1584 to A (Trp 498 to Stop) with both mutations in GP1Bα. One patient was compound heterozygous for the Karlstad mutation as well as an uncharacterized mutation. Of the 21 carriers, 14 had the Icelandic mutation and 6 had the Karlstad mutation and one had the uncharacterized mutation. As expected all patients had very significant macrothrombocytopenia with no expression of the GP1B complex on flow cytometry and absolute absence of ristocetin aggretion response. Compared to normal controls, the BS patients reported excessive bleeding manifested by markedly increased bruisability, prolonged small cut bleeding and increased mucosal bleeding (epistaxis, oral cavity, menstruation). Heterozygote carriers also reported moderately increased bruisability compared to normal controls, longer bleeding from small cuts and increased mucosal bleeding (epistaxis, oral cavity). On blood analysis the carriers had significantly lower platelets counts (189 vs. 266 × 109/L, p<0.0001 ) and larger platelets (9.6 fl vs. 7.8 fl, p<0.0001 ) than controls. The PFA-100 closure times (both CT c/epi and CT c/ADP) were markedly prolonged in all patients while carriers and controls had similar normal values. In this nation-wide study of Bernard Soulier syndrome we have characterized the clinical and hematological symptoms of patients and carriers. As expected the patients have significant clinical mucosal bleeding symptoms and hematological abnormalities. Interestingly we find evidence of mild platelet dysfunction in heterozygote carriers marked by symptoms of increased mucosal bleeding and lower platelet counts as well as larger platelets than controls.
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Bastida, Jose Maria, Mónica del Rey, Rocío Benito, Isabel Sanchez-Guiu, Susana Riesco, Maria Jesús Peñarrubia, Rosa Fisac, et al. "Design and Validate of Next-Generation Sequencing Panel for Inherited Platelet Disorders." Blood 124, no. 21 (December 6, 2014): 4210. http://dx.doi.org/10.1182/blood.v124.21.4210.4210.
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Abstract Introduction The inherited platelet disorders (IPD) are a heterogeneous group of rare diseases including quantitative and/or qualitative platelet defects. Classically, patients with IPD are first functionally tested to know the possible defect before sequencing a single or a few genes. Phenotipyc diagnostic of IPD often requires light transmission aggregometry, quantitative analysis of receptors by flow cytometry and fluorescence and electron microscopy. This diagnostic strategy is complex, poorly standardised and time consuming. In addition, the phenotype can seldom guide the singles candidates genes for conventional Sanger squencing. Therefore, many patients remain without a accurate diagnosis of their IPD. Next generation sequencing (NGS) enables the simultaneous analysis of large groups of candidate genes in IPD and may be useful for rapid genetic diagnosis. The aim of this study was to design and validate a NGS panel for IPD. Patients & Methods We describe a strategy for rapid genetic diagnosis of IPD with Illumina sequencing of 60 candidates genes previously associated with IPD (table1). The baits were designed to tile 400 kb of gDNA sequence corresponding to the exons and splice sites in all known transcripts of the candidate genes identified. The bait library was tested by enriching the candidate IPD genes from 50 ng DNA obtained and sequencing by Nextera Rapid Custom Enrichment system. Results were analysed by Variant Studio system and Sequencing Analysis Viewer. A total of 21 patients were studied. For the validation process, DNA samples of 9 unrelated patients with IPD and their mutation known were used: two patients with Glanzmann Thrombasthenia (ITGA2B, p.Ala989Thr, p.Val982Met and p.Glu538Stop; ITGB3, p.Leu222Pro and p.Tyr216Cys), one Hermansky-Pudlak Sd. (HPS1, p.Glu204 Stop), another with Bernard-Soulier Sd. (GPIX, p.Phe71Stop), a case of Congenital Amegakaryocytic Thrombocytopenia (MPL, p.Arg102Cys), and 2 patients with Chediak Higashi Sd. (LYST, p.Gly3725Arg and p.Cys258Arg). Once validated, the NGS panel was used for genetic diagnostic of 8 patients with suspected IPD. Results Eleven mutations, previously identified in another center by conventional sequencing, were detected by our panel NGS (100% success in the validation process). We then tested this strategy for patients with suspected of IPD without diagnosis: I. a 13 years old girl with agenesis of the corpus callosum, facial dysmorphia, renal agenesis and thrombocytopenia was diagnosed of Thrombocytopenia FLNA-related and Periventricular Nodular Heterotopia (PNHV)[mutation in the FLNA was detected (p.Thr1232Ile)]. II. A two years old patient with severe thrombocytopenia and recurrent infections was diagnosed of Wiskott-Aldrich Sd (WAS, p.Arg268Gly fs Stop40). III. A patient with deafness, macrothrombocytopenia, and Döhle bodies was diagnosed by MYH9 deletion (MYH9; p.Asp1925Thr fs Stop23). IV. Six members of a family (2 of them with symptoms of mucocutaneous bleeding, and macrothrombocytopenia), in which an insertion in NBAL2 (p.Gly1142Arg fs Stop49) gene was found. Therefore, Gray Platelet Sd was diagnosed. Moreover, one patient with aspirin-like syndrome showed a P2RY12 mutation (p.Val279Met). Finally, mother and son with mild Hemophilia A (F8; p.Gln2208Arg) were detected. Conclusions This NGS panel enables a rapid genetic diagnostic of IPD. The use of NGS-based strategy is a feasible tool for the diagnosis of IPD that could be added to the screening of these disorders. Five mutations have not previously been described in the literature. Table 1: Sixty candidates' genes previously associated with IPD: Inherited Platelet Disorders Genes = 60 Cytoskeletal Assembly and Structural Proteins GP1BA, GP1BB, GP5, A2M, GP9, VWF, ITGA2, ITGA2B, ITGB3, ABCA1, ANO6,FERMT3, ACTN1, MASTL Disorders of agonist platelet receptors P2RX1, P2RY1, P2RY12, TBXA2R, TBXAS1, ADRA2A, GP6, CD36 o GP4, DTNBP1 Disorders signal transduction GNAI3, GNAQ, GNAS, PLA2G7, PLCB2PTS, GGCX, DPAGT1, DHCR24 Disorders of platelet granules NBEAL2, GFI1B, PLAU, HPS1, HPS3, HPS4, HPS5, HPS6, LYST, MLPH, BLOC1S3, BLOC1S6, AP3B1, VIPAS39, VPS33B, RAB27A, MYO5A, USF1 Thrombocytopenias and syndromes WAS, MYH9, FLNA, FLI1, STIM1, HOXA11, ANKRD26, MPL, RBM8A RUNX1, GATA1 Disclosures No relevant conflicts of interest to declare.
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Kitamura, Katsumasa, Yusuke Okuno, Kenichi Yoshida, Masashi Sanada, Yuichi Shiraishi, Hideki Muramatsu, Ryoji Kobayashi, et al. "Functional Characterization of a Novel GFI1B Mutation Causing Congenital Macrothrombocytopenia." Blood 126, no. 23 (December 3, 2015): 75. http://dx.doi.org/10.1182/blood.v126.23.75.75.
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Abstract Congenital macrothrombocytopenia (CMTP) is a heterogeneous group of disorders characterized by abnormally large platelets and low platelet counts. So far, genes responsible for CMTP have been identified in approximately half of the cases. The genes currently known are MYH9, GP1BA, GP1BB, GP9, ITGA2B, ITGB3, ACTN1 and other less frequent genes. However, in the remaining half, CMTP-causing genes are unknown. To identify genes causing CMTP, we first performed whole exome sequencing and target sequencing in 46 Japanese CMTP families and identified 1 family with a heterozygous mutation in GFI1B gene. Several groups have recently reported GFI1B as a causative gene for CMTP with alpha-granule deficiency and red cell anisopoikilocytosis. Here, we report on the identification of a novel mutation in GFI1B gene and its functional characterization. We performed whole exome sequencing and additional targeted sequencing in genetically undefined 46 CMTP families and identified 1 family with GFI1B variant (Figure), which co-segregated with CMTP. The proband (arrow in the Figure) of this family was a 15 years old boy with a history of easy bruising and platelet count of 70-115x109/L. His mother also had low platelet count, although details of her bleeding complications are unknown. Morphologic analysis of peripheral blood smear from the affected family members showed gray appeared enlarged platelets and red cell anisocytosis. Subsequent sequencing analysis confirmed a heterozygous point mutation (c.G814+1C, p.G272fsX274) at the splice donor site flanking exon 6 of GFI1B. GFI1B contains six zinc fingers (Znfs), of which Znf 3 to 5 are essential for DNA binding. The mutated transcript predicted a truncation of 58 C-terminal amino acids, which resulted in complete deletion of Znf 5. Since previous reports on GFI1B mutations have also described mutations of Znf 5 (p.H294fsX307 and p.Q287X mutations by Stevenson et al and Monteferrario et al, respectively), we performed biological analyses of these 3 patient-derived mutations affecting essential DNA-binding domain. GFI1B is an essential transcriptional factor for megakaryocyte and erythroid development. Therefore, we tested the effects of the GFI1b mutations on the transcriptional repression of firefly luciferase reporter constructs containing GFI1B promoter as a validated GFI1B target. Whereas wild-type GFI1B repressed the expression of reporter gene, the G272fsX274, H294fsX307 and Q287X mutants were unable to repress the expression. To test whether these patient-derived mutations acted in dominant-negative manner, we measured the effect of increasing the amount of mutant GFI1B cDNA, cotransfected into cells with a set amount of wild-type GFI1B cDNA. All 3 mutants inhibited gene repression mediated by wild-type GFI1B in a dose-dependent manner. To investigate biological effects of mutant GFI1B on proplatelet formation from megakaryocytes, we expressed the mutant GFI1B in mouse fetal liver cells (FLCs) by retrovirus-mediated gene transfer. FLCs harvested from embryonic day 13.5 embryos were transfected with pGCDNsamIRES/EGFP vector containing wild-type or the 3 patient-derived mutant GFI1B and were cultured with thrombopoietin. And we observed proplatelet formation of EGFP positive megakaryocytes. The number of proplatelet-producing megakaryocytes was reduced in mutant GFI1B transduced FLCs on day 3 and 4 after transfection, suggesting that mutant GFI1B inhibited the rate of proplatelet production from megakaryocytes. Next, we investigated the size and number of proplatelet tips from megakaryocytes, and found that proplatelet tips produced from G272fsX274, H294fsX307 and Q287X-transduced megakaryocytes were significantly reduced in numbers, but were increased in the size of the tips. This was compatible with decreased platelet count and enlarged platelet seen in GFI1B mutated patients. These findings indicated that patient-derived mutant GFI1B disrupted proplatelet formation of megakaryocyte. In summary, we have identified a novel GFI1B mutation that causes autosomal dominant macrothrombocytopenia with gray platelets and red cell anisocytosis. The mutant GFI1B acted in a dominant-negative manner over wild-type GFI1B. In addition, we first showed that the mutant-transduced FLCs produced abnormally enlarged proplatelet tips, which was reduced in numbers. Disclosures No relevant conflicts of interest to declare.
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Nagy, Zoltan, Timo Vögtle, Mitchell J. Geer, Jun Mori, Silke Heising, Giada Di Nunzio, Ralph Gareus, et al. "The Gp1ba-Cre transgenic mouse: a new model to delineate platelet and leukocyte functions." Blood 133, no. 4 (January 24, 2019): 331–43. http://dx.doi.org/10.1182/blood-2018-09-877787.
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Abstract Conditional knockout (KO) mouse models are invaluable for elucidating the physiological roles of platelets. The Platelet factor 4-Cre recombinase (Pf4-Cre) transgenic mouse is the current model of choice for generating megakaryocyte/platelet-specific KO mice. Platelets and leukocytes work closely together in a wide range of disease settings, yet the specific contribution of platelets to these processes remains unclear. This is partially a result of the Pf4-Cre transgene being expressed in a variety of leukocyte populations. To overcome this issue, we developed a Gp1ba-Cre transgenic mouse strain in which Cre expression is driven by the endogenous Gp1ba locus. By crossing Gp1ba-Cre and Pf4-Cre mice to the mT/mG dual-fluorescence reporter mouse and performing a head-to-head comparison, we demonstrate more stringent megakaryocyte lineage-specific expression of the Gp1ba-Cre transgene. Broader tissue expression was observed with the Pf4-Cre transgene, leading to recombination in many hematopoietic lineages, including monocytes, macrophages, granulocytes, and dendritic and B and T cells. Direct comparison of phenotypes of Csk, Shp1, or CD148 conditional KO mice generated using either the Gp1ba-Cre or Pf4-Cre strains revealed similar platelet phenotypes. However, additional inflammatory and immunological anomalies were observed in Pf4-Cre-generated KO mice as a result of nonspecific deletion in other hematopoietic lineages. By excluding leukocyte contributions to phenotypes, the Gp1ba-Cre mouse will advance our understanding of the role of platelets in inflammation and other pathophysiological processes in which platelet-leukocyte interactions are involved.
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Rabitzsch, G., J. Mair, P. Lechleitner, F. Noll, U. Hofmann, E. G. Krause, F. Dienstl, and B. Puschendorf. "Immunoenzymometric assay of human glycogen phosphorylase isoenzyme BB in diagnosis of ischemic myocardial injury." Clinical Chemistry 41, no. 7 (July 1, 1995): 966–78. http://dx.doi.org/10.1093/clinchem/41.7.966.
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Abstract With a new immunoenzymometric assay we measured human glycogen phosphorylase isoenzyme BB (GPBB) in 116 healthy individuals, 14 patients with stable angina, 107 nontraumatic chest pain patients on admission to the emergency department [45 acute myocardial infarction (AMI), 49 unstable angina, 13 other diseases], and in serial samples from 41 AMI patients. GPBB was compared with creatine kinase (CK), CKMB mass, myoglobin, and cardiac troponin T. Receiver-operating characteristic plots demonstrated the significantly greater (P < or = 0.012) discriminatory power of GPBB to detect acute ischemic coronary syndromes compared with all other tested markers. GPBB was the most sensitive marker for detection of AMI during the first 4 h after onset of chest pain, and only GPBB was increased above the upper reference limit (7 micrograms/L) on admission in patients who had unstable angina at rest and reversible ST-T alterations. This and the high early sensitivity of GPBB are most likely explained by its function as a key enzyme of glycogenolysis.
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Park, Kwang-Yeol, Ilknur Ay, Ross Avery, Juan Alfredo Caceres, Matthew S. Siket, Octavio M. Pontes-Neto, Hui Zheng, et al. "New biomarker for acute ischaemic stroke: plasma glycogen phosphorylase isoenzyme BB." Journal of Neurology, Neurosurgery & Psychiatry 89, no. 4 (October 13, 2017): 404–9. http://dx.doi.org/10.1136/jnnp-2017-316084.
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BackgroundGlycogen phosphorylase is the key enzyme that breaks down glycogen to yield glucose-1-phosphate in order to restore depleted energy stores during cerebral ischaemia. We sought to determine whether plasma levels of glycogen phosphorylase BB (GPBB) isoform increased in patients with acute ischaemic stroke (AIS).MethodsWe studied plasma GPBB levels within 12 hours and again at 48±24 hours of symptom onset in 172 patients with imaging-confirmed AIS and 133 stroke-free individuals. We determined the ability of plasma GPBB to discriminate between cases and controls and examined the predictive value of plasma GPBB for 90-day functional outcome, 90-day survival and acute lesion volumes on neuroimaging.ResultsThe mean (SD) GPBB levels were higher in cases (46.3±38.6 ng/mL at first measurement and 38.6±36.5 ng/mL at second measurement) than in controls (4.1±7.6 ng/mL, p<0.01 for both). The area under the receiver operating characteristic (ROC) curve for case–control discrimination based on first GPBB measurement was 0.96 (95% CI 0.93 to 0.98). The sensitivity and specificity based on optimal operating point on the ROC curve (7.0 ng/mL) were both 93%. GPBB levels increased in 90% of patients with punctate infarcts (<1.5 mL) and in all patients admitted within the first 4.5 hours of onset. There was no correlation between GPBB concentration and either clinical outcome or acute infarct volume.ConclusionGPBB demonstrates robust response to acute ischaemia and high sensitivity for small infarcts. If confirmed in more diverse populations that also include stroke mimics, GPBB could find utility as a stand-alone marker for acute brain ischaemia.
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Bray, Paul F., Timothy D. Howard, Eric Vittinghoff, David C. Sane, and David M. Herrington. "Effect of genetic variations in platelet glycoproteins Ibα and VI on the risk for coronary heart disease events in postmenopausal women taking hormone therapy." Blood 109, no. 5 (November 14, 2006): 1862–69. http://dx.doi.org/10.1182/blood-2006-03-013151.
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Abstract Millions of women still use postmenopausal hormone therapy (HT). We genotyped 2090 women in Heart and Estrogen/progestin Replacement Study for functional polymorphisms in GP1BA and GP6 and assessed the coronary heart disease (CHD) event rate over 5.8 years of follow-up. In patients receiving placebo, there was an increased CHD death/myocardial infarction (MI)/unstable angina (UA) event rate in carriers of the GP1BA −5C allele (adjusted [adj] P = .006). HT increased the hazard ratio (HR) of CHD events in patients with the GP1BA −5TT genotype by 16% and reduced the HR in patients with the TC+CC genotypes by 46% (adj interaction P < .001). HT reduced the HR in patients with the GP6 13254TT genotype by 17% but increased the HR in patients with the TC+CC genotypes by 35% (adj interaction P < .001). Furthermore, HT increased the HR of CHD events in patients with the GP1BA −5TT plus GP6 13254TC+CC genotypes by 57% and reduced the HR in patients with the GP1BA −5TC+CC plus GP6 13254TT genotypes by 55% (adj interaction P < .001). In postmenopausal women with established CHD, these polymorphisms of platelet genes were predictors of CHD events and significantly modified the effects of HT on CHD risk. It will be important to replicate these findings in other studies.
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Uddin, Md Main, Mostafa M. H. Ibrahim, and Karen P. Briski. "Glycogen Phosphorylase Isoform Regulation of Ventromedial Hypothalamic Nucleus Gluco-Regulatory Neuron 5′-AMP-Activated Protein Kinase and Transmitter Marker Protein Expression." ASN Neuro 13 (January 2021): 175909142110350. http://dx.doi.org/10.1177/17590914211035020.
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Brain glycogen is remodeled during metabolic homeostasis and provides oxidizable L-lactate equivalents. Brain glycogen phosphorylase (GP)-brain (GPbb; AMP-sensitive) and -muscle (GPmm; norepinephrine-sensitive) type isoforms facilitate stimulus-specific control of glycogen disassembly. Here, a whole animal model involving stereotactic-targeted delivery of GPmm or GPbb siRNA to the ventromedial hypothalamic nucleus (VMN) was used to investigate the premise that these variants impose differential control of gluco-regulatory transmission. Intra-VMN GPmm or GPbb siRNA administration inhibited glutamate decarboxylate65/67 (GAD), a protein marker for the gluco-inhibitory transmitter γ--aminobutyric acid (GABA), in the caudal VMN. GPbb knockdown, respectively overturned or exacerbated hypoglycemia-associated GAD suppression in rostral and caudal VMN. GPmm siRNA caused a segment-specific reversal of hypoglycemic augmentation of the gluco-stimulatory transmitter indicator, neuronal nitric oxide synthase (nNOS). In both cell types, GP siRNA down-regulated 5′-AMP-activated protein kinase (AMPK) during euglycemia, but hypoglycemic suppression of AMPK was reversed by GPmm targeting. GP knockdown elevated baseline GABA neuron phosphoAMPK (pAMKP) content, and amplified hypoglycemic augmentation of pAMPK expression in each neuron type. GPbb knockdown increased corticosterone secretion in eu- and hypoglycemic rats. Outcomes validate efficacy of GP siRNA delivery for manipulation of glycogen breakdown in discrete brain structures in vivo, and document VMN GPbb control of local GPmm expression. Results document GPmm and/or -bb regulation of GABAergic and nitrergic transmission in discrete rostro-caudal VMN segments. Contrary effects of glycogenolysis on metabolic-sensory AMPK protein during eu- versus hypoglycemia may reflect energy state-specific astrocyte signaling. Amplifying effects of GPbb knockdown on hypoglycemic stimulation of pAMPK infer that glycogen mobilization by GPbb limits neuronal energy instability during hypoglycemia.
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Di, Jia-Yin, Zong-Xin Zhang, and Shao-Jun Xin. "Glycogen Phosphorylase Isoenzyme Bb, Myoglobin and BNP in ANT-Induced Cardiotoxicity." Open Life Sciences 13, no. 1 (December 31, 2018): 561–68. http://dx.doi.org/10.1515/biol-2018-0067.
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AbstractAnthracyline (ANT) has been demonstrated as a useful treatment for leukemia and solid tumors. However, ANT has previously reported cardiotoxic effects, which can reduce the therapeutic index for cancer treatment. This study aimed to investigate the associations of glycogen phosphorylase isoenzyme BB (GPBB), myoglobin (Mb), and brain natriuretic peptide (BNP) with anthracycline (ANT-induced cardiotoxicity (AIC)) amongst the Chinese population. Patients suffering from leukemia were recruited. Electrocardiogram and echocardiography were used along with chemotherapy to determine left ventricular ejection fraction (LVEF), mitral ratio of peak early to late diastolic filling velocity (E/A), E-wave deceleration time (EDT), and isovolumic relaxation time (IVRT). Double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was employed to examine and compare serum GPBB, Mb, and BNP levels. Following chemotherapy, the patients presented higher levels of serum GPBB, Mb, and BNP than before chemotherapy treatment. The levels of LVEF (%), E/A, and IVRT were significantly decreased after chemotherapy, while EDT was markedly increased. The cumulative ANT dose was positively corelated to serum GPBB, Mb, and BNP levels while it was negatively corelated to LVEF levels. In conclusion, serum GPBB, Mb, and BNP levels in combination might provide higher diagnostic accuracy in the early detection of AIC compared with other single indicators.
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Skalníková, Magdalena, Kateřina Staňo Kozubík, Jakub Trizuljak, Zuzana Vrzalová, Lenka Radová, Kamila Réblová, Radka Holbová, et al. "A GP1BA Variant in a Czech Family with Monoallelic Bernard-Soulier Syndrome." International Journal of Molecular Sciences 23, no. 2 (January 14, 2022): 885. http://dx.doi.org/10.3390/ijms23020885.
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Bernard-Soulier syndrome (BSS) is a rare inherited disorder characterized by unusually large platelets, low platelet count, and prolonged bleeding time. BSS is usually inherited in an autosomal recessive (AR) mode of inheritance due to a deficiency of the GPIb-IX-V complex also known as the von Willebrand factor (VWF) receptor. We investigated a family with macrothrombocytopenia, a mild bleeding tendency, slightly lowered platelet aggregation tests, and suspected autosomal dominant (AD) inheritance. We have detected a heterozygous GP1BA likely pathogenic variant, causing monoallelic BSS. A germline GP1BA gene variant (NM_000173:c.98G > A:p.C33Y), segregating with the macrothrombocytopenia, was detected by whole-exome sequencing. In silico analysis of the protein structure of the novel GPIbα variant revealed a potential structural defect, which could impact proper protein folding and subsequent binding to VWF. Flow cytometry, immunoblot, and electron microscopy demonstrated further differences between p.C33Y GP1BA carriers and healthy controls. Here, we provide a detailed insight into its clinical presentation and phenotype. Moreover, the here described case first presents an mBSS patient with two previous ischemic strokes.
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Gollomp, Kandace, and Mortimer Poncz. "Gp1ba-Cre or Pf4-Cre: pick your poison." Blood 133, no. 4 (January 24, 2019): 287–88. http://dx.doi.org/10.1182/blood-2018-11-887513.
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Kruse, Alexandra, Alexa M. Sughroue, Patrick Morrissey, Claudia Tchatchouang, Guangheng Zhu, Marina Izak, Heyu Ni, and James B. Bussel. "Response to TPO-Receptor Agonists: Role of Immature Platelet Fraction and Anti-GP1b." Blood 124, no. 21 (December 6, 2014): 4190. http://dx.doi.org/10.1182/blood.v124.21.4190.4190.
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Abstract Background: Thrombocytopenia in Immune Thrombocytopenia (ITP) results from a combination of increased platelet destruction and reduced production, both often secondary to anti-platelet antibodies. Absolute immature platelet fraction (A-IPF) is a measure of young reticulated platelets in peripheral blood, and therefore provides an assessment of platelet production. Decreased platelet production in patients with ITP has been addressed by the recently developed Thrombopoietin Receptor Agonists (TPO-RA), which stimulate megakaryopoiesis and thereby, in responders, increase platelet production to a level which overcomes platelet destruction. The majority of ITP patients will respond to these agents, but which ones will respond is not known. Aims: To explore A) whether baseline A-IPF levels are associated with platelet response to TPO–RA, and B) whether antibodies to platelet glycoproteins 1b or β3 influence the platelet response to TPO-RA. Methods: Platelet counts and A-IPF values were collected from patients treated with TPO-RA (n=91) at Weill Medical College of Cornell University until 2013. All available counts were included, and the median count for each month was determined pre-treatment, and at 1, 2, 3, 4, 5, and 6 months. Patients were divided by whether (n=20) or not (n=71) they often received rescue therapy (i.e. IVIG and/or daily steroids >10mg). The 71 patients who received minimal or no rescue treatment were deemed responders if the average of their six median monthly platelet counts doubled from baseline and was >50x109/L. Based on pretreatment A-IPF, patients were stratified into three cohorts: low (0-25), middle (26-40), and high (41+). Clinical variables such as age, splenectomy status, duration of ITP, and gender were also studied. The 20 “rescue” patients were similarly analyzed. The 84 patients with available sera had their antibody levels measured to GP1b and β3 by Elisa by Dr. Ni. Samples were sent to the lab and analyzed without knowledge of response status or associated A-IPF level. Patients were divided into four categories for analysis of their antiplatelet antibodies: positive for anti-ß3 antibody, positive for anti-GP1b, positive for both, or negative for both. Fisher’s exact, student t-, and chi-square tests were used to analyze differences in response to TPO-RA among patient groups, A-IPF levels, and anti-platelet antibodies. Results: 71 Patients with Minimal Rescue Therapy Fifty-nine of the 71 patients responded to TPO-RA. There was no significant difference in A-IPF values for responders and non-responders (p=0.95; Figure 1). A significant positive correlation was observed between increase in A-IPF over the 6-month period and response rate to TPO-RA (p=0.009). Age, gender, duration of ITP, and splenectomy status neither correlated with response rate, nor trended with A-IPF cohorts. Figure 1 Figure 1. 20 Patients with Rescue Therapy For the 20 patients who often received rescue therapy, low response rates were seen in the low A-IPF cohort and high response rates in the higher A-IPF cohorts. The percent of responders to treatment was higher in patients with chronic ITP than with newly diagnosed and persistent ITP (p= 0.04). Age, gender, and splenectomy status showed no relationship with response to TPO-RA. In comparison with the 71 patient group, the 20 patients presented lower response rates in all groups, with the exception of females. Anti-platelet Antibodies There was a weak correlation between A-IPF and anti-GP1b antibody levels (R=0.225), demonstrating that antibodies to GP1b may have a specific impact on platelet production. Patients who did not have detectable antibody to either GP1b or β3 responded better to TPO-RA than patients who tested positive for both Anti-GP1b and Anti-ß3 (p=0.003). The strongest correlation was observed between level of anti-GP1b and response to TPO-RA (p<0.001; Figure 2). Figure 2 Figure 2. Conclusion: Rather than baseline A-IPF levels or clinical variables, the best predictor of good response to TPO-RA therapy was the absence of anti-GP1b and anti- β3 platelet antibodies, especially anti-GP1b. An explanation for the dominant effect of antibodies to GP1b is that stimulating megakaryopoiesis with TPO-RA would not effect a platelet increase as these antibodies may block proplatelet extension, preventing platelet release into the circulation. This may also explain the effect of anti-GP1b on response to steroids and IVIG. Disclosures Off Label Use: Eltrombopag is a thrombopoietin receptor agonist approved for the treatment of thrombocytopenia in adults with chronic ITP. Use in children and adolescents will be discussed.. Bussel:Amgen: Equity Ownership; GSK: Equity Ownership; Amgen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; GSK: Research Funding; Sysmex: Research Funding.
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Hamilton, Alexander, Margareth Ozelo, Jayne Leggo, Colleen Notley, Hannah Brown, Juan Pablo Frontroth, Anne Angelillo-Scherrer, et al. "Frequency of Platelet type versus Type 2B von Willebrand Disease." Thrombosis and Haemostasis 105, no. 03 (2011): 501–8. http://dx.doi.org/10.1160/th10-08-0523.
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SummaryLess than 50 patients are reported with platelet type von Willebrand disease (PT-VWD) worldwide. Several reports have discussed the diagnostic challenge of this disease versus the closely similar disorder type 2B VWD. However, no systematic study has evaluated this dilemma globally. Over three years, a total of 110 samples/data from eight countries were analysed. A molecular approach was utilised, analysing exon 28 of the von Willebrand factor (VWF) gene, and in mutation negative cases the platelet GP1BA gene. Our results show that 48 cases initially diagnosed as putative type 2B/PT-VWD carried exon 28 mutations consistent with type 2B VWD, 17 carried GP1BA mutations consistent with a PT-VWD diagnosis, three had other VWD types (2A and 2M) and five expressed three non-previously published exon 28 mutations. Excluding 10 unaffected family members and one acquired VWD, 26 cases did not have mutations in either genes. Based on our study, the percentage of type 2B VWD diagnosis is 44% while the percentage of misdiagnosis of PT-VWD is 15%. This is the first large international study to investigate the occurrence of PT-VWD and type 2B VWD worldwide and to evaluate DNA analysis as a diagnostic tool for a large cohort of patients. The study highlights the diagnostic limitations due to unavailability/poor application of RIPA and related tests in some centres and proposes genetic analysis as a suitable tool for the discrimination of the two disorders worldwide. Cases that are negative for both VWF and GP1BA gene mutations require further evaluation for alternative diagnoses.
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Annarapu, Gowtham K., Rashi Singhal, Avinash Gupta, Sheetal Chawla, Harish Batra, Tulika Seth, and Prasenjit Guchhait. "HbS Binding to GP1bα Activates Platelets in Sickle Cell Disease." PLOS ONE 11, no. 12 (December 9, 2016): e0167899. http://dx.doi.org/10.1371/journal.pone.0167899.
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Ghalloussi, Dorsaf, Noémie Saut, Denis Bernot, Xavier Pillois, Philippe Rameau, Gérard Sébahoun, Marie-Christine Alessi, Hana Raslova, and Véronique Baccini. "A new heterozygous mutation in GP1BA gene responsible for macrothrombocytopenia." British Journal of Haematology 183, no. 3 (October 30, 2017): 503–6. http://dx.doi.org/10.1111/bjh.14986.
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Othman, Maha, and Jonas Emsley. "Gene of the issue: GP1BA gene mutations associated with bleeding." Platelets 28, no. 8 (September 29, 2017): 832–36. http://dx.doi.org/10.1080/09537104.2017.1361526.
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Bury, Loredana, Emanuela Falcinelli, Haripriya Kuchi Bhotla, Anna Maria Mezzasoma, Giuseppe Guglielmini, Alexander Tischer, Laurie Moon-Tasson, Matthew Auton, and Paolo Gresele. "A p.Arg127Gln variant in GPIbα LRR5 allosterically enhances affinity for VWF: a novel form of platelet-type VWD." Blood Advances 6, no. 7 (April 1, 2022): 2236–46. http://dx.doi.org/10.1182/bloodadvances.2021005463.
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Abstract Gain-of-function (GOF) variants in GP1BA cause platelet-type von Willebrand disease (PT-VWD), a rare inherited autosomal dominant bleeding disorder characterized by enhanced platelet GPIbα to von Willebrand factor (VWF) interaction, and thrombocytopenia. To date, only 6 variants causing PT-VWD have been described, 5 in the C-terminal disulfide loop of the VWF-binding domain of GPIbα and 1 in the macroglycopeptide. GOF GP1BA variants generate a high-affinity conformation of the C-terminal disulfide loop with a consequent allosteric conformational change on another region of GPIbα, the leucine-rich-repeat (LRR) domain. We identified a novel GP1BA variant (p.Arg127Gln) affecting the LRR5 domain of GPIbα in a boy with easy bruising and laboratory test results suggestive of PT-VWD. We thus aimed to investigate the impact of the p.Arg127Gln variant on GPIbα affinity for VWF and GPIbα structure. Chinese hamster ovary cells expressing p.Arg127Gln GPIbα showed increased binding of VWF induced by ristocetin and enhanced tethering on immobilized VWF as compared with cells expressing wild-type GPIbα. Surface plasmon resonance confirmed that p.Arg127Gln enhances the binding affinity of GPIbα for VWF. Hydrogen-deuterium exchange mass spectrometry showed that p.Arg127Gln of LRR, while having little effect on the dynamics of the LRR locally, enhances the conformational dynamics of the GPIbα C-terminal disulfide loop structure. Our data demonstrate for the first time that GOF variants outside the GPIbα C-terminal disulfide loop may be pathogenic and that aminoacidic changes in the LRR may cause allosterically conformational changes in the C-terminal disulfide loop of GPIbα, inducing a conformation with high affinity for VWF.
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Woods, Adriana, Analia Sanchez-Luceros, Emilse Bermejo, Juvenal Paiva, Maria Alberto, Silvia Grosso, Ana Kempfer, and Maria Lazzari. "Identification of p.W246L As a Novel Mutation in the GP1BA Gene Responsible for Platelet-Type von Willebrand Disease." Seminars in Thrombosis and Hemostasis 40, no. 02 (January 28, 2014): 151–60. http://dx.doi.org/10.1055/s-0033-1364183.
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Platelet-type von Willebrand disease (PT-VWD) and type 2B von Willebrand disease (2B-VWD) are rare bleeding disorders characterized by increased ristocetin-induced platelet aggregation (RIPA) at low concentrations of ristocetin. Diagnosis of either condition is not easy and the differential diagnosis between the two entities is especially challenging as evidenced by high levels of misdiagnosis of both conditions, but particularly PT-VWD. Five mutations in the GP1BA gene related to PT-VWD and less than 50 patients are currently reported worldwide. We herein describe a patient with severe bleeding symptoms, macrothrombocytopenia, mild spontaneous platelet aggregation, positive RIPA at 0.3 and 0.4 mg/mL, von Willebrand factor ristocetin cofactor (VWF:RCo) to antigen (VWF:Ag) < 0.2, normal VWF propeptide/VWF:Ag ratio, and RIPA mixing tests and cryoprecipitate challenge positive for PT-VWD. GP1BA gene was studied in the patient, in his mother, and in 100 healthy control subjects. We identified a heterozygous substitution G > T located at nucleotide 3805 in the g.DNA of the patient's GP1BA gene, resulting in a Trp to Leu amino acid change at residue 246 (p.W246L). This mutation was absent in his unaffected mother and also in the 100 controls, and was predicted as damaging by in silico analysis. The residue W246 is located within the VWF-binding region and exists in a strongly conserved position in the phylogenetic tree, which is expected to be unable to tolerate substitutions without changing its functional characteristics. These findings argue strongly in favor of the view that this substitution does not represent a polymorphism and is therefore responsible for the PT-VWD phenotype of the patient.
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Sullivan, Spencer K., Jason A. Mills, Sevasti B. Koukouritaki, Karen K. Vo, Randolph B. Lyde, Prasuna Paluru, Guoha Zhao, et al. "High-level transgene expression in induced pluripotent stem cell–derived megakaryocytes: correction of Glanzmann thrombasthenia." Blood 123, no. 5 (January 30, 2014): 753–57. http://dx.doi.org/10.1182/blood-2013-10-530725.
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Key Points When targeted to a single allele of the AAVS1 locus, the Gp1ba promoter drives a high level of expression specifically to megakaryocytes. Transgene rescue in iPSCs provides a model for the return of surface αIIbβ3 expression to near-normal levels in patients with type I GT.
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Burdorf, L., T. Zhang, E. Rybak, I. I. Salles, K. Broos, E. Welty, C. Avon, et al. "295 Blocking GP1b-vWF Interaction by Anti-GP1b Fab Reduces Activation and Sequestration of Platelets in a Xenogeneic Pig Lung Perfusion Model." Journal of Heart and Lung Transplantation 30, no. 4 (April 2011): S103. http://dx.doi.org/10.1016/j.healun.2011.01.302.
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Mazzeffi, Michael, Shaheer Hasan, Ezeldeen Abuelkasem, Michael Meyer, Kristopher Deatrick, Bradley Taylor, Zachary Kon, Daniel Herr, and Kenichi Tanaka. "Von Willebrand Factor-GP1bα Interactions in Venoarterial Extracorporeal Membrane Oxygenation Patients." Journal of Cardiothoracic and Vascular Anesthesia 33, no. 8 (August 2019): 2125–32. http://dx.doi.org/10.1053/j.jvca.2018.11.031.
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Özdemir, Zeynep C., Yeter Düzenli Kar, Serdar Ceylaner, and Özcan Bör. "A novel mutation in the GP1BA gene in Bernard–Soulier syndrome." Blood Coagulation & Fibrinolysis 31, no. 1 (January 2020): 83–86. http://dx.doi.org/10.1097/mbc.0000000000000868.
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Stavnichuk, Mariya, Zoltan Nagy, Yotis A. Senis, and Svetlana V. Komarova. "Megakaryocyte-Driven Myelofibrosis Leads to Progressive Osteosclerosis in G6b-B Knockout Mice." Blood 134, Supplement_1 (November 13, 2019): 4199. http://dx.doi.org/10.1182/blood-2019-124186.
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Bone and bone marrow are not only anatomically, but also functionally interdependent. In a systematic review, we examined bone health in patients with hematopoietic disorders and demonstrated that an increased hematopoietic cell proliferation, such as in patients with hemolytic anemias, was associated with bone loss, while bone marrow hypocellularity, such as in patients with chronic myelofibrosis (CMF), was associated with bone gain [Steer K et al. J Bone Miner Res 2017]. Since bone mass in CMF increases at the expense of bone marrow, it contributes to patients' morbidity as it is associated with bone pain, and mortality as it may lead to bone marrow failure. A mouse model with a global knockout of the megakaryocyte (MK) lineage specific inhibitory receptor G6b-B was shown to develop myelofibrosis secondary to aberrant platelet production and function [Mazharian A et al Sci Signal 2012]. Moreover, a group of patients with primary myelofibrosis was identified to have loss-of-function mutations in the G6b-B gene [Hofmann I et al Blood 2018]. The objective of this study was to characterize temporal changes in the skeleton of the G6b-B knockout mice. We examined age- and sex-related changes in 4, 8, 16, and 32 week-old G6b-B+/+, G6b-B-/- female and male mice. Starting from 8 weeks-of-age, spleen progressively increased in female G6b-B-/- mice compared to corresponding G6b-B+/+ mice, reaching 2.9-fold increase at 32 weeks (p < 0.001) (Fig.1A). Micro-computed tomography analysis of femur demonstrated that starting at 8 weeks of age female G6b-B-/- mice had a significantly higher proportion of cortical bone and a respectively lower proportion of marrow (Fig.1B). Starting at 16 weeks of age, female G6b-B-/- mice developed trabecula in the medullary cavity normally occupied by the bone marrow, which by 32 weeks led to a 38-fold increase (p < 0.001) in the proportion of bone to tissue volume compared to G6b-B+/+ (Fig.1C,D). At 32-weeks of age, female G6b-B-/- mice also demonstrated a 7-fold increase in BV/TV (p < 0.001) in the region of metaphysis. While some abnormalities were found in male G6b-B-/- mice, they were considerably less severe compared to females. To establish whether the observed bone phenotype is due to MK and platelet functional defects, we performed microcomputed tomography analysis on femurs of 22 week-old G6b-Bfl/fl;Gp1ba-Cre-/- mice with a MK/platelet-specific knockout of G6b-B. Changes in trabecular bone of G6b-Bfl/fl;Gp1ba-Cre-/- mice recapitulated changes of G6b-B-/- mice. However, periosteal perimeter in male G6b-Bfl/fl;Gp1ba-Cre-/- mice was significantly larger, and in female G6b-Bfl/fl;Gp1ba-Cre-/- mice - significantly smaller than in corresponding control mice, while in global G6b-B-/- mice periosteal perimeter was not affected. Female G6b-B-/- mice demonstrated severe splenomegaly as well as progressive osteosclerosis, which was confirmed in G6b-Bfl/fl;Gp1ba-Cre-/- mice, indicating that trabecular bone gain in G6b-B-/- mice is consequent to a MK disfunction. Dramatic sexual dimorphism suggests that sex-related factors play an important role in the development of osteosclerosis. The differences in cortical bone phenotype between the global and conditional knockout of G6b-B suggest the potential role of G6b-B signaling in osteoclasts or osteoblasts. This study demonstrates that MK-associated myelofibrosis is sufficient to induce osteosclerosis of bone marrow, and that sex hormones play an important role either in protecting male mice from osteosclerosis or in exacerbating osteosclerosis in female mice. Disclosures No relevant conflicts of interest to declare.
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Napit, Prabhat R., Abdulrahman Alhamyani, Khaggeswar Bheemanapally, Paul W. Sylvester, and Karen P. Briski. "Sex-Dimorphic Glucocorticoid Receptor Regulation of Hypothalamic Primary Astrocyte Glycogen Metabolism: Interaction with Norepinephrine." Neuroglia 3, no. 4 (November 17, 2022): 144–57. http://dx.doi.org/10.3390/neuroglia3040010.
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Astrocyte glycogen is a critical metabolic variable that affects hypothalamic control of glucostasis. Glucocorticoid hormones regulate peripheral glycogen, but their impact on hypothalamic glycogen is not known. A hypothalamic astrocyte primary culture model was used to investigate the premise that glucocorticoids impose sex-dimorphic independent and interactive control of glycogen metabolic enzyme protein expression and glycogen accumulation. The glucocorticoid receptor (GR) agonist dexamethasone (DEX) down-regulated glycogen synthase (GS), glycogen phosphorylase (GP)–brain type (GPbb), and GP–muscle type (GPmm) proteins in glucose-supplied male astrocytes, but enhanced these profiles in female. The catecholamine neurotransmitter norepinephrine (NE) did not alter these proteins, but amplified DEX inhibition of GS and GPbb in male or abolished GR stimulation of GPmm in female. In both sexes, DEX and NE individually increased glycogen content, but DEX attenuated the magnitude of noradrenergic stimulation. Glucoprivation suppressed GS, GPbb, and GPmm in male, but not female astrocytes, and elevated or diminished glycogen in these sexes, respectively. Glucose-deprived astrocytes exhibit GR-dependent induced glycogen accumulation in both sexes, and corresponding loss (male) or attenuation (female) of noradrenergic-dependent glycogen build-up. Current evidence for GR augmentation of hypothalamic astrocyte glycogen content in each sex, yet divergent effects on glycogen enzyme proteins infers that glucocorticoids may elicit opposite adjustments in glycogen turnover in each sex. Results document GR modulation of NE stimulation of glycogen accumulation in the presence (male and female) or absence (female) of glucose. Outcomes provide novel proof that astrocyte energy status influences the magnitude of GR and NE signal effects on glycogen mass.
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Badolia, Rachit, John Kostyak, Carol Dangelmaier, and Satya Kunapuli. "Syk Activity Is Dispensable for Platelet GP1b-IX-V Signaling." International Journal of Molecular Sciences 18, no. 6 (June 9, 2017): 1238. http://dx.doi.org/10.3390/ijms18061238.
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