Academic literature on the topic 'GP1BB'

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.

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.

SummaryWe describe a syndrome of thrombocytopenia, bleeding episodes, congenital heart disease and facial dysmorphism in a newborn infant, and trace the cause to mutations on chromosome 22 that involve the gene for platelet glycoprotein Ibβ (GPIbβ, Human Genome Organisation gene symbol GPIBB), a critical component of the von Willebrand factor (vWF) receptor. Fluorescence in situ hybridization in transformed lymphoblasts revealed hemizygous microdeletion of 22q11.2 containing the GP1BB locus. DNA sequencing revealed a C to T transition in the patient’s remaining GP1BB allele, predicting a novel proline to serine substitution (Pro96Ser) in the carboxyterminal flanking domain of a leucine-rich repeat. We characterized the mutant GP1BB allele by expression in a cell line (CHOαIX) that stably expresses two other components of the vWF receptor, GPIbα and GPIX. Flow cytometry and confocal imaging of transfected CHOαIX cells demonstrated that P96S GPIbβ abrogates surface assembly of the complex, consistent with platelet flow cytometry studies in the patient. Based on sequence homology to the known crystal structures of two other leucine-rich repeat proteins, the human Nogo receptor and GPIbα, we propose a new structural model of GPIbβ. The model refutes earlier assumptions about cysteine-cysteine interactions in the amino-terminal region of GPIbβ, and predicts a hydrophobic patch the burial of which may contribute to proper conformation of the fully assembled vWF receptor complex.

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.

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.

In view of the proven link between adult hippocampal neurogenesis (AHN) and learning and memory impairment, we generated a straightforward adult neurogenesis in vitro model to recapitulate DNA methylation marks in the context of Alzheimer’s disease (AD). Neural progenitor cells (NPCs) were differentiated for 29 days and Aβ peptide 1–42 was added. mRNA expression of Neuronal Differentiation 1 (NEUROD1), Neural Cell Adhesion Molecule 1 (NCAM1), Tubulin Beta 3 Class III (TUBB3), RNA Binding Fox-1 Homolog 3 (RBFOX3), Calbindin 1 (CALB1), and Glial Fibrillary Acidic Protein (GFAP) was determined by RT-qPCR to characterize the culture and framed within the multistep process of AHN. Hippocampal DNA methylation marks previously identified in Contactin-Associated Protein 1 (CNTNAP1), SEPT5-GP1BB Readthrough (SEPT5-GP1BB), T-Box Transcription Factor 5 (TBX5), and Nucleoredoxin (NXN) genes were profiled by bisulfite pyrosequencing or bisulfite cloning sequencing; mRNA expression was also measured. NXN outlined a peak of DNA methylation overlapping type 3 neuroblasts. Aβ-treated NPCs showed transient decreases of mRNA expression for SEPT5-GP1BB and NXN on day 9 or 19 and an increase in DNA methylation on day 29 for NXN. NXN and SEPT5-GP1BB may reflect alterations detected in the brain of AD human patients, broadening our understanding of this disease.

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.

SummaryThe bleeding disorder Bernard-Soulier syndrome (BSS) is caused by mutations in the genes coding for the platelet glycoprotein GPIb/IX receptor. The septin SEPT5 is important for active membrane movement such as vesicle trafficking and exocytosis in non-dividing cells (i.e. platelets, neurons). We report on a four-year-old boy with a homozygous deletion comprising not only glycoprotein Ibβ (GP1BB) but also the SEPT5 gene, located 5’ to GP1BB. He presented with BSS, cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect. The homozygous deletion of GP1BB and SEPT5, which had been identified by PCR analyses, was confirmed by Southern analyses and denaturing HPLC (DHPLC). The parents were heterozygous for this deletion. Absence of GPIbβ and SEPT5 proteins in the patient’s platelets was illustrated using transmission electron microscopy. Besides decreased GPIb/IX expression, flow cytometry analyses revealedimpaired platelet granule secretion. Because the bleeding disorder was extremely severe, the boy received bone marrow transplantation (BMT) from a HLA-identical unrelated donor. After successful engraftment of BMT, he had no more bleedingepisodes. Interestingly, also his mental development improved strikingly after BMT. This report describes for the first time a patient with SEPT5 deficiency presenting with cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect.

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.

2013/2014
2013/2014
La sindrome di Bernard-Soulier (BSS) è una rara piastrinopenia ereditaria causata da alterazioni a livello del complesso glicoproteico GPIb-IX-V, presente sulla membrana piastrinica e responsabile della adesione delle piastrine in seguito a danno vascolare. La BSS si trasmette come malattia autosomica recessiva (BBSA1) e i pazienti affetti presentano piastrine giganti e severi episodi di sanguinamento. Tuttavia in tempi recenti sono state descritte delle famiglie con una forma dominante nota come BSSA2. In questi pazienti la piastrinopenia è moderata e le piastrine presentano un volume leggermente aumentato. Finora sono state individuate solo 5 varianti in eterozigosi nel BSSA2:, 4 nel gene GP1BA e 1 in GP1BB. Fatta eccezione per p.Ala172Val del gene GP1BA che è relativamente frequente nella la popolazione Italiana, le altre 4 sono state descritte in singole famiglie. I pochi casi di cui disponiamo, soprattutto per la forma recessiva non ci permettono di avere informazioni sui meccanismi patogenetici e sulla sua evoluzione nel tempo. Per questo motivo è stato istituito un Consorzio Internazionale per lo studio della BSS grazie al quale è stato possibile raccogliere i dati clinici e molecolari di 132 famiglie. Tutte le informazioni sono state inserite in un database (BSS Consortium database) attualmente gestito dal nostro laboratorio e consultabile dai gruppi di studio che hanno aderito al Consorzio. Inoltre per aumentare le informazioni sulle varianti identificate nel BSSA1 abbiamo incrementato i dati molecolari delle famiglie del Consorzio con i dati di altre 79 famiglie descritte in letteratura, raggiungendo un totale di 211 famiglie. Tutte le mutazioni identificate in queste famiglie sono state poi inserite in un database pubblico disponibile in rete (LOVD: Leiden Open Variation Database). La raccolta e l’elaborazione dei dati ci ha permesso di chiarire alcuni aspetti clinici e molecolari della malattia. Tuttavia data l’eterogeneità genetica e l’elevata espressione fenotipica gli studi genotipo-fenotipo si sono rivelati difficili da eseguire. Nonostante le molte informazioni acquisite, il database risulta ancora incompleto e limitato; per questo motivo è necessario raccogliere nuovi casi e inserire assieme alle varianti anche i relativi studi funzionali che si rivelano indispensabili per poter definire l’effetto delle varianti sul complesso GPIb-IX-V. Nell’ambito invece dello studio e caratterizzazione della forma meno grave di BSS (BSSA2) sono stati selezionati 120 pazienti piastrinopenici senza diagnosi caratterizzati da piastrine grandi. In questi pazienti sono stati analizzati i geni GP1BA, GP1BB e GP9 e sono state identificate 11 diverse varianti: 1 nonsense, 2 mutazioni di framshift, 1 mutazione nel codone di inizio e 5 varianti missense. Gli studi funzionali eseguiti sulle varianti missense per stabilire il loro ruolo patogenetico sono ancora in corso. Tuttavia se gli studi dovessero confermare la loro patogenicità 11 pazienti su 120 risulterebbero BSSA2 e questa forma dovrebbe essere considerata una tra le piastrinopenie ereditarie più frequenti in Italia. In conclusione grazie a questo studio è stato possibile raccogliere la più ampia casistica di pazienti affetti da BSSA1 fin’ora descritta e ottenere numerose informazioni sia sulla clinica che sulle mutazioni coinvolte. Il BSS Consortium database permetterà ai clinici che hanno partecipato allo studio di osservare nel tempo l’andamento della malattia nei pazienti e di ottenere informazioni utili per stabilire un corretto protocollo per la presa in carico dei pazienti. Infine la caratterizzazione di nuove forme di BSSA2 rappresenta il punto di partenza per descrivere al meglio la malattia BSSA2 sia dal punto di vista clinico che molecolare. In futuro sarà quindi indispensabile estendere il BSS Consortium database anche alla forma BSSA2.
XXVII Ciclo
XXVII Ciclo
1979

Background: Pharmacogenetics promises better control of diseases, such as Cardiovascular disease (CVD). Acetylsalicylic acid, aspirin, prevents the formation of an activating agent of platelet aggregation and vasoconstriction, and it is used to prevent CVD. Nevertheless, patients may have treatment failure, causing recurrence and increased mortality, due to medication adherence, drug- drug interactions, aspirin-independent thromboxane A2 synthesis or genetic variants. In this sense, genetic variants have been related with aspirin resistance (AR). Objective: To evaluate the evidence of impact of genetic variants on AR through systematic literature review. Design and setting: Systematic review. Methods: Articles published since 2009 in MEDLINE/PubMed, Cochrane, Scopus, LILACS and SCIELO were systematically screened. Results: The genetic variants rs1126643 (ITGA2), rs3842787 (PTGS1), rs20417 (PTGS2) and rs5918 (ITGB3) were the most studied. As for the relevance of the genetic variants studied, of the 64 evaluated, 14 had statistical significance (p <0.05, 95% CI) in at least one article. Among them, the following have had unanimous. Results: rs1371097 (P2RY1), rs1045642 (MDR1), rs1051931 and rs7756935 (PLA2G7), rs2071746 (HO1), rs1131882 and rs4523 (TBXA2R), rs434473 (ALOX12), rs9315042 (ALOX5AP) and rs662 (PON1). While these differ in real interference in AR: rs5918 (ITGB3), rs2243093 (GP1BA), rs1330344 (PTGS1) and rs20417 (PTGS2). Conclusion: As limitations of our study, we highlight the non-uniform methodologies of the analyzed articles, as well as population differences. It is also noteworthy that pharmacogenetics is an expanding area. Therefore, further studies are needed to better understand the association between genetic variants and AR, as well as the practical application.