Linking aberrant glycosylation of plasma glycoproteins with progression of myelodysplastic syndromes: a study based on plasmonic biosensor and lectin array.

Aberrant glycosylation of glycoproteins has been linked with various pathologies. Therefore, understanding the relationship between aberrant glycosylation patterns and the onset and progression of the disease is an important research goal that may provide insights into cancer diagnosis and new therapy development. In this study, we use a surface plasmon resonance imaging biosensor and a lectin array to investigate aberrant glycosylation patterns associated with oncohematological disease-myelodysplastic syndromes (MDS). In particular, we detected the interaction between the lectins and glycoproteins present in the blood plasma of patients (three MDS subgroups with different risks of progression to acute myeloid leukemia (AML) and AML patients) and healthy controls. The interaction with lectins from Aleuria aurantia (AAL) and Erythrina cristagalli was more pronounced for plasma samples of the MDS and AML patients, and there was a significant difference between the sensor response to the interaction of AAL with blood plasma from low and medium-risk MDS patients and healthy controls. Our data also suggest that progression from MDS to AML is accompanied by sialylation of glycoproteins and increased levels of truncated O-glycans and that the number of lectins that allow discriminating different stages of disease increases as the disease progresses.

Diagnosing Czech Patients with Inherited Platelet Disorders.

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.

Structural and Functional Characterization of Four Novel Fibrinogen Mutations in FGB Causing Congenital Fibrinogen Disorder.

Congenital fibrinogen disorders are caused by mutations in genes coding for fibrinogen and may lead to various clinical phenotypes. Here, we present a functional and structural analysis of 4 novel variants located in the FGB gene coding for fibrinogen Bß chain-heterozygous missense BßY416C and BßA68S, homozygous nonsense BßY345*, and heterozygous nonsense BßW403* mutations. The cases were identified by coagulation screening tests and further investigated by various methods. Fibrin polymerization had abnormal development with decreased maximal absorbance in all patients. Plasmin-induced fibrin degradation revealed different lytic phases of BßY416C and BßW403* than those of the control. Fibrinopeptide cleavage measured by reverse phase high pressure liquid chromatography of BßA68S showed impaired release of fibrinopeptide B. Morphological properties, studied through scanning electron microscopy, differed significantly in the fiber thickness of BßY416C, BßA68S, and BßW403*, and in the fiber density of BßY416C and BßW403*. Finally, homology modeling of BßA68S showed that mutation caused negligible alternations in the protein structure. In conclusion, all mutations altered the correct fibrinogen function or structure that led to congenital fibrinogen disorders.

Thrombosis-associated hypofibrinogenemia: novel abnormal fibrinogen variant FGG c.8G>A with oxidative posttranslational modifications.

Here, we present the first case of fibrinogen variant FGG c.8G>A. We investigated the behaviour of this mutated fibrinogen in blood coagulation using fibrin polymerization, fibrinolysis, fibrinopeptides release measurement, mass spectrometry (MS), and scanning electron microscopy (SEM). The case was identified by routine coagulation testing of a 34-year-old man diagnosed with thrombosis. Initial genetic analysis revealed a heterozygous mutation in exon 1 of the FGG gene encoding gamma chain signal peptide. Fibrin polymerization by thrombin and reptilase showed the normal formation of the fibrin clot. However, maximal absorbance within polymerization was lower and fibrinolysis had a longer degradation phase than healthy control. SEM revealed a significant difference in clot structure of the patient, and interestingly, MS detected several posttranslational oxidations of fibrinogen. The data suggest that the mutation FGG c.8G>A with the combination of the effect of posttranslational modifications causes a novel case of hypofibrinogenemia associated with thrombosis.

A Novel Nonsense Mutation in FGB (c.1421G>A; p.Trp474Ter) in the Beta Chain of Fibrinogen Causing Hypofibrinogenemia with Bleeding Phenotype.

Congenital hypofibrinogenemia is a rare bleeding disorder characterized by a proportional decrease of functional and antigenic fibrinogen levels. Hypofibrinogenemia can be considered the phenotypic expression of heterozygous loss of function mutations occurring within one of the three fibrinogen genes (FGA, FGB, and FGG). Clinical manifestations are highly variable; most patients are usually asymptomatic, but may appear with mild to severe bleeding or thrombotic complications. We have sequenced all exons of the FGA, FGB, and FGG genes using the DNA isolated from the peripheral blood in two unrelated probands with mild hypofibrinogenemia. Coagulation screening, global hemostasis, and functional analysis tests were performed. Molecular modeling was used to predict the defect of synthesis and structural changes of the identified mutation. DNA sequencing revealed a novel heterozygous variant c.1421G>A in exon 8 of the FGB gene encoding a Bß chain (p.Trp474Ter) in both patients. Clinical data from patients showed bleeding episodes. Protein modelling confirmed changes in the secondary structure of the molecule, with the loss of three ß sheet arrangements. As expected by the low fibrinogen levels, turbidity analyses showed a reduced fibrin polymerisation and imaging difference in thickness fibrin fibers. We have to emphasize that our patients have a quantitative fibrinogen disorder; therefore, the reduced function is due to the reduced concentration of fibrinogen, since the Bß chains carrying the mutation predicted to be retained inside the cell. The study of fibrinogen molecules using protein modelling may help us to understand causality and effect of novel genetic mutations.

Hsp70 Trap Assay for Detection of Misfolded Subproteome Related to Myelodysplastic Syndromes.

The onset and progression of numerous serious diseases (e.g., various types of malignancies, neurodegenerative diseases, and cardiac diseases) are, on a molecular level, associated with protein modifications and misfolding. Current methods for the detection of misfolded proteins are not able to detect the whole misfolded subproteome and, moreover, are rather laborious and time consuming. Herein, we report on a novel simple method for the detection of misfolded proteins employing a surface plasmon resonance (SPR) biosensor and heat shock protein 70 (Hsp70) that recognizes and traps misfolded proteins in a nucleotide-dependent manner. We use this method for the detection of misfolded proteins in blood plasma of patients with various subtypes of myelodysplastic syndromes (MDS) and healthy donors. Our results reveal significantly elevated levels of misfolded proteins in the two stages of MDS that are most affected by oxidative stress: low-risk (RARS) and intermediate-risk (RCMD) patients. This approach can be extended to a variety of diseases and provides unique insights into the thus far unexplored area of blood proteome.