Functional and molecular characterization of inherited platelet disorders in the Iberian Peninsula: results from a collaborative study

Abstract

BackgroundThe diagnostic evaluation of inherited platelet disorders (IPDs) is complicated and time-consuming, resulting in a relevant number of undiagnosed and incorrectly classified patients. In order to evaluate the spectrum of IPDs in individuals with clinical suspicion of these disorders, and to provide a diagnostic tool to centers not having access to specific platelets studies, we established the project “Functional and Molecular Characterization of Patients with Inherited Platelet Disorders” under the scientific sponsorship of the Spanish Society of Thrombosis and Haemostasis.Patients/methodsSubjects were patients from a prospective cohort of individuals referred for clinical suspicion of IPDs as well as healthy controls. Functional studies included light transmission aggregation, flow cytometry, and when indicated, Western-blot analysis of platelet glycoproteins, and clot retraction analysis. Genetic analysis was mainly performed by sequencing of coding regions and proximal regulatory regions of the genes of interest.ResultsOf the 70 cases referred for study, we functionally and molecularly characterized 12 patients with Glanzmann Thrombasthenia, 8 patients with Bernard Soulier syndrome, and 8 with other forms of IPDs. Twelve novel mutations were identified among these patients. The systematic study of patients revealed that almost one-third of patients had been previously misdiagnosed.ConclusionsOur study provides a global picture of the current limitations and access to the diagnosis of IPDs, identifies and confirms new genetic variants that cause these disorders, and emphasizes the need of creating reference centers that can help health care providers in the recognition of these defects.

Novel mutations in RASGRP2, which encodes CalDAG-GEFI, abrogate Rap1 activation, causing platelet dysfunction

“…Platelet-poor plasma (PPP) was prepared by a further centrifugation step of the same tube at 1000g for 20 minutes. Light transmission aggregometry was performed essentially as described 3 by using undiluted PRP in an Aggrecorder II aggregometer (Menarini Diagnostics, Florence, Italy). Time course changes in the maximal percentage of light transmission of PRP over baseline (PPP) were recorded for 300 seconds upon addition of various platelet agonists (1.5 mM arachidonic acid, 25 mM thrombin receptor-activating peptide (such as protease-activated receptor 1 agonist peptide [PAR1p]), 2 and 10 mg/mL collagen, 10 mM ADP, or 100 nM phorbol 12-myristate 13-acetate (PMA).…”

“…3,4 Impaired platelet aggregation and bleeding can also be caused by mutations in genes for signaling proteins that are critical to the inside-out activation of a IIb b 3 . RASGRP2 was recently identified as one such gene affected in patients with a platelet function defect and a bleeding complication.…”

Allogeneic hematopoietic cell transplantation in an adult patient with Glanzmann thrombasthenia

“…Platelet phenotype and genotype of the index case. The platelet phenotype and genotype of the index case were assessed essentially as described elsewhere . (A) Platelet‐rich plasma ( PRP ) from the index case and a parallel control were prepared from citrated blood, and the platelet aggregation response to different agonists was assessed by standard light transmission aggregometry.…”

Speaker Abstracts

“…4 The laboratory and molecular heterogeneity of IPDs challenges their diagnosis, which may result in poor clinical management of the patients and the inappropriate use of invasive therapies such as splenectomy. 2,3,5 Other difficulties for IPDs diagnosis are poor reproducibility and specificity of platelet function tests and limited access to molecular. 6,7 Multicenter projects, such as our project "Functional and molecular characterization of patients with IPDs" under the scientific coverage of the Spanish Society of Thrombosis and Hemostasis, helps these limitations.…”

“…6,7 Multicenter projects, such as our project "Functional and molecular characterization of patients with IPDs" under the scientific coverage of the Spanish Society of Thrombosis and Hemostasis, helps these limitations. 5,8 Until recently, the molecular diagnosis of IPDs was addressed by sequencing of candidate genes "identified" by the clinical and biological phenotype of the patients. This approach led to molecular diagnosis in less than 50% of patients.…”