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1
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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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Shen, Jun, Xiao-Chang Chen, Wang-Jun Li, Qiu Han, Chun Chen, Jing-Min Lu, Jin-Yu Zheng, and Shou-Ru Xue. "Identification of Parkinson’s disease-related pathways and potential risk factors." Journal of International Medical Research 48, no. 10 (October 2020): 030006052095719. http://dx.doi.org/10.1177/0300060520957197.
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Objective To identify Parkinson’s disease (PD)-associated deregulated pathways and genes, to further elucidate the pathogenesis of PD. Methods Dataset GSE100054 was downloaded from the Gene Expression Omnibus, and differentially expressed genes (DEGs) in PD samples were identified. Functional enrichment analyses were conducted for the DEGs. The top 10 hub genes in the protein–protein interaction (PPI) network were screened out and used to construct a support vector machine (SVM) model. The expression of the top 10 genes was then validated in another dataset, GSE46129, and a clinical patient cohort. Results A total of 333 DEGs were identified. The DEGs were clustered into two gene sets that were significantly enriched in 12 pathways, of which 8 were significantly deregulated in PD, including cytokine–cytokine receptor interaction, gap junction, and actin cytoskeleton regulation. The signature of the top 10 hub genes in the PPI network was used to construct the SVM model, which had high performance for predicting PD. Of the 10 genes, GP1BA, GP6, ITGB5, and P2RY12 were independent risk factors of PD. Conclusion Genes such as GP1BA, GP6, P2RY12, and ITGB5 play critical roles in PD pathology through pathways including cytokine−cytokine receptor interaction, gap junctions, and actin cytoskeleton regulation.
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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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Othman, M. "Differential identification of PT-VWD from type 2B VWD and GP1BA nomenclature issues." British Journal of Haematology 142, no. 2 (July 2008): 312–14. http://dx.doi.org/10.1111/j.1365-2141.2008.07171.x.
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Mohamed, Sara, Martina Di Palma, Michela Faleschini, Daniela De Benedittis, Maria Luisa Moleti, Luisa Cardarelli, Anna Maria Testi, Giovanna Palumbo, Anna Savoia, and Fiorina Giona. "Chronic Thrombocytopenia in Children: What Could It Hide?" Blood 136, Supplement 1 (November 5, 2020): 33–34. http://dx.doi.org/10.1182/blood-2020-138651.
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Immune thrombocytopenic purpura (ITP) is one of the most common hemorrhagic disorders in childhood, often caused by an acute self-limiting event. However, 30% of these children develop chronic ITP. Identification of the underlying causes in ITP is an important challenge. Inherited thrombocytopenia (IT) is a rare, underdiagnosed disease, included among the chronic platelet disorders. Next-Generation-Sequencing (NGS) could be an efficient way of discovering potential IT-associated mutations in children with chronic ITP. The purpose of this retrospective study was to investigate children with chronic ITP using a targeted NGS, in order to identify IT-associated mutations. Between June 2017 and April 2020, mutational screening by a targeted NGS was performed on 19 children, either with a familial history of IT [4 unrelated patients (pts)], or with chronic ITP (15 pts), after all other causes of thrombocytopenia were excluded. Nineteen relatives were also investigated. This study was carried out in collaboration with the Laboratory of Genetics, IRCCS Burlo Garofolo in Trieste, that developed a targeted NGS method for the simultaneous analysis of 28 IT genes. The cost of the NGS tests were supported by the public healthcare service. We retrospectively divided our cohort of 19 pts, into three subgroups: Group I included 4 unrelated pts with familial IT; Group II consisted of 6 pts with chronic ITP and a clinical history and/or laboratory features associated with familial IT; and, Group III included 9 pts with chronic ITP refractory to several treatments (Table 1). The median age at the initial diagnosis of thrombocytopenia was lower in Group I than in Groups II and III (19/12 years vs 1310/12 years and 9 years, respectively, p=0.33). The median time between the diagnosis of thrombocytopenia, and the time of the study, was shorter in Group I compared to Groups II and III (11.7 months vs 45.3 and 51.7 months, respectively, p=0.16). Median platelet count at the disease onset was lower in Group III than in Groups I and II (21 x 109/L vs 99 x 109/L and 38 x 109/L, respectively, p=0.28). The median MPV values were 12.5 fL, 9.85 fL and 8.8 fL in Groups II, III, and I respectively. Bleeding symptoms requiring treatment were present at diagnosis in 1/6 (16%) and in 5/9 (55%) children of Groups II and III, respectively. Genetic variants, usually detected in IT, were found in heterozygosity in all children in Groups I and II, and in 7/9 (78%) in Group III. Two out of 4, 2/6 and 2/9 children in Groups I, II, and III, respectively, presented ≥2 variants. Among the 4 children of Group I, ANKDR26 variant was found in 2 pts, together with GP1BA and NBEAL2 (pt#1) and TUBB1 (pt#2). ANKDR26 variant was also recorded as a single mutation in their relatives. Two different variants involving GP1BA (c.98T>A and c.515C<T) were detected in the remaining 2 children of Group I and in their relatives. Pt #4 with GP1BA c.515C>T mutation with mild macrothrombocytopenia had relatives with a previous diagnosis of monoallelic Bernard-Souliers syndrome. As shown in table 1, ABCG8, ACTN1, ETV6, GP1BA, MYH9, SLFN14, or WAS variants, found in combination in 2 pts (pt#5, pt#8), were also detected in the children in Group II, as well as, at least one of the relatives (for a total of 7 cases). ABCG5, ABCG8, ETV6, FLNA, GP1BA, NBEAL2, or SLFN14 were found as variants in patients of Group III. The peripheral blood smear evaluation confirmed the diagnosis of grey platelet syndrome with two NBEAL2 mutations in pt #16. SLFN14, as a single variant, was associated with macrothrombocytopenia in one pt (#10). Three pts (#5, #6, #17), with variants of ABCG8 had hypercholesterolemia. In the cohort of pts with chronic ITP, 4 (#12, #13, #14, #15) had relatives with thrombocytopenia, and 2 (#11, #13) had a familial history of hematological malignancies. Segregation analysis in families, and functional studies to evaluate the pathogenic role of the variants reported, are still in progress. The clinical significance of IT-associated mutations in chronic ITP is uncertain, and yet to be clarified. However, our experience has shown that an in-depth clinical history, and accurate peripheral blood smear examinations, are important to better characterize chronic ITP in children. A targeted NGS method for the simultaneous analysis of different IT genes, has demonstrated to be an effective approach to explore in-depth the IT-associated mutations in children with chronic ITP, refractory to treatment. Table 1. Disclosures Giona: Novartis: Research Funding; Takeda: Speakers Bureau; Sanofi Genzyme: Research Funding, Speakers Bureau.
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Lavenu-Bombled, Cécile, Corinne Guitton, Arnaud Dupuis, Marie-Jeanne Baas, Céline Desconclois, Marie Dreyfus, Renhao Li, et al. "A novel platelet-type von Willebrand disease mutation (GP1BA p.Met255Ile) associated with type 2B “Malmö/New York” von Willebrand disease." Thrombosis and Haemostasis 116, no. 12 (November 2016): 1070–78. http://dx.doi.org/10.1160/th16-06-0438.
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SummaryInteraction between von Willebrand factor (VWF) and platelet GPIbα is required for primary haemostasis. Lack or loss-of-function in the ligand-receptor pair results in bleeding complications. Paradoxically, gain-of-function mutations in VWF or GPIbα also result in bleeding complications as observed in type 2B von Willebrand disease (VWD) and platelet-type- (PT-) VWD, respectively. A similar phenotype is observed with increased ristocetin-induced platelet agglutination and disappearance of the highest molecular weight multimers of VWF. We evaluated a patient with a bleeding disorder and a biological presentation compatible with type 2B VWD. VWF and platelet functional assays, sequencing of the VWF and GP1BA genes, and expression studies in HEK cells were performed. Sequencing of the VWF gene in the propositus revealed a heterozygous p.Pro1266Leu mutation previously found in type 2B VWD Malmö/New York. These variants are characterised by a mild phenotype and a normal VWF multimer composition suggesting the presence of a second mutation in our propositus. Sequencing of the GP1BA gene revealed a heterozygous c.765G>A substitution changing Met at position 255 of GPIbα to Ile. This new mutation is located in the β-switch domain where five other gain-of-function mutations have been reported in PT-VWD. Expression of GPIbα Ile255 in HEK GPIb-IX cells resulted in enhanced VWF binding compared to wild-type, similar to known PT-VWD mutations (p.Val249, p.Ser249 and p.Val255) indicating that it contributes to the propositus defects. This first report associating PT-with type 2B VWD illustrates the importance of combining biological assays with genetic testing to better understand the clinical phenotype.
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Trizuljak, Jakub, Kateřina Staňo Kozubík, Lenka Radová, Michaela Pešová, Karol Pál, Kamila Réblová, Olga Stehlíková, et al. "A novel germline mutation in GP1BA gene N-terminal domain in monoallelic Bernard-Soulier syndrome." Platelets 29, no. 8 (October 17, 2018): 827–33. http://dx.doi.org/10.1080/09537104.2018.1529300.
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Ali, Shahnaz, Shrimati Shetty, and Kanjaksha Ghosh. "A novel mutation in GP1BA gene leads to mono-allelic Bernard Soulier syndrome form of macrothrombocytopenia." Blood Coagulation & Fibrinolysis 28, no. 1 (January 2017): 94–95. http://dx.doi.org/10.1097/mbc.0000000000000530.
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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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Monteiro, Manuel, Luis Almeida, Mariana Morais, and Luis Dias. "Bernard Soulier syndrome: a rare, frequently misdiagnosed and poorly managed bleeding disorder." BMJ Case Reports 14, no. 8 (August 2021): e243518. http://dx.doi.org/10.1136/bcr-2021-243518.
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Bernard Soulier syndrome is a rare, congenital platelet bleeding disorder, with autosomal recessive inheritance. It is characterised by macrothrombocytopenia and platelet dysfunction, leading to mucocutaneous bleeding noted in early childhood. This entity poses an important diagnostic challenge, and blood smear and DNA sequencing are paramount for the correct diagnosis. Differential diagnosis includes May-Hegglin anomaly, Glanzmann Thrombasthenia and von Willebrand disease; it is also often misdiagnosed as idiopathic thrombocytopenic purpura. We report a 68-year-old man diagnosed with von Willebrand disease for three decades, admitted with gastrointestinal bleeding, anaemia and severe thrombocytopenia. Replacement with von Willebrand factor did not stop the haemorrhage, suggesting another aetiology for the bleeding disorder. Corticosteroids and intravenous immune globulin were also ineffective. Genetic sequencing showed a homozygous mutation in GP1BA gene, thus establishing the correct diagnosis.
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Kanaji, Taisuke, Susan Russell, Janet Cunningham, Kenji Izuhara, Joan E. B. Fox, and Jerry Ware. "Megakaryocyte proliferation and ploidy regulated by the cytoplasmic tail of glycoprotein Ibα." Blood 104, no. 10 (November 15, 2004): 3161–68. http://dx.doi.org/10.1182/blood-2004-03-0893.
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Abstract We have investigated the ability of glycoprotein (GP) Ibα, a megakaryocytic gene product, to sequester the signal transduction protein 14-3-3ξ and to influence megakaryocytopoiesis. Using a Gp1ba–/– mouse colony, we compared the rescued phenotypes produced by a wild-type human GP Ibα allele or a similar allele containing a 6-residue cytoplasmic tail truncation that abrogates binding to 14-3-3ξ. The observed phenotypes illustrate an involvement for GP Ibα in thrombopoietin-mediated events of megakaryocyte proliferation, polyploidization, and the expression of apoptotic markers in maturing megakaryocytes. We developed a hypothesis for the involvement of a GP Ibα/14-3-3ξ/PI-3 kinase complex in regulating thrombopoietin-mediated responses. An observed increase in thrombopoietin-mediated Akt phosphorylation in the truncated variant supported the hypothesis and led to the development of a model in which the GP Ibα cytoplasmic tail sequestered signaling proteins during megakaryocytopoiesis and, as such, became a critical regulator in the temporal sequence of events that led to normal megakaryocyte maturation.
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Stavnichuk, Mariya, and Svetlana V. Komarova. "Megakaryocyte-bone cell interactions: lessons from mouse models of experimental myelofibrosis and related disorders." American Journal of Physiology-Cell Physiology 322, no. 2 (February 1, 2022): C177—C184. http://dx.doi.org/10.1152/ajpcell.00328.2021.
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Over the years, numerous studies demonstrated reciprocal communications between processes of bone marrow hematopoiesis and bone remodeling. Megakaryocytes, rare bone marrow cells responsible for platelet production, were demonstrated to be involved in bone homeostasis. Myelofibrosis, characterized by an increase in pleomorphic megakaryocytes in the bone marrow, commonly leads to the development of osteosclerosis. In vivo, an increase in megakaryocyte number was shown to result in osteosclerosis in GATA-1low, Nf-e2−/−, TPOhigh, Mplf/f; PF4cre, Lnk−/−, Mpig6b−/−, Mpig6bfl/fl; Gp1ba-Cr+/KI, and Pt-vWD mouse models. In vitro, megakaryocytes stimulate osteoblast proliferation and have variable effects on osteoclast proliferation and activity through soluble factors and direct cell-cell communications. Intriguingly, new studies revealed that the ability of megakaryocytes to communicate with bone cells is affected by the age and sex of animals. This mini-review summarizes changes seen in bone architecture and bone cell function in mouse models with an elevated number of megakaryocytes and the effects megakaryocytes have on osteoblasts and osteoclasts in vitro, and discusses potential molecular players that can mediate these effects.
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Shlebak, Abdul, Anthony Poles, Richard Manning, Shaikha Almuhareb, Josu De La Funte, Mike Mitchell, and Geoff Lucas. "A Novel Homozygous c.800C>G Substitution in GP1BA Exon 2 in a Kuwaiti Family with Bernard-Soulier Syndrome." Acta Haematologica 134, no. 3 (2015): 193–98. http://dx.doi.org/10.1159/000381328.
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Background: Bernard-Soulier syndrome (BSS) is a congenital bleeding disorder characterised by thrombocytopenia, giant platelets and decreased platelet adhesion resulting from genetic alterations of the glycoprotein (GP) Ib/IX/V complex. Objectives: Three sisters with a lifelong bleeding history and a provisional diagnosis of BSS were referred for further characterisation of their bleeding diathesis. The siblings' symptoms varied in severity from skin and gum bleeding to menorrhagia associated with iron-deficiency anaemia requiring regular transfusion of red cells and platelets. The parents were consanguineous but did not demonstrate any bleeding disorder. Methods: The family were investigated using standard haematological techniques, platelet aggregometry, platelet membrane GP analysis and DNA sequencing of the genes encoding the GPIb/IX complex. Results: All 3 sisters had thrombocytopenia and giant platelets. Platelet aggregation and flow cytometry studies confirmed the lack of aggregation with ristocetin and a markedly reduced GPIb/IX surface expression. Molecular analysis demonstrated a novel homozygous c.800C>G substitution in GP1BA exon 2 leading to a serine 267 Ter stop codon in all 3 siblings. Conclusions: A novel, nonsense mutation was identified as the cause of the bleeding disorder in this family. This is the first reported BSS mutation identified in a family from Kuwait.
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V. Anisimova, A., A. S. Gunchenko, A. Yu. Ikonnikova, S. S. Galkin, M. A. Avdonina, T. V. Nasedkina, and Z. Abdukhalikova. "Study of the Role of Polymorphism of ACE, GP1BA, PDE4D Genes and Clinical Features in the Development of Cerebrovascular Diseases." International Journal of Sciences 8, no. 03 (2019): 97–101. http://dx.doi.org/10.18483/ijsci.1975.
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Li, Xingchuan, Song Wang, Jiusi Wu, Haidong Wang, Jing Wang, Xiangyu Dong, and Ni Zhang. "A Case of Bernard-Soulier Syndrome due to a Novel Homozygous Missense Mutation in an Exon of the GP1BA Gene." Acta Haematologica 143, no. 1 (July 12, 2019): 60–64. http://dx.doi.org/10.1159/000500797.
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Bernard-Soulier syndrome (BSS) is an extremely rare autosomal recessive bleeding disorder clinically characterized by macrothrombocytopenia and a mucocutaneous bleeding tendency. A 1-year-old Chinese patient who was born to consanguineous parents was diagnosed with early onset of BSS. Gene sequencing and bioinformatics analysis were conducted. We identified a novel homozygous missense mutation (c.790T>C) in the GP1BAgene that causes an amino acid residue substitution of a cysteine with an arginine that might have a deleterious effect on the protein function as predicted by bioinformatics analysis. If a patient has clinical manifestations that include recurrent mucocutaneous bleeding, a mean platelet volume and platelet-large cell ratio above normal levels, and giant platelets on a peripheral smear and has consanguineous parents, a diagnosis of BSS can be suspected. In these situations, gene sequencing for mutations in the GPIb-IX-V complex is necessary.
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Skvortsova, V. I., E. A. Koltsova, Ekaterina Igorevna Kimelfeld, S. A. Limborskaya, P. A. Slominsky, and T. V. Tupitsyna. "Analysis of the contribution of -5t/c polymorphism in the GP1BA gene to the development of ischemic stroke in young patients." Neurology, neuropsychiatry, Psychosomatics, no. 4 (December 15, 2012): 39. http://dx.doi.org/10.14412/2074-2711-2012-419.
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Shorikov, E. "C0376: Platelets Aggregation in Relation to Polymorphism of GP1BA-Trombocyte Receptor Gene at Patients with Arterial Hypertension and Diabetes Mellitus Type 2." Thrombosis Research 133 (May 2014): S82. http://dx.doi.org/10.1016/s0049-3848(14)50266-6.
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Gindele, Réka, Adrienne Kerényi, Judit Kállai, György Pfliegler, Ágota Schlammadinger, István Szegedi, Tamás Major, et al. "Resolving Differential Diagnostic Problems in von Willebrand Disease, in Fibrinogen Disorders, in Prekallikrein Deficiency and in Hereditary Hemorrhagic Telangiectasia by Next-Generation Sequencing." Life 11, no. 3 (March 5, 2021): 202. http://dx.doi.org/10.3390/life11030202.
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Diagnosis of rare bleeding disorders is challenging and there are several differential diagnostics issues. Next-generation sequencing (NGS) is a useful tool to overcome these problems. The aim of this study was to demonstrate the usefulness of molecular genetic investigations by summarizing the diagnostic work on cases with certain bleeding disorders. Here we report only those, in whom NGS was indicated due to uncertainty of diagnosis or if genetic confirmation of initial diagnosis was required. Based on clinical and/or laboratory suspicion of von Willebrand disease (vWD, n = 63), hypo-or dysfibrinogenemia (n = 27), hereditary hemorrhagic telangiectasia (HHT, n = 10) and unexplained activated partial thromboplastin time (APTT) prolongation (n = 1), NGS using Illumina platform was performed. Gene panel covered 14 genes (ACVRL1, ENG, MADH4, GDF2, RASA1, F5, F8, FGA, FGB, FGG, KLKB1, ADAMTS13, GP1BA and VWF) selected on the basis of laboratory results. We identified forty-seven mutations, n = 29 (6 novel) in vWD, n = 4 mutations leading to hemophilia A, n = 10 (2 novel) in fibrinogen disorders, n = 2 novel mutations in HHT phenotype and two mutations (1 novel) leading to prekallikrein deficiency. By reporting well-characterized cases using standardized, advanced laboratory methods we add new pieces of data to the continuously developing “bleeding disorders databases”, which are excellent supports for clinical patient management.
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Kunicki, Thomas J., Augusto B. Federici, Daniel R. Salomon, James A. Koziol, Steven R. Head, Tony S. Mondala, Jeffrey D. Chismar, Luciano Baronciani, Maria Teresa Canciani, and Ian R. Peake. "An association of candidate gene haplotypes and bleeding severity in von Willebrand disease (VWD) type 1 pedigrees." Blood 104, no. 8 (October 15, 2004): 2359–67. http://dx.doi.org/10.1182/blood-2004-01-0349.
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Abstract von Willebrand disease (VWD) type 1 is difficult to diagnose because of bleeding variability and low heritability of von Willebrand factor (VWF) levels. We compared a bleeding severity score and bleeding times to candidate gene haplotypes within pedigrees of 14 index cases, using a covariance components model for multivariate traits (Mendel: QTL Association). These pedigrees included 13 affected and 40 unaffected relatives, as defined by plasma ristocetin cofactor (VWF:RCo) levels. The bleeding severity score was derived from a detailed history. Donors were genotyped using a primer extension method, and 9 candidate genes were selected for analysis. VWF:RCo levels had the strongest influence on bleeding severity score and bleeding time. ITGA2 haplotype 2 (807C) and ITGA2B haplotype 1 (Ile843) were each associated with increased bleeding severity scores (P < .01 and P < .01, respectively). GP6 haplotype b (Pro219) was also associated with increased scores (P = .03) after adjustment for donor age. No association was observed with 6 other candidate genes, GP1BA, ITGB3, VWF, FGB, IL6, or TXA2R. Increased plasma VWF:Ag levels were associated with VWF haplotype 1 (–1793G; P = .02). These results establish that genetic differences in the adhesion receptor subunits α2, αIIb, and GPVI can influence the phenotype of VWD type 1.
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Enayat, Said, Shirin Ravanbod, Maryam Rassoulzadegan, Mohammad Jazebi, Shirin Tarighat, Fereydoun Ala, Jonas Emsley, and Maha Othman. "A novel D235Y mutation in the GP1BA gene enhances platelet interaction with von Willebrand factor in an Iranian family with platelet-type von Willebrand disease." Thrombosis and Haemostasis 108, no. 11 (2012): 946–54. http://dx.doi.org/10.1160/th12-04-0189.
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SummaryPlatelet-type von Willebrand disease (PT-VWD) is a rare bleeding disorder with an intrinsic defect in platelets rather than von Willebrand factor (VWF), but has clinical and laboratory features similar to the more common type 2B VWD. The intriguing nature of the pathophysiology and molecular genetics of PT-VWD has created lengthy debate in literature regarding its discrimination from type 2B VWD, and essentially confirming DNA analysis as the gold standard in diagnosis and revealing pathologic mutations. In this report we identify a novel Asp235Tyr mutation in the GP1BA gene of two Iranian patients showing the PT-VWD phenotype who were originally misdiagnosed as type 2B VWD. By structural modelling of the mutant by introducing Tyr235 into the available crystal structure of the glycoprotein (GP)Ibα N-terminal domain, we observed the mutant Tyr235 generates a hydrophobic tip to the extended β-switch loop of GPIbα. Further modelling of the resulting complex with VWFA1 indicates this could result in an enhanced interface compared to wild-type Asp235. This data provides an update to the present knowledge about this rare disorder, and confirms the necessity of genetic testing for accurate diagnosis, and the importance of studying natural mutations to better understand molecular aspects of GPIbα-VWFA1 interaction.
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Wandall, Hans H., Anne Louise Sørensen, Sunita Patel, Jennifer Richardson, Joseph E. Italiano, Victoria Rumjantseva, Eric P. Bennett, Henrik Clausen, Thomas P. Stossel, and John H. Hartwig. "Megakaryocytes Package and Deliver Golgi-Associated Glycosyltransferases into Platelets and to Platelet Surfaces Using Dense Granules." Blood 106, no. 11 (November 16, 2005): 1643. http://dx.doi.org/10.1182/blood.v106.11.1643.1643.
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Abstract We recently presented evidence for the presence of a β4 galactosyl transferase (β4GalT) on the surface of platelets that catalyzes the coupling of galactose in a β1,4 linkage to exposed N-acetylglucosamine residues on the N-linked glycans of GP1ba and that this reaction improved the circulation of chilled murine platelets. Since an externally-disposed glycosyltransferase activity contradicts the dogma that such enzymes reside solely within the Golgi apparatus, we verified it by demonstrating that intact platelets glycosylate 2.8 μm beads coated with the acceptor substrate GlcNAc-β-R. We then explored the origin of this enzyme in megakaryocytes and its mechanism of delivery to platelets. We report that Golgi marker GM130 as well as a YFP tagged Golgi marker construct based on the polypeptide transferase GalNAc-T2 expressed in cultured mouse megakaryocytes transports from the cell body of to nascent platelets in discrete packets. In immature megakaryocytes, the Golgi organizes, as expected, into perinuclear arrays. However, once proplatelet extension begins, the perinuclear apparatus disassembles, and Golgi-associated proteins transport through the proplatelets in vesicular/granular structures. Labeling with granule markers reveals correspondence of Golgi markers only with dense granules. These findings suggest that Golgi membrane becomes packaged into dense granules, which deliver Golgi-associated glycosyltransferases into the nascent platelets and from there to the platelet surface.
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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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Tupitsyna, T. V., E. A. Bondarenko, S. A. Kravchenko, P. F. Tatarskyy, I. M. Shetova, N. A. Shamalov, S. M. Kuznetsova, et al. "Comparative analysis of associations between polymorphic variants of the F2, F5, GP1BA, and ACE genes and the risk of developing stroke in Russian and Ukrainian populations." Molecular Genetics, Microbiology and Virology 28, no. 1 (January 2013): 8–14. http://dx.doi.org/10.3103/s0891416813010072.
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Frontroth, Juan Pablo, Mirta Hepner, Gabriela Sciuccati, Aurora Feliú Torres, Graciela Pieroni, and Mariana Bonduel. "Prospective study of low-dose ristocetin-induced platelet aggregation to identify type 2B von Willebrand disease (VWD) and platelet-type VWD in children." Thrombosis and Haemostasis 104, no. 12 (2010): 1158–65. http://dx.doi.org/10.1160/th10-04-0213.
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SummaryType 2B von Willebrand disease (VWD2B) and platelet-type von Wille-brand disease (PT-VWD) are rare bleeding disorders characterised by an increased ristocetin-induced platelet aggregation (RIPA) at low dose of ristocetin. It was the objective of this study to detect children with VWD2B and PT-VWD using RIPA at low dose of ristocetin (0.5 mg/ml) in the screening evaluation of bleeding disorders, and to analyse the phenotypic data along with the molecular findings. Over a 14-year period, 641 children with personal and family bleeding symptoms or bleeding from birth with previously uncharacterised haemostatic disorders were prospectively studied. Six unrelated patients (0.93%) showed RIPA at low dose of ristocetin. RIPA-based mixing studies identified that the plasma of the six probands and at least one parent from five unrelated families induced aggregation of normal platelets with the addition of low-dose ristocetin. None of the probands’ platelets showed aggre-gation with cryoprecipitate. Low ristocetin cofactor activity/VWF antigen ratio with absent collagen binding activity or thrombocytopenia were detected respectively in only two patients. Molecular analysis of exon 28 of the VWF gene identified mutations in only three patients. No mutation in the GP1BA gene was found. In this large prospective paediatric study, the screening approach including RIPA at low dose of ristocetin permitted the detection of patients with VWD2B that would otherwise have been missed. No patient with phenotype or genotype of PT-VWD was identified. Heterogeneity of bleeding symptoms and phenotypic parameters were found among members of the same family.
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Li, Yang, Guoqing Li, Fu Wang, Xiaoshan Wu, Zhifang Wu, Jinsong Wang, Chunmei Zhang, Junqi He, Hao Wang, and Songlin Wang. "Integrated Analysis of LncRNA-mRNA Coexpression in the Extracellular Matrix of Developing Deciduous Teeth in Miniature Pigs." BioMed Research International 2019 (January 23, 2019): 1–9. http://dx.doi.org/10.1155/2019/6159490.
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Miniature pigs, a valuable alternative model for understanding human tooth development, have deciduous teeth from all four tooth families that are replaced once by permanent molars. The extracellular matrix (ECM) supports cells and maintains the integrity of tooth germs during tooth development. However, details on the role of the ECM in tooth development are poorly understood. Here, we performed long noncoding RNA (lncRNA) and messenger RNA (mRNA) expression profiles in the ECM components of deciduous tooth germs by RNA sequencing in miniature pigs. From the early cap to the late bell stages, we identified 4,562 and 3,238 differentially expressed genes (DEGs) from E40 to E50 and E50 to E60, respectively. In addition, a total of 1,464 differentially expressed lncRNAs from E40 to E50 and 969 differentially expressed lncRNAs from E50 to E60 were obtained. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEGs were enriched significantly for multiple signaling pathways, especially for the ECM pathway. We then outlined the detailed dynamic gene expression profiling of ECM components during deciduous molar development. Comparison of the cap and bell stages revealed that the structure and functions of the ECM dynamically changed. The ECM-related genes, including THBS1, COL4A5, COL4A6, COL1A1, CHAD, TNR, GP1BA, and ITGA3, were significantly changed, and some were shown to enrich during the bell stage development. Finally, we outlined the coexpression of lncRNAs and ECM properties during tooth development. We showed that the interplay of key lncRNAs could change ECM processes and influence the ECM establishment of tooth patterns to accomplish full tooth formation. These results might provide information to elucidate the regulation network of the lncRNA and ECM in tooth development.
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Веремеева, В. В., Н. А. Бухвальд, Э. В. Дашкевич, and Н. Г. Седляр. "Development of the Integrated Algorithm for Examining Women with a Risk of Hereditary Thrombophilia in Planning Pregnancy." Гематология. Трансфузиология. Восточная Европа, no. 2 (July 6, 2020): 208–16. http://dx.doi.org/10.34883/pi.2020.6.2.006.
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В настоящее время установлено, что некоторые аллельные варианты генов связаны со значительным повышением риска развития тромбозов, что может привести к нежелательной потере беременности.В ходе работы были исследованы образцы крови 124 женщин-доноров РНПЦ трансфузиологии и медицинских биотехнологий. В исследование входило определение гемостазиологических параметров: активированное частичное тромбопластиновое время (АЧТВ), выраженное через индекс Ratio (R), протромбиновое время (ПВ), выраженное через международное нормализованное отношение (МНО), а также концентрация фибриногена. А также генетическое типирование по 31 гену (в скобках указан исследованный полиморфизм): FII (G20210А), FV (Arg506Gln), MTHFR (С677Т), F11 С/T rs2289252, GP1BA C/T rs2243093, AGTR1 A/C rs5186, BDKRB2 I/D, PPARD +294T/C, eNOS (4a/4b), F13 (Val34Leu), VEGF (G-634C), ACE (Alu Ins/Del), eNOS (G894T), EPO G3876T, FI (Thr312Ala), F11 T/C rs2036914, FGG C/T rs2066865, MTHFR (А1298С), MTR (А2756G), HIF1A (C1772T), APOE (Cys112Arg; Arg158Cys), CYBA C/T rs4673, Gp6 A/G rs1613662, Gp6 A/G rs1654419, Gp6 G/T rs1671153, ITGA2 C/T rs1126643, ITGB3 T/C rs5918, PPARA G2528C, PPARG Pro12Ala, PPARGC1A G1564A.После проведения лабораторных тестов был проведен статистический анализ результатов, который заключался в поиске достоверных различий в частоте встречаемости конкретных полиморфизмов в различных группах пациенток. На основании полученных данных были сформулированы рекомендации, которые легли в основу алгоритма обследования женщин с риском наследственных тромбофилий при планировании беременности.В соответствии с алгоритмом для стандартного исследования пациенток рекомендуется проведение стандартного гемостазиологического исследования по клиническому протоколу. При наличии отягощенного акушерского анамнеза после исключения акушерско-гинекологических причин рекомендуется проведение углубленного изучения гемостаза с определениемактивности противосвертывающей системы крови, а именно активности протеинов С, S, AT III. В случае наличия каких-либо отклонений может быть рекомендовано проведение генетического типирования по определенным генам.Также в ходе работы была проанализирована зависимость отклонения в показателях гемостаза и характера полиморфизма (благоприятная гомозигота, гетерозигота, неблагоприятная гомозигота) всех генов панели. It has now been established that some allelic variants of the genes are associated with a significant increase of the risk of thrombosis, which can lead to undesirable loss of pregnancy.Firstly, blood samples of 124 female donors of the Republican scientific practical center of transfusiology and medical biotechnology were studied. The study included determination of the following hemostasiological parameters: activated partial thromboplastin time (APTT), expressed through the Ratio index (R), prothrombin time (PT), expressed through the international normalized ratio (INR), as well as fibrinogen concentration. The second part was genetic testing for 31 genes (the studied polymorphism is indicated in parentheses): FII (G20210A), FV (Arg506Gln), MTHFR (C677T), F11 C / T rs2289252, GP1BA C / T rs2243093, AGTR1 A / C rs5186, BDKB / D, PPARD + 294T / C, eNOS (4a/ 4b), F13 (Val34Leu), VEGF (G-634C), ACE (Alu Ins / Del), eNOS (G894T), EPO G3876T, FI (Thr312Ala), F11 T / C rs2036914, FGG C / T rs2066865, MTHFR (A1298C), MTR (A2756G), HIF1A (C1772T), APOE (Cys112Arg; Arg158Cys), CYBA C / T rs4673, Gp6 A / G rs1661966 , Gp6 G / T rs1671153, ITGA2 C / T rs1126643, ITGB3 T / C rs5918, PPARA G2528C, PPARG Pro12Ala, PPARGC1A G1564A.After laboratory tests, the statistical analysis of the results was carried out. The aim of analysis was to search for significant differences in the frequency of occurrence of specific polymorphisms in different groups of patients. On the base of the obtained data, there were formulated the recommendations that formed the basis of the algorithm for examining women with the risk of hereditary thrombophilia during pregnancy planning.In accordance with the algorithm for a standard examination of patients, it is recommended to perform the standard hemostasiological testing, according to the clinical protocol. In the presence of a burdened obstetric history after the exclusion of obstetric and gynecological reasons, it is recommended to conduct in-depth study of hemostasis with determination of the activity of the anticoagulant blood system, namely the activity of proteins C, S, AT III.If there are any abnormalities, genetic testing of certain genes may be recommended.Also, during the work, the dependence of the deviation in hemostasis indices and the nature of polymorphism (favorable homozygote, heterozygote, unfavorable homozygote) of all panel genes was analyzed.
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Van der Reijden, Bert A., Davide Monteferrario, Nikhita Bolar, Anna Marneth, Konnie Hebeda, Saskia Bergevoet, Hans Veenstra, et al. "A Dominant-Negative GFI1B Mutation in Gray Platelet Syndrome." Blood 122, no. 21 (November 15, 2013): LBA—3—LBA—3. http://dx.doi.org/10.1182/blood.v122.21.lba-3.lba-3.
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Abstract Gray platelet syndrome (GPS) is a hereditary, usually autosomal recessive bleeding disorder caused by defective production of α-granules in platelets. GPS patients show reduced numbers of platelets that are larger and have a typical gray appearance under light microscopy, caused by the lack of α-granules. We describe a large family with an autosomal dominant type of GPS characterized by mild to severe bleeding complications. In addition to large gray platelets, other GPS-associated phenomena like myelofibrosis, thrombocytopenia, and low platelet factor 4 expression were observed in affected individuals. Histopathological examination of a BM biopsy from a patient showed a cellular marrow with increased numbers of megakaryocytes that were pleomorphic in size and shape. Megakaryocytes clustered along BM sinuses and showed dysmorphic stretched features. To determine the disease causing mutation we performed linkage analysis and identified a candidate locus on chromosome 9q34 with a LOD score of 3.9. We considered GFI1B (Growth Factor Independence 1B), located within this region, an excellent candidate gene because of its function as a transcriptional repressor in megakaryocyte development. Sequence analysis identified a nonsense mutation in GFI1B exon 6 (c.859C>T, p.Gln287*) that completely co-segregated with the GPS disease in this family. The mutated transcript predicts a 44 amino acid C-terminally truncated protein, GFI1BTr. The truncation is located within zinc finger 5 of GFI1B, deleting all of its four amino acids that directly interact with DNA. Luciferase gene reporter assays showed that GFI1BTr was unable to repress gene expression. Importantly, GFI1BTr inhibited gene repression mediated by wild type GFI1B, indicating that the mutant interferes with wild type GFI1B in a dominant-negative manner. To validate that GFI1BTr adversely affects normal GFI1B, we expressed the mutant in mouse bone marrow cells followed by induction of megakaryocytic differentiation. Compared to control cells, GFI1BTr-positive megakaryocytes showed dysplastic features including hypolobulation of the nuclei, irregular contours and multiple separate nuclei, that were very similar to those observed in patient cells. This indicates that GFI1BTr causes megakaryocytic abnormalities and that it functions in a dominant-negative manner. GFI1B silencing inhibits the development of human megakaryocyte colonies in vitro. We observed that megakaryocyte colony forming cells were significantly more frequent in patient bone marrow compared to controls. In addition, patient-derived megakaryocyte colonies were significantly larger compared to controls. Immunophenotypic analyses of peripheral blood showed no differences in myeloid and erythroid lineages and the platelet markers GP3B, ITGA2B and ITGB3 among affected an non-affected individuals. However, within the ITGA2B/CD41-positive platelet population, 5 of 6 affected members showed a marked decrease in the platelet surface membrane glycoprotein 1b-α (GP1BA/CD42b), compared to unaffected members. In addition, a strong expression of CD34, which is usually confined to immature hematopoietic progenitors, was detected on platelets from all studied affected individuals. Immunostaining of a BM biopsy from a patient showed the presence of ITGB3/CD61 positive megakaryocytes that intensely expressed CD34. Electron microscopy analysis showed megakaryocytes with few, small, irregularly shaped and centrally located α-granules characterized by an extensive peripheral cytoplasm with irregular proplatelets, largely devoid of cell organelles. To test whether these abnormalities were cell intrinsic, we stimulated CD34+ cells from two patients to differentiate along the megakaryocytic lineage in vitro. Megakaryocytic cells showed dysplastic features reminiscent of those observed in the bone marrow aspirates. In addition, increased CD34 and decreased GP1BA/CD42b expression were observed on megakaryocytes, indicating that GFI1BTr-induced abnormalities are intrinsic to the cell. In summary, we have identified GFI1B as a causative gene in autosomal dominant GPS. GFI1BTr acts in a dominant-negative manner over wild type GFI1B and affects the development of megakaryocytes and platelets, demonstrating a pivotal role of GFI1B in governing normal megakaryopoiesis and platelet production. Disclosures: No relevant conflicts of interest to declare.
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Semashchenko, K. S., T. S. Mongush, A. A. Kosinova, T. N. Subbotina, and Y. I. Grinshtein. "Study the Association of Nucleotide Polymorphisms in Platelet Receptor and Cytochrome P450 Genes with the Development of Resistance to Antiplatelet Drugs in Patients with Coronary Artery Disease." Rational Pharmacotherapy in Cardiology 18, no. 3 (July 6, 2022): 289–96. http://dx.doi.org/10.20996/1819-6446-2022-06-15.
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Aim. To study the association of nucleotide polymorphisms in platelet receptor and cytochrome P450 genes with the development of resistance to antiplatelet drugs in CHD patients.Material and Methods. The study included 243 patients diagnosed with CHD after coronary artery bypass surgery (CABG), including 140 patients in the acetylsalicylic acid (ASA) treatment group and 103 patients in the dual antiplatelet therapy (DAT) group. All patients were tested for platelet aggregation using an optical aggregometer with inducers: 5 mM ADP and 1 mM arachidonic acid (AA). DNA samples were analyzed by allele-specific PCR for the presence of polymorphisms rs2046934, rs1126643, rs5918, rs6065, rs4244285 in the platelet receptor and cytochrome P450 genes.Results. No statistically significant differences were found during comparison of the prevalence of the studied polymorphisms in the platelet receptor and cytochrome P450 genes between the groups of aspirin-sensitive and aspirin-resistant patients, as well as between the groups of clopidogrelsensitive and clopidogrel-resistant patients. No association between carriage of the minor and major alleles of the polymorphisms studied and the development of antiplatelet drug resistance was found. In the group of patients on ASA therapy, carriers of the C allele of the T1565C (rs5918) ITGB3 polymorphism had a higher rate of AA-induced platelet aggregation compared to carriers of the T allele (18,49±25,92 vs 10,43±17,34, р=0,004).Conclusion. Polymorphisms of P2RY12 (rs2046934), ITGA2 (rs1126643), ITGB3 (rs5918), GP1BA (rs6065), CYP2C19*2 (rs4244285) genes are not associated with antiplatelet drug resistance in both patients on ASC therapy and on DAT. The presence of minor alleles of the rs2046934, rs1126643, rs6065, rs4244285 polymorphisms are not associated with increased platelet aggregation activity before CABG.However, in the group of patients on ASA therapy C-allele carriers of the rs5918 polymorphism of the ITGB3 gene had a higher rate of AA-induced platelet aggregation compared to T-allele carriers.
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Kunicki, Thomas J., Daniel Diaz, Shirley A. Williams, Richard W. Farndale, and Diane J. Nugent. "The Integrin α2 Dimorphism E534K Modulates Platelet Binding to Decorin but Not Collagen I,." Blood 118, no. 21 (November 18, 2011): 3256. http://dx.doi.org/10.1182/blood.v118.21.3256.3256.
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Abstract Abstract 3256 Background. Integrin a2b1-mediated adhesion to collagens supports cellular attachment, while decorin binding by a2b1 often attenuates adhesion. Collagen I binds to the a2 I-domain via the triple-helical sequence GFOGER, but the decorin binding site is not within the a2 I-domain and has not yet been identified. A single nucleotide polymorphism in the a2 gene ITGA2 (rs1801106) (G1600A) resulting in the amino acid substitution glutamate-534 to lysine-534 (E534K) is the basis for one of the most important human platelet alloantigen (HPA) systems, HPA-5, yet HPA-5 alleles do not influence the binding of a2b1 to collagen I, and the effect of HPA-5 on platelet function, if any, has not been determined. We sought to determine whether the minor allele HPA-5b (534K) might influence the a2b1-mediated adhesion of platelets to a physiologically relevant ligand other than collagen I. One such alternative ligand is decorin, an extracellular matrix small leucine (Leu)-rich proteoglycan (SLRP). Methods/Results. Two groups of normal subjects were compared. The first group (GG) consisted of ten donors homozygous for the major allele rs1801106G. The second group (GA+AA) included one donor homozygous for the minor allele rs1801106A and nine donors heterozygous for these alleles. Aside from this, there were no significant differences between the two groups with respect to platelet count, mean platelet volume, surface expression of four selected platelet receptors, GPIba, GPVI, aIIbb3 or a2b1, or the allelic distribution of five receptor genes, ITGA2B rs5911, ITGB3 rs5918, GP1BA rs6065, GP6 rs1613662 and P2RY1 rs1065776. In direct platelet adhesion assays, we determined that platelets from GG donors bound more strongly to decorin than those from GA+AA donors (p < 0.01), while platelets from either group bound equally well to collagen I (p = 0.73). Using platelets from donors homozygous for the major allele rs1801106G, adhesion to decorin was significantly inhibited by human alloantibodies specific for HPA-5a but not by GFOGER or an a2-specific monoclonal antibody 6F1 known to inhibit cell adhesion to collagen I. Conversely, GFOGER and 6F1 inhibited adhesion to collagen I but not decorin. Conclusions. The simplest explanation of our findings is that 534E, located in the a2 b-propeller domain, is necessary for maximal binding of a2b1 to decorin but not collagen I. These results suggest that 534E contributes to the decorin binding site and that this amino acid represents a potential target for interventions that can modify cell adhesion to decorin and perhaps other proteoglycans. This also represents the first instance of a functional difference attributable to the HPA-5 alleles. These results expand our understanding of the decorin and collagen I interactions described in murine and human studies of cell adhesion and cancer biology, where decorin and collagen I often have opposite effects that are both mediated by a2b1. Decorin knockout (Dcn(−/−)) murine embryonic fibroblasts exhibit greater adhesion to collagen and greater migration on collagen substrates, while decorin attenuates the aggressiveness and metastasis of tumor cells with diverse histologic backgrounds. Further studies of the relationship between the collagen I and decorin binding sites on integrin a2 are warranted, particularly regarding its influence on thrombosis and hemostasis. 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.