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

BACKGROUND: The 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/METHODS: Subjects 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. RESULTS: Of 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. CONCLUSIONS: Our 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.

Molecular characterization of 16 hemophilia B families in Aragon, Spain.

Molecular characterization of hemophilia B at gene level has become an indispensable tool for a proper genetic counseling in carriers and for a closer surveillance of inhibitor development in several severe forms. Our study was aimed at characterizing the molecular defects in the factor IX (FIX) gene in hemophilia B families in Aragon, a center-east region of Spain. Direct sequencing of all regions of likely functional significance of the FIX gene was performed in the screened 18 hemophilia B families. Quantitative techniques, such as multiplex ligation-dependent prove amplification reaction, were carried out only in patients in whom no mutation was found. We have identified the molecular events responsible for hemophilia B in 16 unrelated families (eight with mild hemophilia B and eight with severe hemophilia B). Out of all families studied, we have found 14 missense mutations and two nonsense mutations; still we were unsuccessful in determining the genetic defects in two severe and unrelated families. Of the 16 characterized mutations, 14 of them lie in the protease domain in which one mutation, A233T, was surprisingly found in three unrelated families. We also report and discuss the pathogenicity of F314L, a novel mutation found in the protease domain. Our molecular data reflect a notable heterogeneity of the mutational spectrum mainly in the protease domain of FIX. This is the first mutation report on the disease in Aragon, Spain.